Archiving genetically altered animals: a review of cryopreservation and recovery methods for genome edited animals.

With the ever-expanding numbers of genetically altered (GA) animals created in this new age of CRISPR/Cas, tools for helping the management of this vast and valuable resource are essential. Cryopreservation of embryos and germplasm of GA animals has been a widely used tool for many years now, allowing for the archiving, distribution and colony management of stock. However, each year brings an array of advances, improving survival rates of embryos, success rates of in-vitro fertilisation and the ability to better share lines and refine the methods to preserve them. This article will focus on the mouse field, referencing the latest developments and assessing their efficacy and ease of implementation, with a brief note on other common genetically altered species (rat, zebrafish, Xenopus, avian species and non-human Primates).

[Impacts of ODF2 gene knockdown on the sperm motility and fertility of male mice].

OBJECTIVE: To investigate the knockdown of the outer dense fiber protein 2 (ODF2) gene on the sperm motility and fertility of male mice. METHODS: We constructed three knockdown vectors with the target gene ODF2 and one control vector without the target gene. After infecting ICR mice, we determined the vector with the best knockdown effect by RT-PCR and Western blot and reinfected the mice with it. Then we obtained and analyzed the sperm motility parameters, pathological changes of the testis issue, and the litter size of the mice with gene knockdown. RESULTS: Compared with the normal controls, the mice infected with the vector with the best knockdown effect showed significantly decreased sperm motility parameters, pathomorphological abnormalities of the testis, and a reduced litter size (10.86 ± 1.28 vs 12.72 ± 2.05, P = 0.001). CONCLUSION: Decreased expression of the ODF2 gene deceases sperm motility parameters, impairs the morphology of the testis and affects the fertility of male mice.

Ability of Ureaplasma parvum to invade mouse sperm, fertilize eggs through infected sperm, and impair mouse sperm function and embryo development.

OBJECTIVE: To examine the effect of Ureaplasma parvum (U. parvum) infection on mouse sperm motility, structure, and fertilizing ability and on embryo development. DESIGN: In vitro model of the effects of U. parvum serovar 3 infection on mouse sperm. SETTING: Basic research laboratory. INTERVENTION(S): None. ANIMALS: Mice. MAIN OUTCOME MEASURE(S): Mouse sperm motility was examined using the swim-up method, and their motility parameters were analyzed using the sperm motility analysis system. Localization and invasion of U. parvum were observed with fluorescence, confocal, and scanning electron microscopy. After in vitro fertilization with U. parvum-infected sperm, the quality of the fertilized egg and embryo development were assessed. RESULT(S): U. parvum was attached and internalized into mouse sperms and localized mainly at the sperm head and midpiece. U. parvum-infected mouse sperms exhibited decreased motility in a dose- and duration-dependent manner. Electron micrographs revealed that U. parvum infection induced the aggregation and morphological destruction of mouse sperm. Infected mouse sperm transported U. parvum into the fertilized egg with reduced fertilization rates, and infected embryo development was impaired. CONCLUSION(S): U. parvum infection caused deterioration of the mouse sperm quality and its functions, which affected the fertilization rate and embryo development.

Molecular Mechanism Underlying the Action of Zona-pellucida Glycoproteins on Mouse Sperm.

Mammalian oocytes are enveloped by the zona pellucida (ZP), an extracellular matrix of glycoproteins. In sperm, stimulation with ZP proteins evokes a rapid Ca2+ influx via the sperm-specific, pH-sensitive Ca2+ channel CatSper. However, the physiological role and molecular mechanisms underlying ZP-dependent activation of CatSper are unknown. Here, we delineate the sequence of ZP-signaling events in mouse sperm. We show that ZP proteins evoke a rapid intracellular pH i increase that rests predominantly on Na+/H+ exchange by NHA1 and requires cAMP synthesis by the soluble adenylyl cyclase sAC as well as a sufficiently negative membrane potential set by the spem-specific K+ channel Slo3. The alkaline-activated CatSper channel translates the ZP-induced pH i increase into a Ca2+ response. Our findings reveal the molecular components underlying ZP action on mouse sperm, opening up new avenues for understanding the basic principles of sperm function and, thereby, mammalian fertilization.

Preclinical evaluation of a new cryopreservation container for a limited number of human spermatozoa.

The aim of this study was to develop a new container for cryopreservation of a limited number of spermatozoa. To evaluate the efficacy and safety of this new container, we performed preclinical evaluations using human sperm or mouse oocytes and sperm. First, using human sperm that was frozen and then thawed, we demonstrated that the sperm recovery rate using the new container was 96.7% (58/60), which was significantly higher (P < 0.05) than the recovery rate of 21.2% (11/52) when using the Cryotop®. Sperm motility rates were 19.2% (10/52) using the Cryotop® and 35.0% (21/60) using the new container. Second, murine epididymal spermatozoa were divided into three groups: fresh spermatozoa, spermatozoa frozen using a straw, and spermatozoa frozen using the new container. Sperm motility, sperm membrane and DNA integrity, in vitro development of fertilized eggs, and offspring development after embryo transfer were assessed. The motility of freeze-thawed sperm was lower in spermatozoa that were frozen using the new container than in fresh spermatozoa or those that were frozen using a straw. After intracytoplasmic sperm injection, the survival rate was 96.7% (145/150), the 2-cell development rate was 90.3% (131/145), and the blastocyst development rate was 77.2% (112/145), when using the new container. There were no differences in the sperm membrane, DNA integrity, or in the embryo development rates to the blastocyst stage among the different frozen groups. Six offspring were derived from spermatozoa freeze-thawed in the new container, and they developed normally. Thus, the new container allows easy handling of a small number of sperms and minimizes sperm loss during cryopreservation.

Nuclear Integrity but Not Topology of Mouse Sperm Chromosome is Affected by Oxidative DNA Damage.

Recent studies have revealed a well-defined higher order of chromosome architecture, named chromosome territories, in the human sperm nuclei. The purpose of this work was, first, to investigate the topology of a selected number of chromosomes in murine sperm; second, to evaluate whether sperm DNA damage has any consequence on chromosome architecture. Using fluorescence in situ hybridization, confocal microscopy, and 3D-reconstruction approaches we demonstrate that chromosome positioning in the mouse sperm nucleus is not random. Some chromosomes tend to occupy preferentially discrete positions, while others, such as chromosome 2 in the mouse sperm nucleus are less defined. Using a mouse transgenic model (Gpx5-/-) of sperm nuclear oxidation, we show that oxidative DNA damage does not disrupt chromosome organization. However, when looking at specific nuclear 3D-parameters, we observed that they were significantly affected in the transgenic sperm, compared to the wild-type. Mild reductive DNA challenge confirmed the fragility of the organization of the oxidized sperm nucleus, which may have unforeseen consequences during post-fertilization events. These data suggest that in addition to the sperm DNA fragmentation, which is already known to modify sperm nucleus organization, the more frequent and, to date, the less highly-regarded phenomenon of sperm DNA oxidation also affects sperm chromatin packaging.

Design, Fabrication, and Testing of a Microfluidic Device for Thermotaxis and Chemotaxis Assays of Sperm.

Infertile couples needing assisted reproduction are increasing, so a fundamental understanding of motile sperm migration is required. This paper presents an advanced microfluidic device for sperm motion analysis utilizing chemotaxis and thermotaxis simultaneously for the first time. The proposed device is a transparent polydimethylsiloxane- and glass-based microfluidic chip system providing a low-cost, useful, and disposable platform for sperm analysis. The concentration gradient of the chemoattractant (acetylcholine) and the temperature difference are formed along the microchannel. The temperature gradient is generated and controlled by a microheater and microsensor. Thermotactic and chemotactic responses of mouse sperm were examined using the proposed device. Experimental results show that motile mouse sperm are attracted more sensitively under integrated conditions of chemotaxis and thermotaxis rather than individual conditions of chemotaxis and thermotaxis. This sperm analysis device is expected to be a useful tool for the study of mammalian sperm migration and the improvement of artificial insemination techniques.

Vitamin C exerts novel protective effects against cadmium toxicity in mouse spermatozoa by inducing the dephosphorylation of dihydrolipoamide dehydrogenase.

Cadmium (Cd) has been reported to inhibit mouse sperm motility by inducing the tyrosine phosphorylation of dihydrolipoamide dehydrogenase (DLD). This study aimed to assess the potential effects of vitamin C (Vc) on ameliorating Cd-induced tyrosine phosphorylation of DLD and the specific underlying mechanism. Vc induced the dephosphorylation of DLD or inhibited the tyrosine phosphorylation of DLD. Accordingly, DLD activity, nicotinamide adenine dinucleotide hydrogen (NADH) levels, ATP levels and motility parameters were all restored to normal levels by Vc. Moreover, the effects of Vc on ameliorating these indicators had striking similarities to the effects of ethylenediaminetetraacetic acid (EDTA). In addition, neither the antioxidant melatonin nor the universal oxidant H2O2 influenced the tyrosine phosphorylation of DLD. Hence, the protective effects of Vc on the tyrosine phosphorylation of DLD might be attributed to its binding to Cd ions outside or inside sperm, and were not due to its antioxidant properties.

Lipid-soluble green tea extract: Genotoxicity and subchronic toxicity studies.

To assess the potential safety of lipid soluble green tea extract, also referred to as lipid soluble tea polyphenols (LSTP), a series of genotoxicity tests were conducted, including an Ames, in vivo mouse micronucleus, and in vivo mouse sperm abnormality test. The toxicity of LSTP was evaluated in 90- and 30-day feeding studies. LSTP did not show mutagenic activity in the Ames test and no genotoxic potential in the in vivo assays at doses up to 10 g/kg body weight (bw). In the 90-day feeding study, LSTP was given in the diet at levels providing 0, 0.125, 0.25, or 0.50 g/kg bw/day. No significant effects were noted on body weight, food consumption, hematology, clinical chemistry, organ weights, and histopathological examination. The no-observed-adverse-effect level (NOAEL) was therefore considered to be 0.50 g/kg bw/day, the highest dose tested. Likewise, dosing of SD rats by gavage for 30 days also showed no adverse effects of growth, hematology, clinical chemistry, organ weights, or histopathology at doses of 0.58, 1.17, and 2.33 g/kg bw/day. The NOAEL in the 30-day study was considered to be the highest dose tested. These data provide evidence to support the safe use of LSTP in food.

Tyrosine phosphorylation of dihydrolipoamide dehydrogenase as a potential cadmium target and its inhibitory role in regulating mouse sperm motility.

Cadmium (Cd) is reported to reduce sperm motility and functions. However, the molecular mechanisms of Cd-induced toxicity remain largely unknown, presenting a major knowledge gap in research on reproductive toxicology. In the present study, we identified a candidate protein, dihydrolipoamide dehydrogenase (DLD), which is a post-pyruvate metabolic enzyme, exhibiting tyrosine phosphorylation in mouse sperm exposed to Cd both in vivo and in vitro. Immunoprecipitation assay demonstrated DLD was phosphorylated in tyrosine residues without altered expression after Cd treatment, which further confirmed our identified result. However, the tyrosine phosphorylation of DLD did not participate in mouse sperm capacitation and Bovine Serum Albumin (BSA) effectively prevented the tyrosine phosphorylation of DLD. Moreover, Cd-induced tyrosine phosphorylation of DLD lowered its dehydrogenase activity and meanwhile, Nicotinamide Adenine Dinucleotide Hydrogen (NADH) content, Adenosine Triphosphate (ATP) production and sperm motility were all inhibited by Cd. Interestingly, when the tyrosine phosphorylation of DLD was blocked by BSA, the decrease of DLD activity, NADH and ATP content as well as sperm motility was also suppressed simultaneously. These results suggested that Cd-induced tyrosine phosphorylation of DLD inhibited its activity and thus suppressed the tricarboxylic acid (TCA) cycle, which resulted in the reduction of NADH and hence the ATP production generated through oxidative phosphorylation (OPHOXS). Taken together, our results revealed that Cd induced DLD tyrosine phosphorylation, in response to regulate TCA metabolic pathway, which reduced ATP levels and these negative effects led to decreased sperm motility. This study provided new understanding of the mechanisms contributing to the harmful effects of Cd on the motility and function of spermatozoa.

Cadmium inhibits mouse sperm motility through inducing tyrosine phosphorylation in a specific subset of proteins.

Cadmium (Cd) has been reported to impair male fertility, primarily by disrupting sperm motility, but the underlying molecular mechanism remains unclear. Here we investigated the effects of Cd on sperm motility, tyrosine phosphorylation, AMP-activated protein kinase (AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, and ATP levels in vitro. Our results demonstrated that Cd inhibited sperm motility, GAPDH activity, AMPK activity and ATP production, and induced tyrosine phosphorylation of 55-57KDa proteins. Importantly, all the parameters affected by Cd were restored to normal levels when incubated with 10µM Cd in the presence of 30µM ethylene diamine tetraacetic acid (EDTA). Interestingly, changes of tyrosine phosphorylation levels of 55-57KDa proteins are completely contrary to that of other parameters. These results suggest that Cd-induced tyrosine phosphorylation of 55-57KDa proteins might act as an engine to block intracellular energy metabolism and thus decrease sperm motility.

Broccoli seed extract: Genotoxicity and subchronic toxicity studies.

Potential health benefits have been attributed to broccoli consumption. Hence, there is potential for use of broccoli seed extract (BSE) in food or for use as a dietary supplement. To assess the potential safety of a BSE product, three genotoxicity experiments, including an Ames, in vivo mouse micronucleus, and in vivo mouse sperm abnormality assay, were carried out. BSE was subject to an acute oral toxicity test and was evaluated in a 30-day feeding study in rats. BSE showed no mutagenic activity in the Ames assay and no evidence of genotoxic potential in the in vivo assays at doses up to 10 g/kg body weight (bw). The LD50 of BSE in rats was >10 g/kg bw/d. In the 30-day feeding study, in which BSE was administered in the diet to provide doses of 0, 0.3, 1.0, or 3.0 g/kg bw/d, no toxicological significant effects were noted on body weight, body weight gain, organ weights, or on the results of hematological, clinical chemistry and histopathological evaluations. The no-observed-adverse-effect level was considered to be 3.0 g/kg bw/d, the highest dose tested. Collectively, these results support the safe use of BSE as a food ingredient or product.

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells.

In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1(-/-)) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1(-/-) mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex--that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.

In vitro fertilization embryo development from caffeine-treated murine sperm.

Purpose: To evaluate the effect of long-term caffeine administration on murine sperm and subsequent in vitro fertilization (IVF). Methods: Male mice were injected with various doses (0, 0.2 and 1.0 mg/mouse/day) of caffeine for 1 month. After sperm collection, the IVF rate and embryo development to the blastocyst stage were evaluated. Results: The mean body weight significantly decreased in the 1.0 mg/day treatment group compared to the control group (P < 0.01). Testicular weight and histological features did not differ, and total blood testosterone was no different in spite of the difference between 0.2 and 1.0 mg/day of caffeine. The IVF rate differed significantly between the control group [100/105 (95.2 %)] and 0.2 mg/day group [106/121 (87.6 %)] (P < 0.05). Furthermore, blastocyst formation was significantly and dose-dependently lower with higher caffeine levels: control group: 85/100 (85.0 %); 0.2 mg/day group: 84/106 (79.2 %) (P < 0.05); 1.0 mg/day group: 64/102 (62.7 %) (P < 0.001). Conclusions: Caffeine treatment affected body weight of male mice. However, testicular weight, histological features and total blood testosterone concentration were not statistically different. In addition, following IVF using sperm from these mice, blastocyst formation decreased in a dose-dependent manner. These findings suggest that embryo development from oocytes fertilized with sperm from caffeine-administered male mice is negatively affected.

Effect of cigarette smoke on DNA damage, oxidative stress, and morphological alterations in mouse testis and spermatozoa.

Although the adverse effects of active smoking on sperm quality and fertilization ability are well established, little is known about possible effects of involuntary exposures to cigarette smoke (CS). We designed an experimental study aimed at evaluating the induction of possible noxious effects on testicular morphology and functions in A/J mice exposed whole-body to CS during the first 70 days of life, from birth to early adulthood. Twenty-five sham-exposed neonatal mice and 23 CS-exposed neonatal mice were used. Exposure to CS caused a variety of interconnected alterations in male gonads, including loss of weight and histomorphological alterations of testis, accompanied by a significant increase in abnormalities affecting epidydimal spermatozoa. Induction of oxidative stress was demonstrated by significantly increased concentrations of both reactive oxygen species and lipid peroxidation products in sperm cells. Occurrence of DNA damage in the same cells was documented by using the single cell gel electrophoresis (comet) assay, which showed a remarkable increase in DNA single- and double-strand breaks in CS-exposed mice, as compared with sham-exposed mice. Since biochemical and molecular alterations of sperm cells are known to be associated with impaired sperm quality, our findings suggest that involuntary smoking is potentially able to impair fertility in subjects exposed early in life.

Effect of metformin on the fertilizing ability of mouse spermatozoa.

Numerous antioxidants have been added to cryopreservation media with varied success. The biguanide, metformin, commonly used for the treatment of type II diabetes, possesses properties impacting metabolism control that have not been yet assessed in cryopreservation protocols. The aim of this experiment was to; (i) determine the effect of metformin on fresh spermatozoa properties; and (ii) to assess positive or negative effects of metformin in post-thaw function and fertilizing capacity of mouse spermatozoa when used in cryopreservation media. The experiments have shown that the presence of metformin in fresh semen did not induce negative effects on spermatozoa quality, except a slight reduction in sperm motility at 5000µM metformin. However, when metformin was included in a cryopreservation protocol, an improvement in the fertilization rate and a reduction in the percentage of abnormal zygotes after in vitro fertilization was observed. In conclusion, metformin did not affect sperm quality at low concentrations (50µM), but its presence in the cryopreservation media could represent a benefit to improve the quality of frozen semen.

Protective effect of melatonin on damage in the sperm parameters of mice exposed to diazinon / Efecto protector de la melatonina sobre el daño en los parámetros espermáticos de ratones expuestos a diazinón

Since normal sperm parameters can be altered by organophosphorous pesticides, this study intended to determine if melatonin is able to prevent the damage on sperm quality after an acute exposure to diazinon. Adult male mice were injected intraperitoneally with melatonin, diazinon (1/3 or 2/3 LD50) or both, and sperm parameters were evaluated on days 1 or 32 post injection. Groups treated with diazinon showed elevated lipid peroxidation levels on day 1 post treatment, while groups pretreated with melatonin before diazinon showed no difference compared to control. Sperm count showed a significant decrease in both diazinon-treated groups only on day 32 post injection; no differences were observed in groups pretreated with melatonin prior to diazinon compared to control. The percentage of abnormal sperm morphology increased in the diazinon-treated groups only on day 32 postinjection. The administration of melatonin prior to exposure to diazinon prevents the alteration of sperm parameters commonly caused by organophosphates, possibly due to its antioxidant properties.

Debido a que los parámetros normales de los espermatozoides pueden ser alterados por algunos contaminantes como los pesticidas organofosforados, este estudio pretende determinar si melatonina es capaz de prevenir o proteger del daño en la calidad espermática, después de una exposición aguda a diazinon. Ratones machos adultos fueron inyectados via intraperitoneal con diazinon 1/3 y 2/3 de la LD50 y otro grupo tratados con melatonina + 1/3 diazinon LD50 y melatonina + 2/3 LD50. Los parámetros espermáticos fueron evaluados al día 1 y al día 32 post tratamiento. Los grupos tratados con diazinon solo o conjugado con melatonina mostraron un incremento significativo en los niveles de lipoperoxidación en el tratamiento después de un día. Al día 32 no se observan diferencias significativas con el grupo control. El recuento espermático al día 1 no presenta diferencias entre los grupos tratados y el control. Sin embargo al día 32 los grupos tratados con diazinon solo, muestran una disminución significativa, solo el grupo de melatonina +1/3 diazinon, presenta valores similares al grupo control. La morfología espermática normal presenta una disminución significativa en grupos tratados con diazinon, pero un aumento significativo al día 32 en los grupos tratados con melatonina. Los mayores porcentajes de anormalidades se presentan en la cabeza y la cola de los espermatozoides. La administración de melatonina antes de la exposición al diazinon evita las alteraciones de los parámetros espermáticos, comúnmente causada por organofosforados, posiblemente debido a sus propiedades antioxidantes.

Cryoprotective effects of various saccharides on cryopreserved mouse sperm from various strains.

Aim: Cryopreservation of mouse sperm commonly uses raffinose, which is a trisaccharide, plus 3% skim milk. Because of the present lack of knowledge of the effectiveness of any other saccharides, we examined the cryoprotective effects of various saccharides on the viability of mouse sperm from various strains to determine which saccharides are the best cryoprotectants for mouse sperm. Methods: Sperm from the caudae epididymides of mature C57BL/6J mice were frozen with monosaccharides (fructose, glucose, rhamnose, xylose), disaccharides (lactose, maltose, sucrose, trehalose) or trisaccharides (melezitose, raffinose) in a range of concentrations (4-33%). After thawing, the optimal concentration was determined to be the concentration in which there was the highest proportion of motile sperm. In addition, sperm of inbred and hybrid mice were frozen with the saccharides at the optimal concentrations and used for in vitro fertilization. Results: The optimal concentration was 12% for the disaccharides and 18% for the trisaccharides. The fertility of all strains, except C57BL/6J, showed the best cryoprotective effects with maltose, melezitose and raffinose when compared with fresh sperm. Conclusion: Maltose, melezitose and raffinose have the best effects when used as a protectant for cryopreservation of mouse sperm. (Reprod Med Biol 2007; 6: 229-233).

The Effect of Pentoxifylline on In Vitro Fertilization and Development of Mouse Normal Sperm / 대한산부인과학회잡지

OBJECT: This study was carried out to investigate the effect of pentoxifylline on in vitro fertillization and developmen of preimplantation stage of mouse embryos. MATERIAL AND METHODS:F1 hybrid mice was superovulated with PMSG/hCG and mouse oocytes were recruited. After the normal sperms were incubated with PTX before in vitro fertilization, it was observed whether the fertilization and embryo development was affected or not by the sperm preparation(washing, dilution and no washing or no dilution). And after 1-cell and 2-cell stage of mouse embryos were incubated with PTX, the development to hatching blastocyst was also observed. RESULTS: When in vitro fertilization was revealed by using the washed normal sperms after 0, 3.6 and 7.2 mM PTX incubation, the fertilization rates were 92.5%, 48.8%, 36.8%, respectively. So 3.6 and 7.2 mM groups presented significantly low fertilizatin rate, but the development rates were 93.9%, 85.0%, 95.2%, respectively. Therefore, there were no significant difference between each group. When in vitro fertilization was revealed by using the diluted normal sperms after 0, 3.6, and 7.2 mM PTX incubation, the fertilization rates were 58.6%, 5.4%, 9.4%, respectively. So 3.6 and 7.2 mM groups presented significantly low fertilization rate. The developmental rates were 88.2%, 100%, 100%. And there were no significant difference between each group. When in vitro fertilization was revealed by using the not washed and not diluted normal sperms after 0, 3.6 and 7.2 mM PTX incubation, the fertilizatin rates were 61.2%, 5.7%, 3.8%, respectively. 3.6 and 7.2 mM group presented significantly low fertilization rate. The development rates were 73.3%, 0%, 0%, respectively. So 3.6, 7.2 mM group presented significantly low developmental rate. After 1-cell stage of mouse embryos were incubated in 0, 5, 10, 50 nM of PTX, the development rates were not significantly different among them. After 2-cell stage of mouse embryos were incubated in 0, 5, 10, 50 nM of PTX, the development rates were not significantly different among them. CONCLUSION: In conclusion, when PTX is used in in vitro fertilization program with normal sperms, it may affect the fertilization and embryo development in high concentration. And if PTX concentration is very low, the developmental rate would not be affected. So PTX must not be used to normal sperms and where use of PTX is indicated, it is recommended that remainder PTX must be removed as completely as possible.