New preservation method for mouse spermatozoa without freezing

The objective of this study was to investigate the preservation of spermatozoa in a simple medium without freezing and to examine the effects of the preserved sperm on fertilization and development after injection into mature mouse oocytes. Mouse spermatozoa were collected from two caudae epididymides of mature B6D2F1 males and stored under various conditions: 1) in KSOMaa medium (potassium simplex optimized medium with amino acids) supplemented with 0, 1, or 4 mg/ml BSA and held at room temperature (RT, 27 degrees C); 2) in KSOMaa medium containing 4 mg/ml BSA (KSOM-BSA) and held at 4 degrees C, RT, or 37 degrees C (CO2 incubator); 3) in KSOM-BSA with osmolarity ranging from 271 to 2000 mOsmol, adjusted by addition of NaCl and held at 4 degrees C; and 4) a two-step preservation system consisting of storage in 800 mOsmol KSOM-BSA for 1 wk at RT followed by storage at -20 degrees C. Preservation of mouse spermatozoa at 4 degrees C in a medium with high osmolarity (700-1000 mOsmol) resulted in the highest frequency of live births after intracytoplasmic sperm injection (ICSI) into mature oocytes. The optimal conditions for preservation of mouse spermatozoa were 800 mOsmol KSOM containing 4 mg/ml BSA and a holding temperature of 4 degrees C. More than 40% of oocytes injected with sperm heads stored under these conditions for 2 mo developed to the morula/blastocyst stage in vitro and 39% of the embryos developed to term after transfer to recipient mice. Our results also indicate that mouse spermatozoa can be stored in 800 mOsmol KSOM-BSA medium at RT for 1 wk and then at -20 degrees C for up to 3 mo and retain their competence for ICSI. These new preservation methods permit extended conservation of viable spermatozoa that are capable of supporting normal embryonic development and the live birth of healthy offspring after ICSI.     

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

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

Sperm viability of canine and caprine semen samples preserved in a dry shipper

This study assessed the efficacy of a dry shipper to preserve canine and caprine semen samples. After equilibration, semen straws from six Majorera bucks and five dogs were frozen and stored in liquid nitrogen (LN). Thirty days after freezing, half of the frozen straws were transferred from LN to a dry shipper (DS). Then, thawing was performed at 1, 2, 3, 5 and 7 days and the percentages of motile spermatozoa, acrosome intact spermatozoa and abnormal spermatozoa were determined. The sperm motility (total and progressive) of canine semen samples preserved with DS was quite similar to those preserved in LN, and no significant differences were observed throughout the experimental period. In addition, no differences were observed in the number of abnormal spermatozoa (range: 13.2-19.0%) or intact acrosome (range 91.3-95%) between both storage protocols. Buck semen samples showed equivalent levels of progressive motility (between 50% and 60%) and intact acrosome membrane (around 70%) during the first 3 days of storage in both procedures; however, from the fifth day of storage onwards, a notable decrease in semen quality was observed in the samples preserved in DS, showing a dramatic fall in the semen viability after 7 days of preservation (12.3% and 36.8%, progressive fast spermatozoa and acrosome integrity, respectively). In dog samples, the present study confirmed that seminal quality did not show modifications for the preservation period (7 days), confirming the efficacy of the dry shipper to preserve frozen samples for a short time. However, under the circumstances reported in this study, the sperm quality of buck samples preserved in the dry shipper only held during the first 3 days of storage, and therefore, its practical application could be more limited.     

Cryopreservation of turbot (Scophthalmus maximus) spermatozoa

The aim of this study was to develop a method for cryopreserving turbot semen and to compare sperm motility characteristics, metabolic status and fertilization capacity of frozenthawed and fresh semen. The best results were obtained when spermatozoa were diluted at a 1:2 ratio with a modified Mounib extender, supplemented with 10% BSA and 10% DMSO. For freezing sperm samples, straws were placed at 6.5 cm above the surface of liquid nitrogen (LN) and plunged in LN. The straws were thawed in water bath at 30 degrees C for 5 sec. Use of this simple method resulted in a 60 to 80% reactivation rate of the thawed spermatozoa. Although the percentage of motile spermatozoa in the frozen-thawed semen samples was significantly lower than in fresh semen, spermatozoa velocity and respiratory rate remained unchanged. The process of cryopreservation significantly decreased intracellular ATP content. The fertilization rate of frozen-thawed spermatozoa was significantly lower than that of fresh spermatozoa, but it increased with sperm concentration.     

Possibility of long-term preservation of freeze-dried mouse spermatozoa

Freeze-dried mouse spermatozoa are capable of participating in normal embryonic development after injection into oocytes. When the freeze-dried spermatozoa are used as a method for storage of genetic materials, however, it is essential to assure the relevance of long-term preservation over several decades or centuries. Thus, we applied the theory of accelerated degradation kinetics to freeze-dried mouse spermatozoa. Thermal denaturation kinetics were determined based on Arrhenius plots derived from transition-state theory analysis at three elevated temperatures: 30, 40, and 50 degrees C. Accelerated degradation kinetics were calculated by extrapolation of Arrhenius plots. This theory also is being applied to the long-term stability of drugs. The estimated rate of development to the blastocyst stage at 3 and 6 mo and at 1, 10, and 100 yr of sperm storage at 4 degrees C were 21.60%, 7.91%, 1.00%, 0%, and 0%, respectively. At -80 degrees C, estimated development rates to the blastocyst stage that would be expected after 100 yr of storage did not decline significantly. In addition, after 3 or 6 mo of storage at 4 or -80 degrees C, preimplantation development of the embryos derived from intracytoplasmic sperm injection (ICSI) was examined. The actual developmental rates to the blastocyst stage from ICSI by freeze-dried sperm stored for 3 mo at 4 and -80 degrees C were 21% and 62%, respectively, and the rates for such sperm stored for 6 mo were 13% and 59%, respectively. These results indicate that the determination of accelerated degradation kinetics can be applied to the preservation of freeze-dried mouse spermatozoa. Furthermore, for long-term preservation, freeze-dried mouse spermatozoa appear to require being kept at lower than -80 degrees C.     

Optimization of procedures for separation of motile and nonmotile bull and rabbit spermatozoa with bovine serum albumin gradients

The effect of equipment design, separatory media, and time and temperature of separation were studied. Discontinuous 4%/10% bovine serum albumin (BSA) gradients were used to isolate highly motile spermatozoa in rabbit and bull semen. For all conditions tested, motility of spermatozoa collected from the 4% BSA gradient layer (top) was less than or equal to the motility of the unseparated controls. Fractions collected from the 10% BSA gradient layer contained highly motile spermatozoa. In experiment 1, washed bull spermatozoa were diluted with phosphate-buffered saline (PBS) containing 2% BSA or 4% BSA before being fractionated on BSA columns contained in test tubes. Inclusion of BSA in PBS tended to reduce loss of motility during washing, but the proportion of sperm recovered was highest in PBS. In experiment 2, motility and recovery of buck spermatozoa collected from the 10% BSA gradient region tended to be higher when fractionation temperature was 30°C as compared to 35°C, and motility was significantly higher when incubation time was 30 min as compared to 1 hr. The proportion of sperm recovered was unaffected by incubation time. In experiments 1 and 2, 41 of 48 separations resulted in at least one fraction containing spermatozoa with motility greater than or equal to 90%. In the third experiment, the surface area on which bull and buck spermatozoa were layered was increased by forming the 4%/10% BSA gradients in conical supports. Separation of sperm on conically shaped columns was not as effective as on cylinders. The use of cylinders to support the BSA gradients and a separation time of 30 min at 30°C is recommended.     

Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at 4 degrees C. Samples frozen without cryoprotection were maintained at -196 degrees C. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%, respectively; P > 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P > 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P < 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P < 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P > 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 1.5 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 2) long-term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple, and efficient but, like all routine sperm storage methods, requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying, but sperm can then be stored at 4 degrees C and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.     

Caprine blastocyst development after in vitro fertilization with spermatozoa frozen in different extenders

The feasibility of using frozen-thawed semen in caprine IVF outside the breeding season was investigated. Electroejaculated spermatozoa from a Nubian buck were washed twice and then frozen in skim milk- or in egg yolk-based extenders. Goat oocytes were matured and inseminated by frozen-thawed spermatozoa selected by swim-up. In vitro fertilization was performed in a modified defined medium (mDM), altered experimentally, for 24 h. Embryos were cultured in 50 microL of c-SOF + NEA for 9 d. The percentages of oocytes exposed to heparin-capacitated spermatozoa, (previously cryopreserved in skim milk-based extender) that cleaved, reached morula, blastocyst and expanded blastocyst stages were 82.8, 57.1, 35.7 and 30.0%, respectively. Without heparin treatment the rates for cleavage, morula, blastocyst and expanded blastocyst stages were 44.3, 31.4, 18.6 and 8.6%, respectively. Therefore, heparin treatment was included in sperm capacitation. Use of spermatozoa with BSA in the IVF medium yielded no cleavage. Although extenders containing 8 to 20% egg yolk enabled good sperm motility after cryopreservation, in vitro fertilizing ability was compromised under our conditions. By contrast, semen commercially processed in season in an egg yolk-based diluent remained effective for IVF. The highest proportion of blastocysts resulted from the use of spermatozoa diluted in a skim milk extender, heparin capacitation, and insemination in medium containing lamb serum.     

Fertilizing potential of mouse spermatozoa cryopreserved in a medium containing whole eggs

Experiments were conducted to improve survival of mouse spermatozoa through the cryopreservation process. In the first experiment, percentages of motile spermatozoa and fertilizing capacities of spermatozoa were evaluated when mouse spermatozoa were cryopreserved using three previously reported cryopreservation media: (1) 18% raffinose in 3% skim milk; (2) Tes/Tris medium containing 25% egg yolk and 1.25% glycerol; and (3) PBS containing 18% raffinose and 1.75% glycerol, each at three different cooling rates (-3, -10, and -50 degrees C/min). Spermatozoa frozen in the skim milk/raffinose medium exhibited the highest percentage of motile spermatozoa (39%) when cells were frozen at -10 degrees C/min (P<0.05). The second experiment evaluated the effects of modifying the Tes/Tris/egg yolk medium, comparing different concentrations of egg yolk, BSA, and sodium dodecyl sulfate. Reducing egg yolk from 25% of the medium volume to 5%, increased percentages of motile spermatozoa after cryopreservation from 29 to 36% (P<0.05). Addition of 1% BSA and sodium dodecyl sulfate to medium containing 5% egg yolk further improved percentages of motile spermatozoa after freezing. In the final experiment, 20% whole egg was substituted for 5% egg yolk and 1% BSA used in previous experiments and resulted in percentages of motile spermatozoa (51%) equal to that of the skim milk-raffinose medium. However, fertility rates were higher (68%) than for spermatozoa frozen in the skim milk-raffinose medium (P < 0.05) and were comparable to the fertility rates of fresh spermatozoa (77%; P>0.05). In conclusion, freezing mouse spermatozoa in a medium containing 20% whole egg, 0.035% sodium dodecyl sulfate, and 1.25% glycerol using a cooling rate of -10 degrees C/min preserves the motility and fertilization capacity of mouse spermatozoa.     

Effects of freeze-drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm

Freeze-drying sperm is an alternative to cryopreservation. Although sperm from various species has been freeze-dried, there are few reports for bovine sperm. The primary objective of this study was to evaluate the protective effect of various freeze-drying media on the structural and functional components of bovine sperm. The media tested were composed of TCM 199 with Hanks salts supplemented with 10% fetal calf serum (FCS) and TCM 199 with Hanks salts supplemented with 10% FCS and 0.2 M trehalose and EGTA solution. The efficiency of each medium on the preservation of freeze-dried sperm structures was evaluated with conventional and electron microscopy, DNA integrity was analyzed by a TUNEL assay, and fertilizing ability of lyophilized sperm was determined with ICSI. Although the plasma membrane was damaged in all media tested, mitochondria were similarly preserved in all freeze-drying treatments. The acrosome was best preserved in the media that contained trehalose (other treatments also conserved this structure). In contrast, media containing EGTA or trehalose most effectively preserved the nuclei in freeze-dried sperm, with only 2 and 5%, respectively, of cells with fragmented DNA. Furthermore, sperm conserved with these media also had higher (P<0.05) rates of sperm head decondensation (32.5 and 27.5%), pronucleus formation (37.5 and 45.0%) and blastocyst formation (19.4 and 18.3%) than medium supplemented with FCS (15.0, 20.0 and 10.2%, respectively). In conclusion, media with EGTA and trehalose adequately protected bovine sperm during freeze-drying by preserving the viability of their nuclei.     

Effects of preservation media on in vitro maturation of porcine oocytes

The effects of preservation media for ovaries on in vitro maturation of porcine oocytes was studied. The cumulus-oocyte complexes (COCs) obtained from ovaries that had been preserved in three different media at various temperatures for different time intervals were cultured in the M199 maturation medium. The preservation media used were 0.9% saline solution, BCS (Braun-Collins solution) and Dulbecco's phosphate buffered saline solution (PBS). Mature oocytes obtained from the ovaries preserved in three preservation media for 8 h were electrically activated. The activated oocytes were then cultured in the NCSU23 embryo culture medium for 16 h to observe activation, or for 144 h to observe embryo development. It was found that the preservation temperature significantly affected maturation of the porcine oocytes. A preservation temperature of about 25 degrees C showed an optimal maturation rate for a preservation time of 8 h for the three preservation media. Although the preservation temperature was a major factor influencing the maturation rate, different preservation media at 25 degrees C for 8 h also significantly affected the maturation rate, activation rate and embryo development. Among these three preservation media, PBS exhibited the highest cleavage rate indicating that PBS should be a better preservation medium for porcine ovaries at 25 degrees C for 8 h or longer periods.     

Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification

The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01). In conclusion: (a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.     

Activation, pronuclear formation, and development in vitro of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa

The fertilization of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa was evaluated. Activation and male pronuclear (MPN) formation were better in oocytes injected with isolated freeze-dried sperm heads than whole freeze-dried spermatozoa, but cleaved embryos were generally difficult to develop to the morula or blastocyst stage. When spermatozoa were freeze-dried for 24 h, oocyte activation and MPN formation in activated oocytes after sperm head injection were inhibited. Embryo development to the blastocyst stage was only obtained after injecting sperm heads isolated from spermatozoa freeze-dried for 4 h and stored at 4 degrees C. The proportion of embryos that developed to the blastocyst stage was not increased by the treatment of injected oocytes with Ca ionophore (5-10 microM). Increasing the sperm storage time did not affect oocyte activation or MPN formation, but blastocyst development was observed only after 1 mo of storage. These results demonstrate that pig oocytes can be fertilized with appropriately freeze-dried spermatozoa and that the fertilized oocytes can develop to the blastocyst stage.     

CRYOPRESERVATION OF SEA URCHIN EMBRYOS AND SPERM

A simple method for preserving live sea urchin embryos and sperm in liquid nitrogen (LN) wasdeveloped through the use of DMSO as a cryoprotective additive. Samples of late embryos in double test tubes were cooled to— I96°C by two-step freezing, first to — 76°C and then by plunging in LN. In the case of fertilized eggs, samples were previously frozen to — 40°C, at which temperature the samples were kept for 15 min; they were then transferred into LN. After preservation in LN for various lengths of time, samples in the double test tubes were thawed in water at 15°C. The post-thaw survival was more than 90% for late embryos, and about 10% for fertilized eggs. Difference in the length of the cryopreservation period did not affect survival. Post-thaw development in cryopreserved embryos often showed abnormalities in structure. Sperm with 1.5 M DMSO was successfully preserved in LN by a similar method to the one used for cryopreservation of late embryos. Fertilizability in cryopreserved sperm was complete, regardless of the length of the preservation period. Nearly all the eggs fertilized by cryopreserved sperm developed to normal plutei.     

Optimal physicochemical conditions for the manipulation and short-term preservation of koala (Phascolarctos cinereus) spermatozoa

Protocols for the successful manipulation and preservation of semen in a given species depend upon a fundamental knowledge of how spermatozoa respond to the physicochemical conditions of the extension media; methods developed for the preservation of eutherian spermatozoa may not necessarily be suitable for marsupial semen. The aim of this study was to investigate the effects on koala sperm motility of serial dilution, changes in temperature, diluent pH and osmolality to establish the optimal physicochemical conditions for short-term semen storage. This study showed that electroejaculated koala semen diluted 1∶1 (v/v) with PBS frequently coagulated after incubation at 35 degrees C, but that further dilution and incubation resulted in a corresponding increase in the percentage of spermatozoa swimming in a non-linear trajectory. The effect of rapid temperature change on the motility of koala spermatozoa was investigated by exposing semen, initially diluted at 35 degrees C, to temperatures of 45, 25, 15 and 5 degrees C. Although sperm motility was reduced after incubation at 45 degrees C, a rapid decrease in temperature of up to 20 degrees C did not result in a significant reduction in sperm motility. However, contrary to evidence in other marsupials, there was a small but significant decrease in sperm motility after rapid cooling of diluted semen from 35 to 5 degrees C. The effects of diluent pH and osmolality on the motility of koala spermatozoa were investigated. These experiments indicated that diluents for koala sperm manipulation should buffer in a pH range of 7-8 and have an osmolality of approximately 300 mmol kg(-1). The final experiment compared the relative effectiveness of Tris-citrate buffer (1% glucose) and PBS to maintain koala sperm motility over a range of incubation temperatures (5-35 degrees C) for up to 8 days. Reduction in sperm motility was directly related to temperature, and motility was sustained for the longest duration when stored at 5 degrees C. The Tris-citrate buffer solution was superior to PBS as a preservation diluent at all temperatures, and koala spermatozoa remained motile even after 42 days storage at 5 degrees C. Spermatozoa diluted in PBS (with Ca(2+) or Mg(2+)) and cooled to 5 degrees C showed evidence of an unusual motility pattern, similar to that of hyperactivated eutherian spermatozoa. This study showed that koala spermatozoa respond to different physicochemical conditions associated with short-term liquid storage in essentially the same way as the spermatozoa of eutherian mammals, although koala spermatozoa appear to be more tolerant of rapid temperature shock. The results of this study can be used to make informed selections with regard to appropriate diluent composition and improved short-term sperm preservation protocols and represent the first such database for any species of marsupial.     

Increasing glucose in KSOMaa basal medium on culture Day 2 improves in vitro development of cloned caprine blastocysts produced via intraspecies and interspecies somatic cell nuclear transfer

This study was conducted to evaluate the efficiency of potassium simplex optimization medium with amino acids (KSOMaa) as a basal culture medium for caprine intraspecies somatic cell nuclear transfer (SCNT) and caprine-bovine interspecies somatic cell nuclear transfer (iSCNT) embryos. The effect of increased glucose as an energy substrate for late stage development of cloned caprine embryos in vitro was also evaluated. Enucleated caprine and bovine in vitro matured oocytes at metaphase II were reconstructed with caprine ear skin fibroblast cells for the SCNT and iSCNT studies. The cloned caprine and parthenogenetic embryos were cultured in either KSOMaa with 0.2 mM glucose for 8 days (Treatment 1) or KSOMaa for 2 days followed by KSOMaa with additional glucose at a final concentration of 2.78 mM for the last 6 days (Treatment 2). There were no significant differences in the cleavage rates of SCNT (80.7%) and iSCNT (78.0%) embryos cultured in KSOMaa medium. Both Treatment 1 and Treatment 2 could support in vitro development of SCNT and iSCNT embryos to the blastocyst stage. However, the blastocyst development rate of SCNT embryos was significantly higher (P < 0.05) in Treatment 2 compared to Treatment 1. Increasing glucose for later stage embryo development (8-cell stage onwards) during in vitro culture (IVC) in Treatment 2 also improved both caprine SCNT and iSCNT embryo development to the hatched blastocyst stage. In conclusion, this study shows that cloned caprine embryos derived from SCNT and iSCNT could develop to the blastocyst stage in KSOMaa medium supplemented with additional glucose (2.78 mM, final concentration) and this medium also supported hatching of caprine cloned blastocysts.     

Sperm cryopreservation of African catfish, Clarias gariepinus: cryoprotectants, freezing rates and sperm:egg dilution ratio

Methods for cryopreserving spermatozoa and optimizing sperm:egg dilution ratio in African catfish Clarias gariepinus were developed. Five percent to 25% DMSO and methanol were tested as cryoprotectants, by diluting semen in Ginzburg fish ringer and freezing in 1-milliliter cryovials in a programmable freezer. To avoid an excess of spermatozoa per egg, post-thaw semen was diluted 1:20, 1:200 or 1:2,000 before fertilization. Highest hatching rates were obtained by spermatozoa frozen in 10% methanol and post-thaw diluted to 1:200. Then, slow freezing rates (-2, -5 or -10 degrees C/min) to various endpoint temperatures (range -25 to -70 degrees C) before fast freezing in liquid nitrogen (LN2) were evaluated. Hatching rates equal to control (P > 0.05) were obtained by spermatozoa frozen at -5 degrees C/min to -45 to -50 degrees C and at -10 degrees C/min to -55 degrees C. In 3-step freezing programs, at -5 degrees C/min, the effect of holding spermatozoa for 0, 2 or 5 min at -30, -35 or -40 degrees C before fast freezing in LN2 was analyzed. Hatching rates equal to control (P > 0.05) were produced by spermatozoa frozen to, and held at, -35 degrees C for 5 min and at -40 degrees C for 2 or 5 min. Finally, frozen spermatozoa (10% methanol, -5 degrees C/min, 5-min hold at -40 degrees C, LN2, post-thaw diluted to 1:200) were tested in on-farm fertilization conditions. Again, no difference (P > 0.05) in hatching rate was observed between frozen and fresh spermatozoa. Cryopreservation offers utility as a routine method of sperm storage and management for catfish.     

Effect of inhibiting nitric oxide production on mouse preimplantation embryo development and metabolism

Nitric oxide (NO) is a free radical that functions as a cell signaling molecule but at high concentrations can be toxic. It is formed from arginine, which is consumed by the mouse blastocyst, but its effect on early embryo development has been little studied. In this study, the role of NO in mouse preimplantation development has been examined in terms of developmental rate and oxidative metabolism. Zygotes were cultured in one of four media; potassium simplex optimization medium (KSOM), KSOM with amino acids (KSOMaa), KSOM without glutamine (KSOM-glut), or KSOM with 0.5 mM arginine (KSOMarg) +/- l-NAME (a specific inhibitor of NO production). End points were Day 4 blastocyst rates, cell counts determined using bisbenzimide and oxygen consumption. In KSOM and KSOM-glut, the blastocyst rate was decreased by 1 mM l-NAME from 50.2% +/- 3.1% and 37.4% +/- 4.5% to 6% +/- 3% and 0%, respectively. In KSOMaa, cavitation rates were unaltered but the blastocysts contained fewer cells (P < 0.001). Blastocysts cultured in KSOM and KSOM-glut consumed significantly more oxygen than those cultured in KSOMaa (P < 0.001 and P < 0.05, respectively). However, the addition of 0.1 mM or 1 mM l-NAME to KSOMaa significantly increased the amount of oxygen consumed (P < 0.05 and P < 0.001, respectively). The data suggest a physiological role for NO in mouse preimplantation metabolism and development. One possibility is that NO may limit oxygen consumption at the blastocyst stage at the level of mitochondrial cytochrome c oxidase.     

The birth of mice from testicular spermatozoa retrieved from frozen testicular sections

Male gamete cryopreservation has been widely used for both human reproduction and animal breeding. We investigated whether testicular spermatozoa retrieved from frozen testicular sections (10 or 25 mum thick) could support the full-term development of normal progeny. For this purpose, frozen testicular sections were prepared from two genetic backgrounds (BDF1 or B6 GFP transgenic mice), and the functional ability of testicular spermatozoa after preservation for 1 day, 1 mo, and 3 mo was assessed by intracytoplasmic sperm injection (ICSI). Testicular spermatozoa were successfully retrieved from frozen testicular sections for the use of ICSI, regardless of the preservation period. The ICSI technique revealed that oocytes (BDF1 or B6 background) injected with testicular spermatozoa prepared from frozen testicular sections developed into normal progeny, even though the sections had been cryopreserved for 3 mo at -30 degrees C. Approximately 15% and 5% of the embryos preserved for 3 mo developed to full term if the testicular spermatozoa were prepared from the 25- and 10-mum sections, respectively. These results clearly indicate that male gametes can be viably preserved in frozen testicular sections. The technique described herein will allow the preservation of male gametes in the form of a "book" or "file" by mounting the sections on a paper-thin sheet. Furthermore, this technique may be of value in the clinical treatment of severe male infertility, since testicular spermatozoa can easily be found through examination of testicular cross sections rather than by attempts to identify them in testicular cell suspension.     

Effect of ionic strength, serum albumin and other macromolecules on the maintenance of motility and the surface of mammalian spermatozoa in a simple medium

The seminal plasma in sperm suspensions from boar, bull, rabbit, ram and stallion was replaced with simple defined media as completely as possible by a combination of centrifugation through Ficoll and dilution. After this process, motility declined and the cells showed a tendency to agglutinate and/or stick to glass. Varying the ionic strength of the medium had little effect upon these parameters but sperm motility was preserved better in the presence of serum albumin. When a number of purified proteins and other macromolecules were tested individually in this way for their motility-preserving ability, bovine or human serum albumin was consistently the most effective. Defatting the albumin or altering its nature by mild reduction, oxidation or alkylation had little detectable effect on its motility-preserving ability; the protein did not appear to be acting as a chelator of metal ions, for it could not be replaced by EDTA. The response of the spermatozoa to replacemrnt of seminal plasma varied between species: bull spermatozoa were particularly sensitive and serum albumin had little effect upon their subsequent motility.