Bovine in vitro fertilization with frozen-thawed semen

A procedure to obtain high and repeatable fertilization frequencies for bovine in vitro fertilization (IVF) with frozen-thawed sperm was developed. IVF frequency of in vitro matured oocytes was increased by a swimup sperm separation procedure (P=0.01) or treatment of sperm with the glycosaminoglycan heparin (P=0.0001), but the two factors did not interact (P=0.23). Heparin was the most important factor in increasing IVF frequencies. The fertilization frequency was not affected by the batch of oocytes used (P=0.38), but bull effects were present (P<0.05). Within a bull, the IVF system was highly repeatable and varied between trials no more than +/- 12% in fertilization frequency with an overall fertilization frequency of 299 379 (79%) on four trials over four bulls. In vivo matured oocytes fertilized in vitro were transferred to ewe or heifer oviducts. Morulae or blastocysts were recovered from ewes after four to five days, while conceptuses were present in the bovine after 25 days (diagnosed by ultrasound). Embryonic development from the IVF system either pre- or postimplantation was normal.     

In vitro development of domestic cat embryos following intra-cytoplasmic sperm injection with testicular spermatozoa

The objective was to assess the ability of testicular spermatozoa to fertilize in vitro matured domestic cat oocytes and support blastocyst formation in vitro following intra-cytoplasmic sperm injection (ICSI). After IVM, oocytes were randomly and equally allocated among treatment groups (ICSI with testicular spermatozoa, ICSI with ejaculated spermatozoa, sham ICSI, and control IVF). At 18 h after either injection or insemination, the percentage of fertilized oocytes (per total metaphase II oocytes) was approximately 65% after ICSI with testicular or ejaculated spermatozoa (P > 0.05), which was less (P < 0.05) than control IVF (approximately 90%). On Day 7, the percentage of cleaved embryos (per total metaphase II oocytes) was approximately 60% after ICSI with testicular or ejaculated spermatozoa (P > 0.05), which also was less (P < 0.05) than control IVF (approximately 85%). After ICSI with testicular spermatozoa, the percentage of blastocysts (per total cleaved embryos) was approximately 11.0%, which was less (P < 0.05) than ICSI with ejaculated spermatozoa (approximately 21.0%); the latter was less (P < 0.05) than control IVF (approximately 43.0%). No blastocyst formation was observed after sham ICSI. For the first time in the domestic cat, this study demonstrated the fertilizing ability and developmental potential of intra-testicular spermatozoa delivered directly into intra-ovarian oocytes matured in vitro.     

Embryo development of prepubertal goat oocytes fertilised by intracytoplasmic sperm injection (ICSI) according to oocyte diameter

The aim of this study was to evaluate embryo development of prepubertal goat oocytes fertilised by ICSI according to their diameter. Three experiments were carried out to achieve this objective. In all experiments, oocytes were matured in TCM199 supplemented with hormones, cysteamine and serum for 27 h at 38.5 degrees C. In Experiment 1, we studied the nuclear stage of goat zygotes produced by conventional ICSI and IVF using 20 nM ionomycin plus 10 microM heparin as sperm treatment. A group of Sham-injected oocytes was used as control. Results showed differences in the percentage of 2 PN (zygotes with male and female pronuclei) between ICSI, IVF and Sham (40.9, 26.6 and 3.0%, respectively; P<0.05). In Experiment 2, we evaluated the embryo development of prepubertal goat oocytes produced by ICSI and IVF after 192 h of culture in SOF medium. The percentage of morulae plus blastocysts obtained was higher in the ICSI than in the IVF group (13.4 and 5.1%, respectively; P<0.05). In Experiment 3, IVM-oocytes were classified in four groups depending on their diameter (Group A: <110 microm; Group B: 110-125 microm; Group C: 125-135 microm; Group D: >135 microm), fertilised by ICSI and cultured for 192 h. Results showed a positive correlation between oocyte diameter and embryo development (morulae+blastocysts: Group A: 0%; Group B: 6.2%; Group C: 46.4% and Group D: 33.3%). In conclusion, sperm treatment with ionomycin plus heparin using the conventional ICSI protocol improved fertilisation rates in comparison to IVF. Oocytes smaller than 125 microm were unable to develop up to blastocyst stage.     

Optimization of cryopreservation of buffalo (Bubalus bubalis) blastocysts produced by in vitro fertilization and somatic cell nuclear transfer

The objective of this study was to optimize cryopreservation conditions for buffalo in vitro produced (IVP) embryos. The in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) blastocysts were vitrified with either 40% ethylene glycol (EG), 25% EG + 25% dimethylsulfoxide (DMSO), or 20% EG + 20% DMSO + 0.5 m sucrose, and the IVF blastocysts produced from abattoir-derived ovaries were also slow-frozen with either 10% EG or 0.05 m trehalose dehydrate + 1.8% EG + 0.4% BSA. Cryosurvival rates of blastocysts harvested on various days or at various developmental stages were also examined. In this study: (1) vitrification with 20% EG + 20% DMSO + 0.5 m sucrose had the best cryopreservation efficiency; (2) IVF and SCNT blastocysts had similar cryotolerance (P > 0.05); (3) after thawing, slow-frozen blastocysts reexpanded earlier than the vitrified blastocysts (P < 0.01); (4) cryosurvival rate of expanded blastocysts was higher than that of early blastocysts (P < 0.05); (5) cryosurvival rates of Days 5 to 7 blastocysts (Day 0 = day of IVF or SCNT) were higher than those of Days 8 to 9 blastocysts (P < 0.01); and (6) after embryo transfer, pregnancy rates for fresh and cryopreserved blastocysts were not different (P > 0.05). In conclusion, vitrification of Days 6 to 7 expanded blastocysts with 20% EG + 20% DMSO + 0.5 m sucrose was optimal for cryopreservation of buffalo IVP embryos.     

Full term development of rabbit oocytes fertilized by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has been applied successfully in the treatment of male infertility in humans and in fertilization research in mice. However, the technique has had limited success in producing offspring in other species including the rabbit. The aim of this research was to test the in vitro and in vivo developmental of rabbit oocytes after ICSI. Sperm used for ICSI were collected from mature Dutch Belted buck and washed 2-3 times with PBS +0.1% polyvinyl alcohol (PVA) and then mixed with 10% polyvinyl pyrrolidone (PVP) prior to microinjection. Oocytes were collected from superovulated does 14-15 hr after hCG injection and were fertilized by microinjection of a single sperm into the ooplasm of each oocyte without additional activation treatment. After ICSI, the presumed zygotes were either cultured in KSOM +0.3% BSA for 4 days or transferred into oviducts of recipient does at the pronuclear or 2-cell stage. A high percentage of fertilization (78%, n = 114) and blastocyst development (39%) was obtained after ICSI. Control oocytes, receiving a sham injection, exhibited a lower activation rate (31%, n = 51) and were unable to develop to the blastocyst stage, suggesting that the blastocysts developed following ICSI were derived from successful fertilization rather than parthenogenetic development. A total of 113 embryos were transferred to six recipient does. Two recipients became pregnant and delivered seven live young. Our results demonstrated that rabbit oocytes can be successfully fertilized and activated by ICSI and can result in the birth of live offspring.     

Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability

To study the effect of partial removal of intracytoplasmatic lipids from bovine zygotes on their in vitro and in vivo survival, presumptive zygotes were delipidated by micromanipulation and cocultured with Vero cells in B2+10% FCS. Blastocyst rates of delipidated (n=960), sham (centrifuged but not delipidated, n=830) and control embryos (n=950) were 42.1, 42.3 and 39.9% respectively (P > 0.05). Day 7 blastocysts derived from delipidated zygotes had a mean of 123.9 +/-45.6 nuclei compared to 137.5+/-32.9 for control blastocysts (P > 0.05). The full-term development of delipidated blastocysts after single transfer to recipients was similar to that of control IVF blastocysts (41.2% vs 45.4% respectively). To assess the effect of delipidation on the embryo tolerance to freezing/thawing, delipidated (n=73), control (n=67) and sham (n=50) Day 7 blastocysts were frozen in 1.36 M glycerol + 0.25 M sucrose in PBS. After thawing, embryos were cocultured for 72 h with Vero cells in B2+10% FCS. Survival rates at 24 h were not significantly different between groups. However, in the delipidated group, the survival rate after 48 h in culture was significantly higher than in the control group (56.2 vs 39.8, P < 0.02), resulting in a higher hatching rate after 3 days in culture (45.2 vs 22.4, P < 0.02). Pregnancy rates for delipidated and control frozen/thawed embryos were respectively 10.5 and 22.2% (P > 0.05). Electron microscopic observations showed much fewer lipid droplets (and smaller) in delipated blastocysts than in controls. Taken together, our data show that delipidation of one cell stage bovine embryos is compatible with their normal development to term and has a beneficial effect on their tolerance to freezing and thawing at the blastocyst stage. This procedure, however, alters the developmental potential of such blastocysts, suggesting that maternally inherited lipid stores interfere with metabolic recovery after thawing.     

Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle

Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is one of the most important pathogens of dairy cattle; it can cause several clinical syndromes, ranging from subclinical to severe disease. The objectives of the current studies were to assess the effects of two biotypes of BVDV on sperm attachment to the zona pellucida (ZP) of oocytes and on fertilization rate in bovine in vitro fertilization (IVF). In two experiments, sperm at two concentrations (10⁵ and 10⁶/mL) and oocytes were incubated with 10⁶ TCID₅₀/mL cythopatic (CP) or noncythopatic (NCP) BVDV. In the first experiment, with the lower sperm concentration (10⁵/mL), male and female gametes were infected with CP or NCP BVDV, whereas in the second experiment, the sperm concentration was 10⁶/mL, and sperm and oocytes were also infected with CP or NCP BVDV. The number of sperm attached to the ZP and the fertilization rate were evaluated with fluorescence microscopy on the ZP of fertile and infertile oocytes. In the first experiment, compared to the control group (n = 97), oocytes infected with CP BVDV and incubated at the lower (10⁵/mL) sperm concentration positively affected sperm attachment (n = 123) to the ZP of fertile oocytes (P < 0.05). In comparison with the control group (n = 115), sperm infected with CP BVDV negatively affected sperm binding (n = 93) to the ZP of infertile oocytes (P < 0.05). In the second experiment (10⁶ sperm/mL), for both fertile and infertile oocyte groups, sperm attachment in the control group was very high and deemed uncountable. However, in treated groups, the number of sperm attached to the ZP was countable. Only sperm infected with CP BVDV negatively affected sperm binding capacity (n = 81) to the ZP of fertile oocytes (P < 0.05). Although CP and NCP BVDV significantly reduced the fertilization rate of oocytes incubated with a higher sperm concentration, with the lower sperm concentration, only NCP BVDV significantly diminished fertilization rate with contaminated sperm and oocytes (P < 0.05). In conclusion, this study supported the detrimental impacts of sperm or ooctyes infected with CP or NCP BVDV on sperm attachment to the ZP of bovine oocytes and on fertilization rate during bovine IVF.     

小鼠精子注入兔卵母细胞受精研究

The methods of intracytoplasmic sperm injection (ICSI) and subzonal injection (SUZI) were used to study heterologous fertilization and embryonic development between the mouse and the rabbit. Results were as follows: 1. The mouse sperm nuclei decondensed and formed pronuclei following microinjection into cytoplasm and perivitelline space (PVS) of rabbit oocytes; 2. The hybrid embryos developed to the stage of 8-cell when cultured in vitro; 3. The karyotype analysis showed a normal complement of rabbit oocyte and mouse sperm chromosomes in the 4-cell hybrid embryos; 4. The ultrastructure of 4-cell hybrid embryos was similar to that of normal 4-cell rabbit embryos; 5. The fertilization rate (32.4%) and cleavage rate (22.2%) when 5-10 mouse spermatozoa were injected were higher than those of injection of a single spermatozoon into PVS of the rabbit oocyte, but the difference was not significant (P > 0.05). The fertilization rate (42.3%) and cleavage rate (30.8%) in rabbit oocytes in vitro matured for 11-12 h were higher than those in the oocytes which were in vitro matured for 24-25 h following microinjection of 1-2 mouse spermatozoa into PVS, but the difference was not significant (P > 0.05).     

In vitro embryo production after microinjection and ovarian dynamics following transvaginal follicular oocyte aspiration

Ultrasound-guided transvaginal follicular aspiration of oocytes from live cows combined with IVM, IVF and in vitro culture (IVC) is a procedure for producing preimplantation-stage bovine embryos and a source of oocytes for pronuclear microinjection of DNA for producing transgenic cattle. This experiment was designed to compare in vitro embryo development rates between oocytes derived from transvaginal follicular aspiration and those obtained from cows at slaughter. Nine cows were subject to a twice-weekly aspiration. Oocytes were aspirated with a 5 MHz ultrasound transducer packaged in a vaginal probe equipped with a dorsal-mounted needle guide (16-ga). All visible follicles (>2 mm) were punctured with a 17-ga, 55-cm needle at each aspiration session and the contents removed under vacuum suction. Oocytes underwent IVM/IVF/IVC. Microinjection of DNA was performed during the pronuclear stage of development, and the zygotes were co-cultured on Buffalo Rat Liver (BRL) cells in modified M199 at 39 degrees C in 5% CO2 and air. After 7 d in culture, embryos were removed and scored for development. A Chi-square analysis was used to compare transvaginal follicular-derived oocytes (microinjected and not) and slaughterhouse-derived, matured in transit oocytes (SHDMT; microinjected and not). Nonmicroinjected embryos resulting from IVF of transvaginal aspiration-derived oocytes developed to blastocysts at a higher rate than SHDMT oocytes (40.0 vs 30.8%; P < 0.05). There was no difference in development rates between the microinjected groups (aspiration = 15.9% vs SHDMT = 12.8%). Higher proportions of the embryos generated from the aspirated oocytes were of excellent or good quality following culture (P < 0.05). In the present experiments the effects of microinjection may overshadow some effects of ova source, but transvaginal follicular aspiration may provide a more consistent, synchronous population of oocytes than those derived from commercial slaughter house sources for use with in vitro systems.     

Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits

The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8-, 16-, and 32-cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate-buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire-polished micropipette following incubation in Ca+(+)-free PBS for 15 min at 39 degrees C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8- or 16-cell blastomere was aspirated into an injection pipette (35 microns or 25 microns at the tip, respectively) and inserted into the perivitelline space of an irradiated or non-irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P greater than .05). When the inserted blastomere was induced to fuse with an intact non-irradiated oocyte under an electric field, 56-57% were fused and 39-45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8-32-cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV-light irradiation, 63-66% of them were fused, but only 15-22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.     

Sperm movement in the ooplasm, dithiothreitol pretreatment and sperm freezing are not required for the development of porcine embryos derived from injection of head membrane-damaged sperm

The present study investigated the correlation of sperm movement in the ooplasm, pretreatment of sperm with dithiothreitol (DTT) and sperm freezing with the development of porcine embryos derived from modified intracytoplasmic sperm injection (ICSI). In vitro, matured gilt oocytes without centrifugation were injected with head membrane-damaged spermatozoa aspirated tail-first. In Exp. 1, frozen-thawed sperm were categorized into three groups: impaired, immotile or motile. Oocytes injected with motile sperm (43.6%) showed a higher (P < 0.05) fertilization rate compared to oocytes injected with impaired or immotile sperm (34.5 or 37.2%). The survival rate was significantly higher (P < 0.05) in oocytes injected with impaired sperm (92.9%) than in oocytes injected with immotile or motile sperm (84.8 or 86.7%). No differences were observed in the rates of cleavage or blastocyst formation, and in total cell number of blastocysts among three groups of oocytes. In Exp. 2, motile frozen-thawed sperm were pretreated with DTT before injection and non-treated sperm served as controls. Higher rates (P < 0.05) of fertilization, male pronucleus (MPN) and decondensed sperm head (DSH) formation were observed in oocytes injected with control sperm (41.1, 50.0 and 91.1%, respectively) than in oocytes injected with DTT-treated sperm (22.1, 30.2 and 72.1%, respectively). No differences in embryo development and total cell number of blastocysts were observed between two groups of oocytes. In Exp. 3, motile frozen-thawed or fresh sperm without DTT pretreatment were injected into oocytes. The rates of fertilization and MPN formation were significantly higher (P < 0.05) in oocytes injected with fresh sperm (59.8 and 73.5%) than in oocytes injected with frozen-thawed sperm (36.7 and 59.2%). No differences in embryo development and total cell number of blastocysts were observed between two groups of oocytes. In conclusion, the present study clearly demonstrated that sperm movement in the ooplasm, use of DTT and fresh spermatozoa did not significantly affect on embryo development in porcine modified ICSI.     

Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization,perivitelline, and intracytoplasmic sperm injections

IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine sperm capacitated with dilauroyl phosphatidylcholine would result in higher fertilization rates than standard IVF in bovine and equine oocytes. In experiment 1, bovine oocytes were used for (1) IVF with bovine sperm, (2) IVF with equine sperm, and (3) intracytoplasmic sperm injections (ICSIs) with equine sperm. Presumptive zygotes were either stained with 4′,6-diamidino-2-phenylindole from 18 to 26 hours at 2-hour intervals or evaluated for cleavage at 56 hours after addition of sperm. Equine sperm fertilized bovine oocytes; however, pronuclei formation was delayed compared with bovine sperm after IVF. The delayed pronuclear formation was not seen after ICSI. In experiment 2, bovine oocytes were assigned to the following five groups: (1) cumulus oocyte complexes (COCs) coincubated with bovine sperm; (2) COC exposed to sucrose then coincubated with bovine sperm; (3) COC coincubated with equine sperm; (4) COC exposed to sucrose, and coincubated with equine sperm; and (5) oocytes exposed to sucrose, and 10 to 15 equine sperm injected into the perivitelline (PV) space. Equine sperm tended (P = 0.08) to fertilize more bovine oocytes when injected into the PV space than after IVF. In experiment 3, oocytes were assigned to the following four groups: (1) IVF, equine, and bovine COC coincubated with equine sperm; (2) PVI of equine and bovine oocytes; (3) PVI with equine oocytes pretreated with sucrose; and (4) ICSI of equine oocytes. Oocytes were examined at 24 hours for cleavage. No equine oocytes cleaved after IVF or PVI. However, ICSI conducted with equine sperm treated with dilauroyl phosphatidylcholine resulted in 85% of the oocytes cleaving. Sperm injected into the PV space of equine oocytes did not appear to enter the ooplasm. This study validated the use of bovine oocytes for equine sperm studies and indicates that failure of equine IVF is more than an inability of equine sperm to penetrate the ZP.     

Potential of hypertonic medium treatment for embryo micromanipulation: I. Survival of rabbit embryos in vitro and in vivo following sucrose treatment

Rabbit zygotes and embryos were exposed to hypertonic sucrose in phosphate-buffered saline (SPBS). In experiment one, 144 zygotes shrank to 32-36% of their initial volume in 1.0 M SPBS within 30 min. Neither hypertonic treatment with 0.5 M or 1.0 M SPBS nor micropuncture of the zona pellucida after shrinkage affected embryo development into blastocysts in vitro (88%, 83%, and 82%, respectively), compared to that of the controls (93%, P greater than .05). In experiment two, 252 two- to four-cell- and 177 morula-stage embryos were exposed to isotonic PBS control or 0.5 M, 1.0 M, or 1.5 M SPBS for 30, 60, 90, 120, and 150 min before transfer to PBS (290 mOsm). Embryo development was significantly reduced (P less than .05) when embryos were exposed in 0.5 M and 1.0 M SPBS for more than 60 min or in 1.5 M SPBS for more than 30 min. In experiment 3, morulae exposed for 60 min to 0.5 M or 1.0 M SPBS shrank to 37-39% or 32-35% of their initial volume and then expanded to 87-94% or 81-90% of their initial volume, respectively, after being returned to isotonic PBS for 60 min, but embryos in 1.5 M SPBS had erratic osmotic behavior. In experiment four, 192 two- to four-cell embryos exposed to 0.5 M SPBS for 0, 30, and 60 min before transfer to oviducts of recipients resulted in the production of 39%, 42% and 31% young, respectively (P greater than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Hybrid embryos produced by transferring panda or cat somatic nuclei into rabbit MII oocytes can develop to blastocyst in vitro

The developmental potential of hybrid embryos produced by transferring panda or cat fibroblasts into nucleated rabbit oocytes was assessed. Both the panda-rabbit and the cat-rabbit hybrid embryos were able to form blastocysts in vitro. However, the rates of attaining the two-cell, four-cell, eight-cell, morula, or blastocyst stages for panda-rabbit hybrids were significantly greater than those of cat-rabbit hybrids (P<0.05). Transferring the rabbit fibroblasts into nucleated rabbit oocytes, 31.0% of the blastocyst rate was obtained, which was significantly higher than that of both the panda-rabbit and the cat-rabbit hybrid embryos (P<0.05). Whether or not the second polar body (PB2) was extruded from the one-cell hybrid embryos (both panda-rabbit and cat-rabbit hybrids) significantly affected their developmental capacity. Embryos without an extruded PB2 showed a higher capacity to develop into blastocysts (panda-rabbit: 19.2%; cat-rabbit: 4.3%), while embryos with extruded PB2 could only develop to the morula stage. The hybrid embryos formed pronucleus-like structures (PN) in 2-4 hr after activation, and the number of PN in one-cell embryos varied from one to five. Tracking of the nucleus in the egg after fusion revealed that the somatic nucleus could approach and aggregate with the oocyte nucleus spontaneously. Chromosome analysis of the panda-rabbit blastocysts showed that the karyotype of the hybrid embryos (2n=86) consisted of chromosomes from both the panda (2n=42) and the rabbit (2n=44). The results demonstrate that (1) it is possible to produce genetic hybrid embryos by interspecies nuclear transfer; (2) the developmental potential of the hybrid embryos is highly correlated to the donor nucleus species; and (3) the hybrid genome is able to support the complete preimplantation embryonic development of the hybrids.     

Viability, acrosome morphology and fertilizing capacity of boar spermatozoa treated with strontium chloride

The positive effect of strontium ions (Sr2+) on sperm motility, capacitation and acrosome reaction has been demonstrated in the mouse, human, guinea pig and hamster. In the present study, we have evaluated the effect of Sr2+ on the viability and acrosome morphology of boar spermatozoa, and on the fertilization and development after the microinjection of Sr(2+)-treated spermatozoa into porcine oocytes. Before incubation, 79% of spermatozoa were classified as propidium iodide (PI)-negative (live) using the LIVE/DEAD Sperm Viability Kit. After incubation with strontium chloride (SrCl2), 39% (0 mM; no divalent cations), 25% (1.9 mM) and 24% (7.5 mM) of them were classified as PI-negative. The proportion of spermatozoa that had initiated the acrosome reaction was higher in Sr(2+)-containing medium than in Sr(2+)-free medium, when assessed by electron microscopy. There was no significant difference in percentage of spermatozoa initiating the acrosome reaction between Sr2+-treated groups (1.9 mM: 22%, 7.5 mM: 33%, p>0.05). After the microinjection of spermatozoa incubated with SrCl2, 67% (1.9 mM) and 61% (7.5 mM) of injected oocytes were successfully fertilized, and then 43% (1.9 mM) and 41% (7.5 mM) contained a fully decondensed sperm head. Sham-injected oocytes were significantly activated at a lower rate than Sr(2+)-treated groups (27%, p<0.05). Next, after microinjection of spermatozoa incubated with 1.9 mM SrCl2 (n=51), 45% of injected oocytes cleaved on day 2, and 18% developed to blastocysts on day 7 (sham-injection, n=48: 15% to cleavage and 0% to blastocyst). These results demonstrate that Sr2+ is likely to positively affect the fertilizing capacity of spermatozoa in the pig.     

In vitro fertilization and subsequent development of porcine oocytes using cryopreserved and liquid-stored spermatozoa from various boars

We had previously developed a porcine IVF system using a chemically defined medium, i.e., porcine gamete medium supplemented with theophylline, adenosine, and cysteine (PGMtac). In the present study, we investigated the utility of this IVF system using different types of semen: (1) cryopreserved ejaculated (n = 8); (2) cryopreserved epididymal (n = 4); and (3) liquid-stored ejaculated (n = 5). Cryopreserved spermatozoa were prepared by three methods. In vitro-matured porcine oocytes were fertilized for 20 h in PGMtac using each type of semen, and the presumptive zygotes were cultured in porcine zygote medium (PZM)-4 for 5 days. In the case of frozen-thawed spermatozoa, the number of spermatozoa per penetrated oocyte (1.1-1.7), rate of blastocyst formation (26-56%), and total number of cells per blastocyst (34-49) differed (P < 0.05) among freezing methods. However, blastocysts were produced using all types of cryopreserved spermatozoa (14-75%). When spermatozoa were liquid-stored for 1-14 days after semen collection, the rate of sperm penetration (P < 0.05) decreased as storage time increased, although there was no significant reduction in sperm motility during storage. In all groups, semen that had been stored within 10 days after collection enabled blastocyst production in vitro (20-48%). In conclusion, this IVF system, which uses a chemically defined medium, had widespread utility with both frozen-thawed and liquid-stored spermatozoa.     

The influence of cumulus cells during in vitro fertilization of buffalo (Bubalus bubalis) denuded oocytes that have undergone vitrification

The aim of this work was to evaluate whether providing a support of cumulus cells during IVF of buffalo denuded oocytes submitted to vitrification-warming enhances their fertilizing ability. In vitro matured denuded oocytes were vitrified by Cryotop in 20% EG + 20% of DMSO and 0.5 M sucrose and warmed into decreasing concentrations of sucrose (1.25 M-0.3M). Oocytes that survived vitrification were fertilized: 1) in the absence of a somatic support (DOs); 2) in the presence of bovine cumulus cells in suspension (DOs+susp); 3) on a bovine cumulus monolayer (DOs+monol); and 4) with intact bovine COCs in a 1:1 ratio (DOs+COCs). In vitro matured oocytes were fertilized and cultured to the blastocyst stage as a control.An increased cleavage rate was obtained from DOs+COCs (60.9%) compared to DOs, DOs+susp (43.6 and 38.4, respectively; P < 0.01) and DOs+monol (47.5%; P < 0.05). Interestingly, cleavage rate of DOs+COCs was similar to that of fresh control oocytes (67.8%). However, development to blastocysts significantly decreased in all vitrification groups compared to the control (P < 0.01).In conclusion the co-culture with intact COCs during IVF completely restores fertilizing capability of buffalo denuded vitrified oocytes, without improving blastocyst development.     

绵羊胞内单精子注射技术

In this study, the possibility of sheep transgenesis by intracytoplasmic sperm injection (ICSI) was assessed. In experiment 1, activation of ovine oocytes matured in vitro in preparation for ICSI has been investigated with 3.42 mmol/L Ca2+ treatment, ionomycin alone and ionomycin followed by 6-dimethylaminopurine (DMAP) after 3-h delay (group 1, 2 and 3, respectively). After activation, the oocytes were then cultured in SOFaaBSA medium. Cleavage rates were significantly (P<0.05) different among three groups (18.4%, 91.8% and 71.7%, respectively). In additional culture, no parthenotes in group 1, whereas 11% and 17.4% in group 2 and 3 developed to the blastocyst stage. Therefore we used the third activation method in the following ICSI tests. In experiment 2, development of ovine oocytes after ICSI was investigated. Thawed semen from two rams was separated by Percoll centrifugation and was used for ICSI or in vitro fertilization (IVF) trails. A total of 71.8% of oocytes reached the 2-cell stage following living sperm injection, which was significantly (p>0.05) different from those following IVF (41.4%) and sham-ICSI (30.2%). After seven days' culture, no sham-injected oocytes developed into the blastocyst stage, although 7% in ICSI and 16.1% in IVF-oocytes developed into the blastocyst stage, but there was no significant difference in ICSI and IVF groups (p>0.05). In the further study, the possibility of sheep transgenesis by ICSI was assessed. After coinjection of ovine oocytes matured in vitro with dead sperm cold to -20 degrees C and exogenous DNA encoding green fluorescent protein (GFP), seventy-three percent of coinjected oocytes developed to 2-cell stage (33/45) and two of them were transgene-expressing embryos. Among ten embryos at the 16-cell stage, all embryonic cells in one transgenic embryo still expressed GFP. Four coinjected blastocysts were thawed and transferred to the uterine of the two progesterone-synchronized recipient ewe. No pregnancies were detected on the 60th day. These results suggested sheep transgenic embryos could be produced by ICSI and further studies should be performed.     

Osteopontin reduces polyspermy during in vitro fertilization of porcine oocytes

This study was designed to determine the role of osteopontin (SPP1) in in vitro fertilization (IVF) in swine. The initial objective was to evaluate the effect of various concentrations of SPP1 (0, 0.001, 0.01, 0.1 and 1 microg/ml) on spermatozoa and oocytes during IVF. The results demonstrate that SPP1 reduced the rate of polyspermy in a dose-dependent manner (P < 0.05). SPP1 also reduced both the number of sperm in oocytes as compared to the control and the number of spermatozoa bound to the zona pellucida (ZP) (P < 0.05). High doses of SPP1 (1 microg/ml) reduced penetration and male pronucleus formation as compared to the control (P < 0.05). Interestingly, compared to the control group, medium doses of SPP1 increased fertilization efficiency (42.6% and 44.6% vs. 31.6%; P < 0.05), representing a 41% improvement for 0.1 microg/ml SPP1). The ZP of 0.1 microg/ml SPP1-treated oocytes was more difficult to digest than control oocytes (P < 0.05). The percentage of acrosome-reacted spermatozoa bound to the ZP during IVF increased after 4 h of 1.0 microg/ml SPP1 treatment compared to 0 or 0.1 microg/ml SPP1. SPP1 did not have an effect on sperm motility, progressive motility, and sperm viability. To confirm that the reduction of polyspermy was specific to SPP1, a mixture of pregnancy-associated glycoproteins was included in the IVF protocol and shown to have no effect on polyspermy. Furthermore, Western blotting demonstrated that a 50-kDa SPP1 form was present in the oviducts on Days 0, 3, and 5 in pregnant and nonpregnant gilts, and the concentration of SPP1 on Day 0 was higher than on Days 3 and 5. The current study represents the first report to demonstrate that SPP1 plays an important role in the regulation of pig polyspermic fertilization; it decreases polyspermy and increases fertilization efficiency during IVF.