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In rats, the success of in vitro fertilization (IVF) was reported 40 years ago. Although it has been demonstrated in papers that these IVF oocytes using sperm freshly collected from cauda epididymides can be developed to term via embryo transfer, successful IVF with cryopreserved rat sperm has never been reported to date. Here, we report establishment of a successful IVF system using frozen/thawed rat spermatozoa. Our data showed that intracellular cAMP and free cholesterol levels in frozen/thawed rat sperm were maintained low, suppressing capacitation-associated tyrosine phosphorylation. The treatment of methyl-beta-cyclodextrin improved removal of free cholesterol from the membrane in frozen/thawed sperm but not induction of capacitation-associated tyrosine phosphorylation in the sperm. Treatment with a phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX), dramatically increased cAMP and tyrosine phosphorylation levels in frozen/thawed rat sperm. When the IBMX-treated frozen/thawed sperm were used for IVF, the proportions of pronuclear formation and blastocyst formation were significantly higher than those of frozen/thawed sperm treated without IBMX (P < 0.05). The embryos were developed to term at a high success rate equivalent to the rate obtained with IVF using fresh sperm. Thus, we established for the first time a successful IVF system in rats using cryopreserved spermatozoa.

When intracytoplasmic sperm injection (ICSI) is applied in the rat, sperm chromatin is introduced into the oocyte together with the acrosome, which does not enter the cytoplasm of the oocyte during normal fertilization, resulting in the rat giving birth to pups. Since successful ICSI was reported in rats, but with low efficiency, it has been observed that the acrosome of the sperm head seems to have detrimental effects on the embryonic development of ICSI oocytes. To improve ICSI in rats, the effects of removal of the acrosomal membrane from rat sperm on the development of ICSI oocytes were examined. While most control (non-treated) sperm had an intact acrosomal membrane, the Triton X-100 (TX)- and lysolecithin (LL)-treated groups showed high percentages of sperm with a removed acrosomal membrane. The timing of pronuclear formation in ICSI-oocytes using TX- or LL-treated sperm was significantly accelerated compared with that of the control sperm (P

The rabbit is considered to be a valuable laboratory animal. We compared glycerol, lactamide, acetamide, and dimethylsulfoxide (DMSO) as cryoprotectants in egg-yolk diluent of ejaculated Japanese white rabbit spermatozoa for improvement of sperm cryopreservation methods. Rabbit semen was frozen with 1.0 M glycerol, lactamide, acetamide, or DMSO in plastic straws. Forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rate of forward progressive motile spermatozoa in lactamide (37.8 +/- 3.0%) was significantly (P

For preservation of rat spermatozoa, the general-purpose method requires that the male be sacrificed for collection of spermatozoa from the epididymides. However, it would be highly useful if the ejaculated spermatozoa could be successfully cryopreserved and the frozen-thawed spermatozoa used for in vitro fertilization, since this would allow the genetically valuable rats to be maintained alive rather than sacrificed. The aim of the present study was to clarify whether ejaculated rat spermatozoa could be successfully cryopreserved and fertilized in vitro. The motility and viability of frozen-thawed ejaculated spermatozoa were similar to those of frozen-thawed epididymal spermatozoa (around 10%). The percentage of acrosomal integrity in epididymal spermatozoa was significantly higher than that in ejaculated spermatozoa after freezing/thawing. The level of capacitation-associated protein tyrosine phosphorylation in frozen-thawed ejaculated sperm was slightly increased at 5h. When the frozen-thawed ejaculated spermatozoa were used for in vitro fertilization, the percentages of fertilization, pronuclear formation, and development to the 2-cell stage (26.5%, 23.0%, and 91.0%, respectively) were similar to those of frozen-thawed epididymal spermatozoa (19.4%, 15.0%, and 84.1%, respectively). However, the rate of blastocyst formation in the ejaculated group was significantly lower than that in the epididymal group (12.0% vs 43.2%). Results from the embryo transfer experiment showed that the proportions of embryos developed to term were similar between the ejaculated (47.7%) and epididymal groups (53.7%). We showed here for the first time that ejaculated spermatozoa can be cryopreserved and the frozen-thawed sperm could be developed to term via in vitro fertilization in rats.

Cryopreservation of matured oocytes is a useful technique because the oocytes can be used for some assisted reproductive technologies after warming. Even though rats, like mice, have been used in various research fields including reproductive technology, information about cryopreservation of rat oocytes is limited. The objective of the present study was to improve the vitrification protocol for matured rat oocytes. To determine the optimal equilibration time, oocytes were equilibrated in 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% fetal calf serum (FCS) for 1, 4, 7 or 10 min at 24 C and then 15% EG + 15% DMSO + 0.5 M sucrose + 20% FCS for 1 min at 24 C before being plunged into liquid nitrogen on Cryotops. Oocytes exposed to equilibration medium for 4 min showed higher survival and cleavage rates after artificial activation than those of oocytes exposed for 1, 7 or 10 min. The survival and cleavage rates of vitrified oocytes after activation were 98.3 and 78.4%, respectively. However, the perivitelline spaces of most of the vitrified/warmed oocytes (6/168, 3.6%) could not be penetrated by sperm after in vitro fertilization, and cortical granule exocytosis (CGE) was observed in the oocytes. Therefore, the inhibitory effect of calcium and cryoprotectants in vitrification medium on CGE was examined. In most of the oocytes vitrified in calcium-free media, CGE was strongly suppressed independent of cryoprotectants. Oocytes vitrified in EG-supplemented calcium-free media showed high survival rates after warming (79.4%). After artificial activation, the cleavage and blastocyst formation rates of the oocytes were also high (72.8 and 23.1%, respectively). The zona penetration rate of vitrified/warmed oocytes was dramatically improved by using EG-supplemented calcium-free media after in vitro fertilization (111/155, 63.9%). Thus, our data suggest that EG-supplemented calcium-free media improve zona penetration of vitrified rat oocytes by sperm cells.

Although in vitro and in vivo fertilization are powerful tools for restoring conserved sperm as well as stocked males in the rat, the techniques have progressively gained importance. However, the techniques are not used extensively for efficient production of rat offspring, because the techniques require a great deal of skill. This chapter describes the protocols for in vitro and in vivo fertilization in the rat. Namely, sperm collection, sperm cryopreservation, pre-incubation of sperm, and insemination (co-culture with sperm and oocytes) for in vitro fertilization and intrauterine insemination for in vivo fertilization with fresh or frozen/thawed spermatozoa are provided.

Embryo cryopreservation is a valuable tool for efficient production of animals as well as banking of genetic resources. Even though the laboratory rat is one of the most important experimental animals for various research fields, it has been reported that survival and developmental ability of cryopreserved rat embryos are generally low, especially at the early stages. The aim of the present study was to establish rapid cooling method that can be applied for cryopreservation of rat pronuclear-stage embryos using Cryotops (a device). First, optimal equilibration time was examined. Pronuclear-stage embryos were equilibrated in 7.5% ethylene glycol (EG) + 7.5% dimethylsulfoxide (DMSO) + 20% fetal calf serum (FCS) for 7, 8 or 9 min at 20–22 °C and then 15% EG + 15% DMSO + 0.5 M sucrose + 20% FCS for 1 min at 20–22 °C, being plunged into liquid nitrogen on Cryotops. This established that development to the 2-cell (82.0 ± 9.7% to 96.1 ± 3.0%) and blastocyst (36.5 ± 2.1% to 40.3 ± 10.2%) stages in vitro was not influenced by the equilibration time. Furthermore development to term in vivo (56.0 ± 4.9%) was equivalent to the rate (54.8 ± 6.6%) obtained with control embryos. Taken together, this demonstrated that this method is suitable for the successful cryopreservation of pronuclear-stage embryos in rats.

SummaryIn nuclear-transferred or round spermatid-injected oocytes, artificial activation is required for further development in mammals. Although strontium chloride is widely used as the reagent for inducing oocyte activation in mice, the optimal method for oocyte activation remains controversial in rats because ovulated rat oocytes are spontaneously activated in vitro before artificial activation is applied. In our previous study, we found that cytostatic factor activity, which is indispensable for arrest at the MII stage, is potentially low in rats and that this activity differs greatly between two outbred rats (Slc: Sprague-Dawley (SD) and Crj: Wistar). Therefore, it is necessary to establish an optimal protocol for oocyte activation independent of strains. Given that comparative studies of the in vitro development of oocytes activated by different activation protocols are very limited, we compared four different protocols for oocyte activation (ethanol, ionomycin, strontium and electrical pulses) in two different SD and Wistar rats. Our results show that oocytes derived from SD rats have significantly higher cleavage and blastocyst formation than those from Wistar rats independent of activation regimes. In both types of rat, ethanol treatment provided significantly higher developmental ability at cleavage and blastocyst formation compared to the other activation protocols. However, the initial culture in a fertilization medium (high osmolarity mR1ECM) for 24 h showed a detrimental effect on the further in vitro development of parthenogenetic rat oocytes. Taken together, our results show that ethanol treatment is the optimal protocol for the activation of rat oocytes in SD and Wistar outbred rats. Our data also suggest that high-osmolarity media are inadequate for the in vitro development of parthenogenetically activated oocytes compared with fertilized oocytes.

The rabbit is considered to be a valuable laboratory animal. We compared 2% acetamide and glycerol as cryoprotectants in egg-yolk diluent for ejaculated Japanese white rabbit spermatozoa to improve sperm cryopreservation methods. Fertility through artificial insemination, forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rates of forward progressive motility and plasma membrane integrity of the spermatozoa frozen with acetamide (27.1 +/- 8.3% and 24.5 +/- 6.5%) were significantly (P0.05) higher than those of the spermatozoa frozen with glycerol (16.3 +/- 10.9% and 14.3 +/- 7.6%). Though there was no significant difference in the kindling rates, the litter size of females inseminated with spermatozoa frozen with acetamide (6.0 +/- 1.1) were significantly (P0.05) higher than those of spermatozoa frozen with glycerol (3.0 +/- 0.4). The results indicate that 2% acetamide has a higher cryoprotective effect than 2% glycerol for sperm cryopreservation in the Japanese white rabbit.

s / Placenta 34 (2013) A1–A99 A22 [1] Stanley et al 2012. Hypertension. 59(5): 1021-8. [2] Constancia et al 2002. Nature. 417, 945-8. http://dx.doi.org/10.1016/j.placenta.2013.06.065 P1.29. THE ROLE OF CDX2 IN TROPHOBLAST DIFFERENTIATION FROM HUMAN PLURIPOTENT TSEM Chi-Wei Lu, Yasunari Seita Rutgers University, New Brunswick, NJ, USA Objective The unique requirement for Cdx2 in the development of mouse placental trophoblasts has been well-studied, in genetic models and through manipulating its expression in embryonic stem cells. The role of CDX2 in human trophoblast differentiation remains elusive. MethodsWe studied the involvement of CDX2 in trophoblast differentiation fromhumanembryonic and induced pluripotent stem cells (ESCs and iPSCs). Results From BMP4-induced trophoblast differentiation, we observed that CDX2 expressed in nearly 100% of cells, and largely overlapping with OCT4 expression, suggesting the model of their mutual transcription repression seen in the mouse is not conserved in humans. We tested whether CDX2 expression alone is associated with a trophoblast progenitor fate, through generating iPS cell lines with ectopic CDX2 expression. Two CDX2 expressing cell lines were analyzed, both expressing telomerase activity and capable of differentiation into syncytiotrophoblast after switching into serum containing media. However, in the cell line expressing CDX2 at a very high level, differentiation into mesoderm and endoderm lineages was also observed, suggesting the effect of CDX2 for trophoblast differentiation is dose dependent. Repressing CDX2 expression by shRNA does not blockade BMP4-induced trophoblast differentiation. Conclusion Altogether, our results suggest that while the mechanism of transcription regulation is different from the mouse, CDX2 in the human plays a conservative role in trophoblast development as being not necessary for differentiation, but sufficient for inducing a trophoblastic-stem cell like phenotype from pluripotent stem cells. http://dx.doi.org/10.1016/j.placenta.2013.06.066 P1.30. ENDOTHELIAL DYSFUNCTION OF THE FETOPLACENTAL CIRCULATION CONTRIBUTES TO THE INCREASED VASCULAR RESISTANCE OBSERVED IN FETAL GROWTH RESTRICTION Sarah Jones, Helen Bischof, Edward Johnstone, Sue Greenwood, Mark Wareing, Colin Sibley, Paul Brownbill University of Manchester, Manchester, UK Fetal growth restriction (FGR) affects 3-8% of pregnancies and is associated with increased risk of neonatalmorbidityandmortality. A hallmark of severe FGR is increased vascular resistance identified clinically using umbilical arteryDopplermeasurements. However, the sites andunderlyingmechanisms of increased vascular resistance in the fetoplacental circulation remain unclear. In this studywe tested the hypothesis that endothelial dysfunction and altered nitric oxide (NO) production in placental vessels contribute to the pathology of FGR. Dual perfusion was performed on placental cotyledons from normal pregnancies and those complicated by idiopathic FGR ( 5th centile). Vascular resistance and shear stress-induced vasodilatation of the fetal vasculaturewasmeasured in response to increasingflow rates (2-12ml/ min), and the role of NOwas examined using the NO synthase inhibitor Nunitro-L-arginine (L-NNA). Incremental increases in fetoplacental flow (212ml/min) in normal placentas elevated vascular resistance in a stepwise manner. This was significantly amplified in perfused cotyledons from FGR pregnancy, compared to those fromnormal pregnancy, particularly at higher flow rates (76.5 9mmHg versus 45.4 6mmHg at 12ml/min respectively; mean sem; n1⁄410 per group). Inhibition of eNOS by L-NNA moderately increased the resistance at high flow rates in normal placentas. In contrast, steady state vascular resistance in FGRplacentaswas augmentedby L-NNAat all flow rates. We propose that this effect of L-NNA may relate to increased levels of NO being produced to compensate for the increased resistance; a theory supported by our work on isolated endothelial cells (see separate abstract). In normal placentas increases in fetal-side flow ratewere followed by significant shear-induced vasodilatationwhichwas inhibited by L-NNA. In FGR placentas responses to shear-mediated vasodilatation were absent. In conclusion, placental vessels from pregnancies complicated by fetal growth restriction exhibit endothelial dysfunction and fail to adapt to increases in flow, resulting in increased vascular resistance. http://dx.doi.org/10.1016/j.placenta.2013.06.067 P1.31. MATERNAL ADMINISTRATION OF SILDENAFIL CITRATE ALTERS FETAL VASCULAR FUNCTION IN A MOUSE MODEL OF FETAL GROWTH RESTRICTION Lewis Renshall, Elizabeth Cowley, Susan Greenwood, Mark Dilworth, Mark Wareing Maternal and Fetal Research Centre, The University of Manchester

In rats, successful IVF with cryopreserved rat sperm has never been reported. The objective of the present study was to establish and improve the IVF protocol using epididymal and ejaculated rat spermatozoa after cryopreservation. At first, we examined whether cryopreserved ejaculated spermatozoa would be useful for IVF in Wistar rats. Capacitation-associated tyrosine phosphorylation was accelerated in frozen-thawed ejaculated sperm in a time-dependent manner. These frozen-thawed spermatozoa were co-cultured with cumulus–oocyte complexes in modified R1ECM for 10 h. The putative zygotes were transferred to R1ECM and then cultured up to 144 h. Although the rates of insemination and 2 pronucleus (2PN) formation were low (26.5 and 23.0%, respectively), most of 2PN oocytes were developed to the 2-cell stage (91.0%). A total of 44 embryos at the 2-cell stage derived from frozen-thawed ejaculated sperm were transferred to 5 recipient females and 21 pups (47.7%) were delivered. Next, we used frozen-thawed epididymal spermatozoa for IVF in Wistar rats. After thawing, intracellular cyclic adenosine monophosphate (cAMP), free cholesterol levels and capacitation-associated protein tyrosine phosphorylation levels of the sperm were assayed. Intracellular cAMP and free cholesterol levels in frozen-thawed epididymal sperm were maintained at a low level, suppressing capacitation-associated tyrosine phosphorylation. Treatment with a phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX) dramatically increased cAMP and capacitation-associated tyrosine phosphorylation levels in frozen-thawed epididymal sperm. When the IBMX-treated frozen-thawed sperm was used for IVF, the proportions of 2PN and the development to blastocysts were significantly higher (approximately 40 and 20%, respectively) than those of frozen-thawed epididymal sperm treated without IBMX (approximately 10 and 3%, respectively). These embryos were developed to term at a high success rate (49%) equivalent to the rate obtained with IVF using fresh sperm (58%). Moreover, we tried to apply our IVF system to inbred rat strains [Fischer 344 (F344) and Brown-Norway (BN)]. We examined whether the IVF protocol was available for F344 and BN rats. Fresh and frozen-thawed sperm collected from cauda epididymides in F344 and BN were used for detection of capacitation-associated tyrosine phosphorylation. In fresh F344 sperm, capacitation-associated tyrosine phosphorylation was induced in a time-dependent manner. Although tyrosine phosphorylation was inhibited in frozen-thawed F344 sperm, it was dramatically accelerated by IBMX treatment as well as frozen-thawed Wistar sperm. However, tyrosine phosphorylation in fresh and frozen-thawed BN sperm was suppressed and the phosphorylation in frozen-thawed sperm was not improved by IBMX. Taken together, we developed an IVF protocol using cryopreserved rat sperm and our data suggest that the IVF system can be applied not only to Wistar rats but also to the F344 strain. This research was also partially supported by a research project grant awarded by the Azabu University Research Services Division.

In rats, the success of in vitro fertilization (IVF) was reported almost 40 years ago. Although it had been demonstrated in papers that these IVF oocytes using sperm freshly collected from cauda epididymides can be developed to term via embryo transfer, successful IVF with cryopreserved rat sperm has never been reported. Very recently, we reported establishment of a successful IVF system using frozen-thawed spermatozoa treated with a phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX), in Wistar rats (Seita et al. 2009 Biol. Reprod.). The objectives in this study were (1) improvement of the IVF system to a more convenient and simple protocol and (2) a preliminary study for applying our IVF system to inbred rat strains Fischer 344 (F344) and Brown-Norway (BN). In experiment 1, we examined the effect of preincubation time for frozen-thawed sperm on fertilization (2 pronuclei formation). Frozen-thawed sperm were preincubated up to 6 h and then used for IVF according to our previous report. Data showed that sperm preincubated for 5 h contributed to higher fertility than those for other preincubation times. In experiment 2, we examined the effect of co-culture time up to 10 h for IVF on fertility and embryonic development in vitro. The oocytes co-cultured with sperm beyond 6 h showed higher fertilization and blastocyst formation rates than those in 2 and 4 h. In experiment 3, we examined the effect of initial culture period in fertilization medium (310 mOsm modified R1ECM; O h et al. 1998 Biol. Reprod.) on embryonic development in vitro. After IVF, oocytes were cultured in fertilization medium for 6, 12, or 24 h and further cultured in R1ECM up to 120 h. Cleavage rates were not affected by initial culture time in fertilization medium. However, oocytes cultured in fertilization medium for 12 h showed higher blastocyst formation rate than those for 0, 6 and 24 h. In experiment 4, we examined whether the IVF protocol can be applied to F344 and BN rats. Fresh and frozen-thawed sperm collected from cauda epididymides in F344 and BN were used for detection of capacitation-associated tyrosine phosphorylation. In fresh F344 sperm, tyrosine phosphorylation was induced in a time-dependent manner. Although tyrosine phosphorylation was inhibited in frozen-thawed F344 sperm, it was dramatically accelerated by IBMX treatment as well as frozen-thawed Wistar sperm. However, tyrosine phosphorylation in fresh and frozen-thawed BN sperm was suppressed and the phosphorylation in frozen-thawed sperm was not improved by IBMX. Our data demonstrate that (1) a more efficient IVF system using frozen-thawed rat sperm was developed and (2) the IVF system can be applied to at least F344 strain. The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J.I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.

The rat, as well as the mouse, is one of the most valuable experimental animals for biomedical and physiological research. There are numerous valuable mutant rats including transgenetic strains. Cryopreservation of rat oocytes and sperm as haploid germ cells is a key technology for banking the genetic resources efficiently. The aim of the present study was to examine survival of vitrified/warmed oocytes and developmental competence of resultant zygotes in the rat. Rats used in the present study were all Wistar rats. Epididymal spermatozoa were frozen as described previously (Seita et al. 2005 Reprod. Fertil. Dev. 18, 256). After thawing, spermatozoa were sonicated to obtain sperm heads for intracytoplasmic sperm injection (ICSI). Oocytes were collected from immature females superovulated with eCG and hCG. Oocytes were equilibrated in 7.5% (v/v) ethylene glycol (EG) + 7.5% (v/v) dimethylsulfoxide (DMSO) + 20% (v/v) FCS in PB1 for 5 min and then transferred into 15.0% EG (v/v) + 15.0% DMSO (v/v) + 20% FCS + 0.5 m sucrose in PB1 (vitrification solution) for 1 min at room temperature (22–24�C). During exposure to the vitrification solution, oocytes were loaded on a Cryotop� (Kitazato Supply Co., Tokyo, Japan). At warming, the film of Cryotop was directly immersed into PB1 containing 0.5 m sucrose and 20% FCS at 37.5�C. The warmed oocytes were washed three times and put into a HEPES-buffered (22 mm) modified R1ECM (310 mOsm) medium. The sperm heads were microinjected intracytoplasmically into the warmed oocytes. Then, presumptive zygotes were transferred surgically into the oviducts of recipient females (Day 0), and Caesarean section of the recipients was performed on Day 22. After vitrification and warming, 245 of 275 (88%) oocytes survived morphologically, 240 of the warmed oocytes were injected, and 156 oocytes (65%) were morphologically normal after the injection. To confirm development to term of zygotes derived from cryopreserved oocytes and sperm, 143 injected oocytes were transferred to 9 recipients, resulting in 3 pregnancies and the generation of one live pup. The results indicate that rat zygotes derived from cryopreserved oocytes and sperm through ICSI can develop to term, and full developmental competence can be preserved in rat oocytes after cryopreservation.

In intracytoplasmic sperm injection (ICSI), sperm chromatin is introduced into the oocyte together with acrosome, which does not enter cytoplasm of oocytes during normal fertilization. In mice, acrosome of sperm head has a detrimental effect on embryonic development of the ICSI oocytes (Morozumi et al. 2006 Proc. Natl. Acad. Sci. USA 103, 17 661–17 666). We examined the effect of acrosome removal of frozen/thawed (F/T) rat sperm on development to term of the ICSI oocytes in order to improve production efficiency of live offspring from F/T rat spermatozoa through ICSI. In experiment 1, epididymal spermatozoa of the Wistar rats were frozen as described previously (Seita et al. 2005 Reprod. Fertil. Dev. 18, 256 abst). The F/T spermatozoa were sonicated to separate sperm heads. The sperm heads were divided into 3 treatment groups; nontreated sperm head (control), sperm heads exposed in 0.02% of Triton X-100 solution for 1 min (TX), sperm heads exposed in 0.02% lysolectin solution for 1 min (LL). Acrosomal status of the sperm heads was then examined by FITC-peanut agglutinin stain. In experiment 2, sperm heads of the control, TX, and LL treatments were microinjected into denuded oocytes obtained from superovulated females (Hirabayashi et al. 2002 Transgenic Res. 11, 221–228). The ICSI oocytes were cultured and observed for the formation of pronuclei (2PN) for 6 h and blastocyst formation at 120 h of culture. In experiment 3, the ICSI oocytes cultured for 6 h were transferred to recipient females to examine development to term. Statistical analyses of the results were carried out by 1-way ANOVA. In experiment 1, the TX (76%) and LL (89%) treatments showed higher rates of acrosome removal than that of the control (24%) group (P < 0.05). In experiment 2, the percentage of 2PN formation at 6 h after ICSI was not significantly different among sperm-treated groups, although the TX (76%) and LL (70%) groups were higher than the control (24%) group at 4 h (P < 0.05). The percentages of blastocyst formation were not significantly different among sperm-treated groups (control: 10%; TX: 25%; and LL: 25%). In experiment 3, the efficiency of development to term of the TX treatment group (21%: 12/57) was significantly higher than the control (5%: 3/55; P < 0.05), although the LL treatment group (16%: 12/75) was not significantly different from the control and TX treatment groups. These results indicate that acrosome removal using TX of F/T rat sperm heads before ICSI improves development to term of the ICSI oocytes. This benefical effect of acrosome removal may be due to the quick release of sperm-activating factors from sperm heads after ICSI in the rat.

The purpose of the National BioResource project is to facilitate the availability of genetically and phenotypically standardized rat strains for life sciences. The BioResource is available to scientists worldwide. To bank genetic resources efficiently in the rat, cryopreservation of both sperm and embryos is a very important technology. The objective of the present study was to confirm the ability of banked and transported rat spermatozoa to fertilize oocytes through intrauterine insemination and for the embryos to develop to term, with the ultimate aim of developing a system for banking rat genetic resources. The epidydimal spermatozoa from the KLM rat, whose body size is small because the Prkg2 gene is partially defective, were frozen with egg yolk medium supplemented with 0.7% Equex Stm (Nakatsukasa et al. 2001 Reproduction 122, 463–467) and banked in the Institute of Laboratory Animals, Kyoto University. The cryopreserved sperm in 0.25-mL straws were transported to the laboratory at Azabu University, Kanagawa. Two straws from different males were thawed in a 37�C water bath for 15 s. Thawed semen was diluted with 1.0 mL of mR1ECM (Miyoshi et al. 1997 Biol. Reprod. 56, 180–185) with 0.4% (w/v) bovine serum albumin (BSA, fraction V; Sigma-Aldrich Japan K.K., Tokyo, Japan) at 37�C and then incubated at 37�C in 5% CO2 in humidified air until insemination. The percentage of motile spermatozoa was assessed visibly and determined by direct observation at 37�C under a light microscope at 100�. The thawed semen (50 �L, 3–4 � 105 sperm cells) was then inseminated into the top of both uterine horns of recipient females that were mated with a vasectomized male. The post-thaw motility of frozen spermatozoa was 10%. Seven of 15 inseminated females became pregnant and 13 live pups were born. It is thought that the low number of pups born in spite of the relatively high pregnancy rate was caused by sperm damage during the freezing and thawing procedure. The results of the present study show that rat spermatozoa cryopreserved in the BioResource have the ability to revive genetic resources through intrauterine insemination.

We previously reported the successful production of live offspring from cryopreserved rat spermatozoa through IVF (2006 Reprod. Fertil. Dev. 18, 256). The objective of the present study was to improve the IVF system with frozen–thawed spermatozoa for more efficient reproduction of live offspring. We examined the effect of caffeine-supplemented medium on post-thaw sperm fertility in vitro. Epididymal spermatozoa of Wistar rats were cryopreserved in 2.0 mL of freezing medium containing 23% (v/v) egg yolk, 8.0% (w/v) lactose monohydrate, and 0.7% (v/v) Equex STM. Post-thaw spermatozoa were diluted to 0.5 to 1.5 � 106 sperm mL-1 in a droplet of 200 �L of R1ECM as the pre-incubation medium. In Experiment 1, the effect of caffeine-supplemented medium on fertility in vitro was examined. The post-thaw spermatozoa were incubated for 5 h in pre-incubation medium with added caffeine at concentrations of 0, 1, 2, and 4 mM. Ovulated oocytes were introduced into droplets of the pre-incubation medium and co-cultured for 10 h. The oocytes were denuded and fixed to examine for 2 pronuclei formation (2PNF). In Experiment 2, the effect of caffeine-supplemented medium on post-thaw sperm function was examined using the chlortetracycline (CTC) fluorescence assessment. The post-thaw spermatozoa were incubated in pre-incubation medium with added caffeine at concentrations of 0 (Caf+) and 2 mM (Caf-), and CTC patterns were examined hourly until 6 h later. Statistical analyses of the results were carried out by one-way ANOVA. In Experiment 1, the percentages of 2PNF were 14 � 6, 32 � 8, 42 � 4, and 27 � 5% for the 0, 1, 2, and 4-mM groups, respectively, and only the 2-mM group was different from the 0-mM group (P < 0.05). In Experiment 2, the rate of capacitated spermatozoa was not significantly different (P > 0.05) among incubation times in the Caf+ group, but the rates at 5 and 6 h were lower (P < 0.05) than the rate at 1 h in the Caf- group. Although the rates of acrosome-reacted spermatozoa were not significantly different (P > 0.05) in the Caf+ group after 3 h of incubation, the accelerated trend of the acrosome reaction was observed in the Caf- group with increasing incubation time. The results demonstrated that frozen–thawed rat spermatozoa pre-incubated in medium containing 2 mM caffeine could improve in vitro fertility. This positive effect of caffeine would be provided to induce and keep sperm capacitation, and to inhibit the spontaneous sperm acrosome reaction in frozen–thawed rat spermatozoa.

We have previously reported successful cryopreservation of epididymal rat spermatozoa (Nakatsukasa et al. 2001 Reproduction 122, 463). However, the procedure for cryopreservation of rat spermatozoa has a disadvantage; a male has to be euthanized for collection of spermatozoa from its epididymides. Obtaining ejaculated spermatozoa repeatedly from the same male could be useful for cryopreservation of invaluable spermatozoa which carry mutations including transgenes. The objective of the present study was to develop a reliable system for cryopreservation of ejaculated rat spermatozoa and efficient production of offspring from the cryopreserved spermatozoa. Matured Wistar females were mated with three males of the same strain, and killed by cervical dislocation after formation of vaginal plugs. The uteri of mated females were excised and flushed with freezing medium containing 23.0% egg yolk, 8.0% lactose, and 0.7% Equex STM to recover ejaculated spermatozoa. The semen samples were loaded into 0.25-mL straws. The straws were cooled to 5.0�C at 0.5�C/min in a programmable freezer and then exposed to liquid nitrogen (LN) vapor at 4 cm (-150�C) above the LN level for 15 min. The straws were then plunged into LN and stored for at least a week. The straws were thawed in a 37.0�C water bath for 10 min. The thawed samples were diluted to 0.5-1.5 � 106 sperm/mL into a 200-�L droplet of R1ECM and then pre-incubated for 5 h. Ovulated oocytes collected from superovulated females were introduced into the droplet and co-cultured for 10 h for in vitro fertilization (IVF). The oocytes were denuded and examined for the presence of two pronuclei (2PN) microscopically. The denuded oocytes with 2PN were transferred into the oviducts of pseudo-pregnant females. The rates of sperm motility at recovery, post-thaw, and the initiation of IVF (after pre-incubation) were 57 � 6%, 24 � 5%, and 18 � 3%, respectively. After co-culture, 46 (14%) of the total 329 co-cultured oocytes were confirmed to contain 2PN. A total of the 44 putative zygotes were transferred to five recipients, and a total of 21 live young (48%) were born from all of the transferred recipients. We were able to produce zygotes and offspring derived from cryopreserved ejaculated spermatozoa of all three males used in the present study. In conclusion, the cryopreservation system for ejaculated rat spermatozoa used in the present study is a workable protocol for banking of valuable genetic resources of laboratory rats. Further studies on the IVF procedure with cryopreserved ejaculated spermatozoa in the rat are needed to improve the fertilization rate.

The aim of the present study was to develop an IVF system with frozen/thawed rat spermatozoa. We examined the effect of cooling rate to 5.0°C on post-thaw sperm motility and membrane integrity, and also investigated the ability of post-thaw spermatozoa to form pronuclei. Under room temperature, epididymal spermatozoa of Wistar rats were collected in 2.0 mL of egg yolk medium containing 8.0% (w/v) lactose monohydrate and 0.7% (v/v) Equex Stem. Samples were loaded into 0.25-mL straws and cooled to 5.0°C in the chamber of a programmed freezer. For cryopreservation, the samples were exposed to liquid nitrogen (LN) vapor for 10 min and then plunged into LN. Straws were thawed in a 37.0°C water bath for 10 s. Ovulated oocytes were collected and the zona pellucidae were removed with 0.1% pronase. One-hundred μL of thawed samples were put into a droplet of 400 μL R1ECM and pre-incubated for 1 h. R1ECM solution was added to the droplet to adjust to 0.5–1.5 × 106 sperm mL−1. The zona-free oocytes were then transferred into the droplet and co-cultured for 10 h. Oocytes were observed for pronuclei formation by means of an inverted phase contrast microscope. In Experiment I, the influence of sperm cooling rate to 5.0°C on sperm motility and membrane integrity was evaluated. Portions of samples were cooled at 54.0°C/min, 0.9°C/min, 0.5°C/min, and 0.3°C/min. The remainders were then frozen. The non-cooled samples were designated as controls. In Experiment II, we examined whether post-thaw spermatozoa have the ability to form pronuclei in vitro or not. All percentage data were arc-sine transformed and then analyzed by the Student's t-test. In Experiment I, the membrane integrity between the spermatozoa cooled at 0.5°C/min and the non-cooled spermatozoa was not different (38.1% vs. 37.2%; P > 0.05), but the integrity of these was higher than in spermatozoa cooled directly at 54.0°C/min (38.1% vs. 25.3%; P < 0.05). After culture for 1 h, the motility of spermatozoa cooled at 0.5°C/min was higher than that of those cooled at 54.0°C/min (61.3% vs. 53.3%; P < 0.05). At 2 h post-thaw the motility of spermatozoa cooled at 0.5°C/min was higher than that of spermatozoa cooled at 54.0°C/min and at 0.9°C/min (11.0% vs. 4.5%, 4.9%; P < 0.05). The membrane integrity of post-thaw spermatozoa cooled at 0.5°C/min was also higher compared to that of spermatozoa cooled at 54.0°C/min (22.5% vs. 8.4%; P < 0.01). In Experiment II, 28 (26.2%) of 107 oocytes had pronuclei when the post-thaw spermatozoa cooled at 0.5°C/min were used. The results indicated that the frozen/thawed spermatozoa cooled to 5.0°C at 0.5°C/min showed higher sperm motility and membrane integrity, and that spermatozoa can form pronuclei in homologous zona-free oocytes in vitro. Although in the rat sperm damage occurred during cooling to 5.0°C, and sperm motility and membrane integrity were also decreased by the cold shock, it is possible to decrease the damage by cooling slowly to 5.0°C at 0.5°C/min.