Effect of epididymis handling conditions on the quality of ram spermatozoa recovered post-mortem

Post-mortem spermatozoa recovery is an important technique for obtaining germplasm reserves from genetically valuable animals or endangered species. However, there are many factors that influence the outcome of this technique. We have studied the effect of the interval between animal's death and sperm recovery (0, 24 or 48 h) on the quality and freezability of ram spermatozoa from cauda epididymidis. Storage temperature of epididymis (room temperature or 5 degrees C) was also analysed. Spermatozoa were diluted with Tes-Tris-Fructose solution supplemented with egg yolk (10%) and glycerol (4%), and frozen using a programmable biofreezer (-20 degrees C/min). Pre-freeze and post-thaw sperm samples showed viable spermatozoa up to 48 h after the animal's death, although their quality declined significantly as post-mortem storage time increased. Epididymis sperm stored at 5 degrees C showed better motility and a lower percentage of abnormal forms than epididymis stored at room temperature after 24 and 48 h. The fertilizing ability of cauda epididymis ram spermatozoa obtained at 0 and 24h after the animal's death is similar to that of ejaculated spermatozoa. Therefore, a good protocol for post-mortem semen collection in rams when epididymal spermatozoa cannot be collected immediately, is to preserve the epididymis at 5 degrees C and process the samples in the first 24h after the animal's death.     

Viable spermatozoa can be recovered from refrigerated mice up to 7 days after death

To develop a model for utilizing germ cells collected from dead animals, male mice were euthanized and refrigerated for various periods, and the viability of the epididymal spermatozoa was examined by in vitro fertilization, embryo culture, and embryo transfer. Higher proportions of fresh oocytes were fertilized when males had been stored at 4-6 or 8-10 degrees C than at 0 degrees C. By partially dissecting the zona of freshly ovulated oocytes, spermatozoa from ICR male mice could fertilize oocytes (21% fertilization rate) after being stored for 5 days at 4-6 degrees C, and spermatozoa from BDF1 male mice could fertilize oocytes (39%) after being stored for 7 days at 4-6 degrees C. The resulting two-cell embryos had the ability to develop into expanded blastocysts in culture (81-100%) and into live young after transfer (34-47%). With further refinement of this system, it should be applicable not only for rescuing valuable genetic variants in laboratory animals or livestock animals but also for wild species in the future.     

Live rats resulting from injection of oocytes with spermatozoa freeze-dried and stored for one year

This study was designed to examine whether rat spermatozoa after freeze-drying and 1-year storage can participate in full-term development following intracytoplasmic sperm injection (ICSI). Cauda epididymal spermatozoa from Crlj:Wistar rats were frozen in liquid nitrogen (LN(2)), first dried for 14 hr at 0.37 hPa and then for 3 hr at 0.001 hPa. The dried spermatozoa were stored for 1 year in a desiccator at +25 degrees C, or in a refrigerator at +4 degrees C, or in LN(2) at -196 degrees C. Controls consisted of sperm that had only been frozen and stored in LN(2). After being stored, spermatozoa were sonicated to dissociate the sperm tail and were injected into oocytes from superovulated Slc:SD rats. The respective fertilization rates of oocytes injected with frozen sperm, or with freeze-dried sperm stored at +25, +4, and -196 degrees C were 79%, 75%, 70%, and 73%. However, the corresponding cleavage rates of injected oocytes were 63%, 1%, 38%, and 36%. After transfer of >80 zygotes of each group into recipients, the respective percentages of full-term normal offspring resulting from frozen sperm or from freeze-dried sperm stored at +25, +4, and -196 degrees C were 36%, 0%, 7%, and 14%. These results demonstrate that the storage temperature significantly influenced the likelihood of term development of rats produced by injection of oocytes with freeze-dried spermatozoa. Chromosomal analysis of the rat spermatozoa in the ICSI oocytes indicated that chromosomal aberration in freeze-dried spermatozoa stored at +25 degrees C (100%) occurred more frequently than in frozen control spermatozoa (41%) and freeze-dried spermatozoa stored at -196 degrees C (35%), and the frequency of chromosomal aberrations in freeze-dried spermatozoa stored at +4 degrees C (65%) was the intermediate. In conclusion, rat spermatozoa freeze-dried and stored at +4 degrees C for 1 year are capable of participating in full-term development after ICSI.     

Fertilization and development in vitro of bovine oocytes following intracytoplasmic injection of heat-dried sperm heads

This study investigated the development of bovine oocytes following intracytoplasmic injection of sperm heads from spermatozoa dried by heating. When sperm suspension was heated in a dry oven at 50, 56, 90, and 120 degrees C, the mean amounts of residual water were about 0.3 g water/g dry weight within 8 h, 6 h, 1.5 h, and 20 min of heating, respectively. Oocyte activation, cleavage of oocytes, and development of cleaved embryos to the morula stage were better in oocytes injected with spermatozoa stored at 25 degrees C for 7-10 days following drying at 50 and 56 degrees C than at 90 and 120 degrees C; however, only a small proportion of oocytes developed to the blastocyst stage. When spermatozoa were dried at 50 degrees C for 16 h, activation, male pronucleus (MPN) formation, cleavage, and development to the morula stage were less good than when spermatozoa were dried for 8 and 10 h and no blastocysts were obtained. The development of oocytes was significantly better when spermatozoa were stored for 7-10 days at 4 degrees C than 25 degrees C after drying at 50 degrees C for 8 h. Longer storage (7 days-12 mo) of heat-dried spermatozoa at 4 degrees C did not affect MPN formation in activated oocytes, but blastocyst development was significantly lower when spermatozoa were stored for 3 mo or more. These results demonstrate that bovine oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop at least to the blastocyst stage.     

Fertilisability of ovine, bovine or minke whale (Balaenoptera acutorostrata) spermatozoa intracytoplasmically injected into bovine oocytes

This study was conducted to investigate the possibility of using bovine oocytes for a heterologous fertility test by intracytoplasmic sperm injection (ICSI) and to compare the pronuclear formation of ram, bull and minke whale spermatozoa after injection into bovine oocytes. Bovine oocytes were cultured in vitro for 24 h and those with a polar body were selected for ICSI. Frozen-thawed semen from the three species were treated with 5 mM dithiothreitol for 1 h and spermatozoa were killed by storing them in a -20 degrees C refrigerator before use. ICSI was performed using a Piezo system. Three experiments were designed. In experiment 1, a higher (p < 0.05) male pronuclear formation rate was found in the oocytes injected with ram (52.6%) or bull (53.4%) spermatozoa than with minke whale spermatozoa (39.1%). In experiment 2, sperm head decondensation was detected at 2 h after ICSI in the oocytes injected with a spermatozoon of each species. Male pronuclei were first observed at 4 h in the oocytes injected with ram or bull spermatozoa and at 6 h in oocytes injected with minke whale spermatozoa. The mean diameters of male pronuclei derived from both whale and bull spermatozoa were larger than those from ram spermatozoa (30.4 microm and 28.3 microm vs 22.4 microm, p < 0.005). The mean diameter of female pronuclei in the oocytes injected with whale spermatozoa was also larger than with ram spermatozoa (29.3 microm vs 24.7 microm, p < 0.05). The development of male and female pronuclei was synchronous. In experiment 3, ethanol-activated oocytes injected with a spermatozoon from any of the three species achieved significantly higher (p < 0.05-0.001) cleavage rates than control oocytes. Blastocyst formation was only observed when bull spermatozoa were used. The results of this study indicate that dead foreign spermatozoa can participate in fertilisation activities in bovine oocytes after ICSI.     

Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C

In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.     

Ability to activate oocytes and chromosome integrity of mouse spermatozoa preserved in EGTA Tris-HCl buffered solution supplemented with antioxidants

Potential methods for cryopreservation of mouse spermatozoa are freeze-drying, desiccation, and suspension in EGTA Tris-HCl buffered solution (ETBS: 50 mM NaCl, 50 mM EGTA, and 10 mM Tris-HCl). To determine the duration that mouse spermatozoa suspended in ETBS-based solutions could retain their normal characteristics without freezing, spermatozoa collected from the cauda epididymis were suspended in ETBS or in ETBS supplemented with the antioxidants, dimethyl sulfoxide (DMSO), or DL-alpha-tocopherol acetate (Vitamin E acetate; VEA) diluted in DMSO, then held at ambient temperature (22-24 degrees C) for up to 9 days. When oocytes were injected with spermatozoa preserved in ETBS alone, activation rates of oocytes and chromosome integrity at the first cleavage metaphase decreased at 1 day (P < 0.001) and 2-4 days (P < 0.01) following treatment. When oocytes were injected with spermatozoa preserved in ETBS supplemented with DMSO or VEA/DMSO, chromosome integrity did not decrease significantly (through 9 days of preservation). Although DMSO maintained sperm chromosome integrity more effectively than VEA/DMSO up to 2-4 days (91 and 67%, normal karyotypes in DMSO and VEA/DMSO, respectively), VEA/DMSO helped to maintain the ability of spermatozoa to activate oocytes, but did not enhance the maintenance of sperm chromosome integrity. These results suggested that deterioration of spermatozoa preserved in ETBS alone was delayed by supplementation with antioxidants.     

Effects of temperature on the immobilization and the initiation of motility of spermatozoa in the male reproductive tract of the domestic fowl, Gallus domesticus

1. The motility of undiluted fowl spermatozoa taken from testis, epididymis and ductus deferens was negligible at 40 degrees C, around the normal avian body temperature. 2. The immobilization was not permanent and motility was restored by decreasing the temperature to 30 degrees C or by suspending in a NaCl/TES buffer with 2 mM Ca2+, 2 mM HCO3- or 10% seminal plasma at 40 degrees C. 3. Demembranated spermatozoa taken from testis, epididymis and ductus deferens were also immotile at 40 degrees C. However, these spermatozoa were restored the motility at 30 degrees C except testicular spermatozoa. 4. These results suggest that the capacity of movement of fowl spermatozoa can be readily obtained from testis, but that these spermatozoa are immotile due to temperature-dependent immobilization in the male reproductive tract. 5. Furthermore, it is possible that changes in environmental temperature at ejaculation are one of the important exogenous physiological factors of the initiation of fowl sperm motility.     

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.     

Morphological and functional characteristics of chilled semen obtained from domestic feline epididymides (Felis catus)

The objective of this study was to determine morphological and functional characteristics of semen retrieved from the feline epididymis before and after cooling. Sixteen adult male cats were orchiectomized. The distal portion of the epididymis and proximal part of the deferent ducts were dissected and squeezed to obtain their content. After centrifugation, the supernatant was removed, sperm were resuspended in a 0.9 mL Tris-fructose-citric acid extender containing 20% egg yolk, aliquoted into three 0.3 mL samples, placed in a refrigerator (4.8 degrees C) and cooled (0.5 degrees C/min). Semen evaluations were performed on four occassions: immediately after epididymal sperm retrieval (T0), and at 24 h (T1), 48 h (T2) and 72 h (T3) after cooling. On each occasion, progressive motility, vigor and sperm morphology were determined. Mean motility and vigor decreased (P < 0.05) between each successive examination. Although the majority of sperm cell damage occurred within the first 24 h, there was a decrease (P < 0.05) in mean percentage of morphologically normal sperm between T0 and each evaluated time (T1, T2, T3) after cooling, due to an increase in coiled and bent sperm tails. Further studies are needed to evaluate the effects of cooling on the fertilizing capacity of cat epididymal spermatozoa in assisted reproduction programs.     

The ability of freeze-dried bull spermatozoa to induce calcium oscillations and resumption of meiosis

The objective was to investigate the ability of freeze-dried (FD) bull spermatozoa to induce calcium oscillations in mouse oocytes and meiosis resumption in in vitro-matured bovine oocytes after intracytoplasmic sperm injection (ICSI). Bull spermatozoa were freeze-dried and stored for 1 y at +25, +4, or -196 degrees C. In the first experiment, rehydrated sperm heads were microinseminated into hybrid mouse oocytes loaded with fluo-3/AM, and the kinetics of intracellular calcium concentration was monitored for 1h. Repetitive increases of intracellular calcium concentration were recorded in the majority of injected oocytes, with exception of a few oocytes injected with FD sperm heads stored at +4 degrees C (11%) and +25 degrees C (8%) that exhibited a single increase or no response (non-oscillated). The proportion of oocytes that oscillated with high frequency (>or=10 spikes/h) was higher in the non-dried control group (79%; P<0.05) than in the FD groups (58, 55, and 54% for storage at -196, +4, and +25 degrees C, respectively). In the second experiment, control and FD spermatozoa were microinseminated into in vitro-matured, denuded bovine oocytes. The oocytes were fixed and stained 12h after ICSI. A higher proportion of bovine oocytes injected with control spermatozoa (70%; P<0.05) resumed meiosis than those injected with +25, +4 and -196 degrees C stored FD spermatozoa (53, 48, and 57%, respectively). The proportion of ICSI oocytes that developed to the pronuclear stage (complete activation) was higher in the control group (64%; P<0.05) than those in all the FD groups (34, 27, and 28% for storage at -196, +4, and +25 degrees C, respectively). Thus, the ability of bull spermatozoa to induce frequent intracellular calcium spikes in mouse oocytes was impaired by the process of freeze-drying, without differences among storage at +25, +4 or -196 degrees C, probably resulting in a lower proportion of bovine oocytes that resumed meiosis and/or developed to the pronuclear stage.     

Relation between storage temperature and fertilizing ability of freeze-dried mouse spermatozoa

The advantage of freeze-dried mouse spermatozoa is that samples can be stored in the refrigerator (+4 degrees C). Moreover, the storage of freeze-dried spermatozoa at ambient temperature would permit spermatozoa to be shipped easily and at low cost around the world. To examine the influence of the storage temperature on freeze-dried spermatozoa, we assessed the fertilizing ability of spermatozoa stored at different temperatures. Cauda epididymal spermatozoa were freeze-dried in buffer consisting of 50 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 50 mM NaCl, and 10 mM Tris-HCl (pH 8.0). Samples of freeze-dried spermatozoa were stored at -70, -20, +4, or +24 degrees C for periods of 1 week and 1, 3, and 5 months. Sperm chromosomes were maintained well at -70, -20, and + 4 degrees C for 5 months, and oocytes fertilized with these spermatozoa developed to normal offspring. Moreover, the chromosomal integrity of spermatozoa stored at -20 or + 4 degrees C did not decrease even after 17 months. In contrast, the chromosomes of spermatozoa stored at +24 degrees C were maintained well for 1 month but became considerably degraded after 3 months. In addition, to investigate the cause of deterioration of sperm chromosomes during storage at +24 degrees C, spermatozoa were freeze-dried in buffer containing DNase I. The chromosomes of spermatozoa freeze-dried with 1 or 0.2 units/ml of DNase I, 100% or 72%, respectively, exhibited chromosomal abnormalities. Our findings suggest that freeze-dried spermatozoa can be stored long-term with stability at +4 degrees C, and the suppression of nucleases present in the buffer or spermatozoa during storage led to the achievement of long-term storage of freeze-dried spermatozoa.     

Lactate dehydrogenase activity of rat epididymis and spermatozoa: effect of constant light

During its passage through the epididymis, the gamete undergoes a process of "maturation" leading to the acquisition of its fertilizing ability. The epididymis displays regional variations in the morphology and metabolic properties of its epithelium which are relevant for the progressive development of mature sperm characteristics. The epididymis has spontaneous peristaltic contractions and receives sympathetic innervation that is modulated by melatonin, a hormone synthesized and released by the pineal gland. Constant lighting disrupts melatonin synthesis and secretion. We have studied the effect of constant light on lactate dehydrogenase (LDH; EC 1.1.1.27) and its isozyme C4 activities and protein content in whole epididymis, epididymal tissue and in spermatozoa from caput and cauda segments. Animals were exposed from birth to an illumination schedule of 14 h light:10 h dark (group L:D). At 60 days of age one group of animals was submitted to constant light over 50 days (group L:L). In order to test the fertilizing ability, the rats of each group were mated with soliciting estrous females. The percentage of pregnancies in females mated with males maintained in L:L was remarkably lower than those in females mated with males maintained in the L:D photoperiod (44% and 88% respectively). Constant light increased protein concentration and LDH activity in caput as well as in cauda of total epididymis. On the contrary, in epididymal tissue, the protein content decreased in both epididymal sections compared with controls. When enzymatic activity was expressed in Units per spermatozoa, constant light induced a significant reduction of total LDH and LDHC4 in caput and cauda spermatozoa while LDH activity of epididymal tissue was not affected. In spite of the decrease in LDH per sperm cell when rats were exposed to constant light, in total epididymis (epididymis tissue plus sperm cells content) and in spermatozoa, values of enzyme activities expressed per weight unit were higher than those of controls. This is explained by the increase in the amount of stored spermatozoa, both in caput and cauda, produced by exposure of animals to constant light. Our results confirm that in rats, chronic exposure to constant light promotes a reduction of fertilizing ability and indicates that continuous lighting reduces the total LDH and LDHC4 activities, possibly due to moderate aging of spermatozoa within the duct by lengthening of the sperm transit through the epididymis.     

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.     

Sperm penetration in vitro of pig follicular oocytes matured in culture.

Boar ejaculated and epididymal spermatozoa were preincubated in modified KRB or the isolated oviduct and uterine horn of an oestrous sow for 4.5-5 h at 37 degrees C before introduction into medium containing ovarian oocytes previously cultured for 24 h. At examination 17-20 h after insemination 60.6% of the total oocytes had reached at least the 2nd metaphase. The proportions of oocytes penetrated (i.e. enlarged sperm head or male pronucleus and corresponding sperm tail) were 0, 10.0 and 16.7% with ejaculated spermatozoa, and 3.3, 19.6 and 26.4% with epididymal spermatozoa preincubated in modified KRB, oviduct and uterus, respectively. Although the proportion of oocytes with morphologically normal male and female pronuclei was low (10/36 = 27.8%), the results suggest that boar spermatozoa can be capacitated in the isolated genital tract of an oestrous sow and that capacitation of epididymal is better than that of ejaculated spermatozoa.     

Recovery of motile,membrane-intact spermatozoa from canine epididymides stored for 8 days at 4 degrees C

The purpose of this study was to determine how long canine spermatozoa remain motile and with intact membranes when maintained within epididymides stored at 4 degrees C, and to determine whether such stored spermatozoa are able to bind to canine zonae pellucidae. Testes with attached epididymides, obtained from 32 dogs (26 purebred; six mixed breeds) at orchiectomy, were refrigerated at 4 degrees C, and spermatozoa were collected from caudae epididymides at nine time intervals ranging from 5 to 192 h. The effects on spermatozoa that had been refrigerated within epididymides for various times were determined by assaying sperm motility, integrity of plasma membranes and of acrosomes, and measuring binding of membrane-intact spermatozoa to canine zonae pellucidae. Membrane integrity was assessed using a double fluorescent dye, and acrosome integrity by staining with Pisum sativum agglutinin. For the zona-binding assay at various refrigeration time points, duplicate sets of six oocytes each, isolated from ovaries retrieved at elective ovariohysterectomy, were placed into 100 microl droplets of sperm capacitation medium containing 5 x 10(6) spermatozoa/ml. One minute later, oocytes were rinsed vigorously by pipetting, and then incubated for 1 h at 38.5 degrees C in a humidified atmosphere of 5% CO2 in air; the number of membrane-intact spermatozoa bound to zonae were counted. There was no significant decrease in membrane integrity and acrosome integrity of spermatozoa recovered from epididymides stored at 4 degrees C within the first 48 h of refrigeration. In contrast, sperm motility decreased significantly within the first 5 h of refrigeration (P < 0.05), but then declined more gradually thereafter. Some spermatozoa recovered from epididymides that had been refrigerated for 192 h retained their capability to bind to zonae pellucidae, although the mean number of refrigerated spermatozoa (0.4) bound to zonae was less than that of fresh samples (9.0). Membrane integrity of spermatozoa recovered from epididymides refrigerated for various times was highly correlated (r = 0.88) with sperm motility. Even after storage for 192 h (8 days) at 4 degrees C, motile spermatozoa could be recovered from the epididymides, and such refrigerated spermatozoa were capable of binding to zonae. We interpreted these data to indicate that it might be possible to recover functional spermatozoa from postmortem specimens of domestic and nondomestic canids.     

Thermostability of sperm nuclei assessed by microinjection into hamster oocytes

Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronuclei. Mature, mammalian sperm nuclei, which are stabilized by protamine disulfide bonds, were moderately heat resistant. For example, they remained capable of pronucleus formation even after pretreatment for 30 min at 90 degrees C. Indeed, a temperature of 125 degrees C (steam) was required to inactivate hamster sperm nuclei completely. On the other hand, nuclei of rooster and tilapia spermatozoa and those of immature hamster and mouse spermatozoa, which are not stabilized by protamine disulfide bonds, were sensitive to heating; although some of them decondensed after exposure to 90 degrees C, none formed male pronuclei. Furthermore, nuclei of mature hamster sperm became heat labile when they were pretreated with dithiothreitol to reduce their protamine disulfide bonds. These observations suggest that the thermostability shown by the nuclei of mature spermatozoa of eutherian mammals is related to disulfide cross-linking of sperm protamines.     

The effect of liposomes on thermotropic membrane phase transitions of bovine spermatozoa and oocytes: implications for reducing chilling sensitivity

We have examined the effects of combinations between egg-phosphatidylcholine (EPC) or dipalmitoylphosphatidylcholine (DPPC) liposomes with either bovine spermatozoa or oocytes on cellular chilling sensitivity, lipid phase transition temperature (T(m)), and the ability of the oocytes to develop to the blastocyst stage. Spermatozoa and oocytes were exposed to EPC and DPPC liposomes at various temperatures (spermatozoa: 4, 12, 16, and 25 degrees C; oocytes: 4, 16, and 32 degrees C). The membrane integrity of the spermatozoa-control group decreased significantly following exposure to 16 or 12 degrees C, compared to ambient temperature (25 degrees C). In contrast, the EPC-sperm group had a greater resistance to chilling at each temperature and showed a decline in membrane integrity only at the lowest temperatures investigated. However, the DPPC-sperm group was injured significantly at all temperatures tested. Similar to the sperm, oocytes from the control group that were exposed to 16 degrees C were injured more severely than oocytes that were electrofused with EPC or DPPC liposomes. The membrane integrity of the oocytes at 16 degrees C that were electrofused with either EPC or DPPC liposomes was approximately the same as the control group held at 32 degrees C (normalized to 100%), compared to 46% in the control group at 16 degrees C (P<0.01). The transition temperatures of the sperm and oocyte membranes revealed different T(m) for the different liposome treatments. All groups had a significantly higher cleavage rate, as well as increased blastocyst formation when oocytes were exposed to temperatures above or below their T(m). We suggest that the T(m) of spermatozoa or oocytes can be changed by spontaneous association or electrofusion of liposomes with cellular membranes and, consequently, the chilling sensitivity can be altered. The resulting possibility is that embryo development after cryopreservation could be improved with such a method.     

Factors affecting the efficiency of producing porcine embryos expressing enhanced green fluorescence protein by ICSI-mediated gene transfer method

This study aims to investigate factors that affect the efficiency of blastocyst development and enhanced green fluorescence protein (EGFP) expression in porcine embryos following intracytoplasmic sperm injection (ICSI)-mediated DNA transfer. Frozen-thawed dead spermatozoa were exposed to different concentrations (0.01 microg/mL, 0.05 microg/mL or 0.1 microg/mL) of EGFP DNA solution, and then microinjected into in vitro matured oocytes. The optimal concentration for EGFP expression of resultant embryos was 0.05 microg/mL. When oocytes were microinjected on a warm stage at 30 degrees C, the percentage of EGFP-expressing embryos was higher than that at 38.5 degrees C (40.1% vs. 20.9%, P<0.01). The efficiency of EGFP expression in embryos following ICSI using linear EGFP DNA-exposed spermatozoa was higher than using circular DNA (40.8% vs. 28.2%, P<0.05). ICSI oocytes treated with 6-DMAP after electro-activation had a higher percentage of embryos expressing EGFP than those not treated (52.5% vs. 26.3%, P<0.01). However, neither incubation temperatures of spermatozoa and DNA (4 degrees C, 24 degrees C or 39 degrees C) nor BSA addition to the incubation medium affected the efficiency of producing EGFP-expressing embryos. Furthermore, treatment with DNase I after preincubation of sperm and DNA prevented the embryos from expressing EGFP. The EGFP expression of ICSI oocytes was affected neither by intracytoplasmic injection using sperm heads or whole spermatozoa, nor by washing of the sperm after preincubation. The above-mentioned factors did not affect embryonic developmental competence, apart from 6-DMAP treatment after electro-activation. In conclusion, most exogenous DNA molecules were tightly bound on the membranes of sperm head after incubation of DNA and sperm, and the temperature during ICSI, 6-DMAP treatment, exogenous DNA concentrations and constructs could significantly affect EGFP expression in porcine embryos following ICSI-mediated DNA transfer.     

Addition of an androgen-free epididymal protein extract increases the ability of immature hamster spermatozoa to fertilize in vivo and in vitro

The fertility of spermatozoa from the different epididymal segments of hamsters was tested by in-vivo insemination. Caput and proximal corpus spermatozoa were non-fertile; spermatozoa from the distal corpus epididymidis fertilized 13% (38/290) oocytes and those from the proximal and distal cauda epididymidis 71 and 87%, respectively. When tested by in-vitro insemination, distal corpus spermatozoa penetrated 44% of oocytes while those from the distal cauda fertilized 87% of oocytes. Spermatozoa from the distal corpus recovered in Medium BMOC fertilized 13% (28/219) of oocytes in vivo, while those mixed with an epididymal protein preparation (0.8 mg protein/ml) fertilized 24% (49/204; P less than 0.01) of oocytes. When distal corpus spermatozoa were inseminated in vivo with 0.8 mg epididymal protein preparation 34% (31/90) oocytes were fertilized and only 22% (23/103; P less than 0.05) oocytes were fertilized when the proteins were obtained from epididymides of animals castrated for 30 days. When distal corpus spermatozoa were preincubated for 5 h in medium without (control) or with protein preparation (0.8 or 1.6 mg protein/ml), a significant increase in in-vitro oocyte penetration was found (25 compared with 45%; P less than 0.05) when the protein was present at 1.6 mg/ml. These results confirm and extend previous observations suggesting a role for androgen-dependent glycoproteins secreted by the epididymis in the acquisition of fertilizing ability that occurs during sperm maturation.