Experiments to improve in vitro fertilization techniques for in vivo-matured porcine oocytes

Three experiments were designed for the in vitro fertilization of in vivo matured oocytes following different sperm treatments: In experiment I, spermatozoa (sperm rich fraction) were capacitated in isolated and ligated uterine horns of estrous gilts (Method A) for at least 3.5 hours and 1x10(6) sperm/ml were exposed to mature oocytes. Of 1586 oocytes 509 (32.1%) developed to the two to eight cell stage. Cleavage stage embryos (n=187) were transferred into nine recipients; 81 embryos (43.3%) were recovered after 72 hours and 21 (26%) had developed to the morula or blastocyst stage. The average number of nuclei (whole mount staining) was 108.4 and indicated normal developmental capacity. Another 127 embryos were transferred into three recipients, two became pregnant and one gilt of them delivered two offspring after 116 days of gestation. In experiment II, two different procedures for the capacitation of spermatozoa using modified TCM 199 medium were compared with that of Method A. Semen was either adjusted to a concentration of 2x10(8) sperm/ml with modified TCM 199E (Method B), or after centrifugation the sperm pellet was diluted in a 1:1 ratio with the same extender (Method C). Motility, and hyperactivity were significantly different (P 0.05) among treatments and were best after uterine incubation (Method A). The percentage of head-to-head aggregation increased significantly (P 0.05) after incubation in the uterine horn, and is suspected to represent a spontaneous acrosome reaction. A total of 492 oocytes were fertilized with semen capacitated by the three methods however, maximal fertilization (34.7%; P 0.05) was obtained with Method B. In experiment III, Method B was repeated and in order to minimize the rate of polyspermy, a total of 298 oocytes were exposed to different sperm concentrations (8x10(3); 4x10(3); 2x10(3) per oocyte). The reduction of spermatozoa exposed to oocytes improved the fertilization rate significantly (P 0.05) from 37.2 to 54.5%.     

Effects of heparin, sperm concentration and bull variation on in vitro fertilization of bovine oocytes in a protein-free medium

The present study was conducted to examine the effects of heparin, sperm concentration and bull variation on the fertilization of bovine oocytes in a protein-free medium supplemented with polyvinyl alcohol and subsequent in vitro development of fertilized embryos. The effects in protein-free medium were compared with those in medium supplemented with bovine serum albumin (BSA). In the presence of heparin (1, 10 and 100 microg/ml), nearly all the oocytes were fertilized with and without BSA. In the absence of BSA, polyspermy was lower (4 to 15%) than in its presence (15 to 48%; P < 0.05). An increase in sperm concentration from 1 x 10(4) cells/ml during insemination enhanced fertilization rate up to 1 x 10(6) cells/ml with and without BSA (14 to 90% and 3 to 77%, respectively). In the absence of BSA, the highest concentration of spermatozoa (1 x 10(7) cells/ml) gave a lower fertilization rate (55%) than that at 1 x 10(6) cells/ml (77%; P < 0.05). Polyspermy neither increased nor decreased sperm concentration without BSA (0 to 8%; P > 0.05). The effects of spermatozoa from 5 different bulls chosen randomly on in vitro fertilization in medium without BSA were examined. Individual bull variation in fertilization rate (36 to 95%) was noted at 3 different heparin concentrations (1, 10 and 100 microg/ml). Polyspermic fertilization was low (0 to 14%) and was the same for all bulls at all heparin concentrations. Embryos fertilized without BSA developed to the blastocyst stage at the same rate (27%) as those with BSA (33%; P > 0.05).     

In vitro fertilization of pig oocytes after different coincubation intervals

The effects of gamete coincubation time on porcine in vitro fertilization for 4, 6 or 8 hours in Trial 1 or for 1, 2, 3 or 4 hours in Trial 2 were studied. The 876 oocytes were recovered from oviducts of prepubertal gilts. Ovulation was induced. Excessive spermatozoa attached to zona pellucida were removed by pippeting after coincubation (1 to 8 hours). Optimum results were obtained after 3 to 4 hours of coincubation, when 23 to 29% of the oocytes were fertilized by a single spermatozoa. Shorter intervals of coincubation were characterized by reduced percentages of fertilized oocytes and longer intervals of coincubation by increased rates of polyspermic fertilization. The employed sperm concentration was deliberately high (2 x 10(6) sperm/ml). The results show that a coincubation time of 4 hours may be optimal for pig in vitro fertilization. Under these assay conditions, modification of other factors such as sperm concentration, medium volume and physiological environment may increase the percentage of monospermic fertilization.     

Effects of bull and heparin and sperm concentrations on in vitro fertilization of buffalo (Bubalus bubalis ) oocytes matured in vitro

A study was undertaken to assess the ability of spermatozoa from 6 buffalo bulls, at different levels of heparin and sperm concentrations, to achieve an acceptable level of fertilization in vitro. Frozen-thawed spermatozoa, 3 dosages of heparin (0, 10 and 100 ug/ml) in the presence and absence of penicillamine, hypotaurine and epinephrine (PHE), and 4 sperm concentrations (1 x 10(6), 2 x 10(6), 3 x 10(6) and 4 x 10(6) /ml) were studied using 3202 buffalo oocytes. The mean proportions of fertilized oocytes in the group treated with 10 ug/ml of heparin were significantly higher (P<0.05) with the semen of Bulls A, B and C (44.7 to 64.3%) than in medium devoid of heparin. An increase in the dosage of heparin from 10 ug/ml to 100 ug/ml reduced the overall fertilization rate. However, optimal fertilization (30.9%) at 100 ug/ml heparin was observed for semen from Bull D. Bulls E and F yielded the lowest fertilization rate (9.6 and 14.2%, respectively) at the above mentioned heparin dosage. Analysis of sperm density revealed that a concentration of 2 x 10(6) spermatozoa yielded optimal fertilization rates in vitro. Higher sperm concentrations (3 x 10(6) or 4 x 10(6)) resulted in higher oocyte penetration rates but gave rise to polyspermy.     

In vitro fertilization and development of llama (Lama glama ) oocytes using epididymal spermatozoa and oviductal cell co-culture

A study was designed to determine the feasibility of developing in vitro maturation, fertilization and culture systems utilizing follicular oocytes and epididymal spermatozoa collected from llamas at slaughter. From a total of 1324 cumulus oocyte complexes (COCs) recovered, 972 were cultured in 50-ul drops of TCM-199 medium with 10% heat inactivated steer serum (DBS) and hormones for 30 h. After maturation, the oocytes were randomly allocated into 4 groups in a 2x2 factorial design: cumulus-enclosed oocytes, 2 ug/ml heparin (Group 1); cumulus-enclosed oocytes, 5 ug/ml heparin (Group 2); denuded oocytes, 2 ug/ml heparin (Group 3); and denuded oocytes, 5 ug/ml heparin (Group 4). Denuded oocytes were obtained for groups 3 and 4 by vortexing. Epididymides were also collected at slaugther and fresh spermatozoa (for each replicate) were obtained by mincing the cauda epididymis with a scalpel blade. A total of 721 oocytes were inseminated with 2-3 x 10(6) epididymal spermatozoa/ml in a 50-ul drop of FERT-TALP medium. After 18 h of in vitro insemination, 234 oocytes were placed in a llama oviductal epithelial cell (LLOEC) co-culture in TCM-199 for 9 d. All cultures were done at 38.5 degrees C under 5% CO(2) in air with high humidity. The rate of fertilization, initial cleavage and development in co-culture were evaluated and compared. Of 192 oocytes examined for signs of fertilization, 56 (29.2%) were penetrated by spermatozoa with 57.1% (32 56 ) of the penetrated oocytes having a male and female pronucleus. There were no differences among treatment groups in total fertilization. However, the frequency of oocytes fertilized normally tended to be higher in the denuded oocytes 67.7% (21 31 ) than the oocytes inseminated with cumulus cells 44.0% (11 25 ) independent of heparin concentration (P<0.06). The total embryo development rate to the 2 cells to blastocyst stage was 32.1% (75 234 ). There was no difference in development rate between groups. From the 234 oocytes co-cultured in LLOEC for 9 d, 15.8% developed into 2 to 16 cells, 5.6% into morulae, 6.0% into early/expanded blastocysts and 4.7% into hatching/hatched blastocysts. The results indicate that an in vitro fertilization system is possible in the llama utilizing slaughterhouse material and that llama oocytes can be fertilized in the presence of heparin and epididymal spermatozoa.     

Effect of oviductal epithelial cells on fertilization of pig oocytes in vitro

The incidence of polyspermy is reduced by co-culture of pig oocytes with oviductal cells. It is not known whether the effect is due to soluble factors secreted into the medium. Oviductal epithelial cell monolayers and cell-conditioned media were prepared and their effects on fertilization of pig oocytes were examined. In vitro matured pig oocytes were inseminated with ejaculated boar spermatozoa at a concentration of 1 x 10(5) or 1 x 10(6) cells/ml and co-cultured in one of 5 culture systems: an oviductal epithelial cell monolayer, a fibroblast monolayer, an oviductal epithelial cell-conditioned medium, or a fibroblast-conditioned medium, and medium alone (modified-TCM199). In all 5 systems, the majority (range 85 to 100%) of the oocytes were penetrated by sperm. When oocytes were inseminated with spermatozoa at a concentration of 1 x 10(5) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with oviductal epithelial cells and fibroblasts than that of the oocytes cultured without these cells. In contrast, when oocytes were inseminated with spermatozoa at a concentration of 1 x 10(6) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with epithelial cells than those cultured with the fibroblasts and in the control medium. The suppressive effect on polyspermy was observed in the oviductal epithelial cells-conditioned medium when oocytes were inseminated with spermatozoa at both concentrations of 1 x 10(5) and 1 x 10(6) cells/ml. The effect was absent in the fibroblasts-conditioned medium. Moreover, the effect of the epithelial cells was maintained during the culture period, whereas the proportion of monospermic oocytes co-cultured with fibroblasts showed a gradual decrease, reaching 0% after 16 h. These results suggest that a soluble factor(s) derived from the oviductal epithelial cells decreased the number of spermatozoa penetrating the oocytes without suppressing the high rate of fertilization.     

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.     

Reduced polyspermic penetration in porcine oocytes inseminated in a new in vitro fertilization (IVF) system: straw IVF

High incidence of polyspermy is still a major problem in the in vitro fertilization (IVF) of porcine oocytes matured in vitro. This study was designed to examine whether embryo cryopreservation straws can be used to conduct IVF in porcine oocytes. The efficiency of this system was further compared with traditional microdrop IVF. Immature oocytes were aspirated from antral follicles and matured in vitro. After maturation, oocytes were inseminated either in straws or in microdrops with frozen-thawed boar spermatozoa. For straw IVF, sperm concentration and the presence of air columns between insemination segment and oil column were examined. Sperm-oocyte binding and cortical granules (CGs) before and after sperm penetration were examined by confocal microscopy. When various sperm concentrations were used for IVF in the straws with air columns, it was found that 5 x 106 cells/ml of sperm concentration was the optimal concentration; a high penetration rate (94.0%) and normal fertilization (oocytes with both male and female pronuclei) rate (38.2%) were obtained. Increasing sperm concentration to 10 x 106 cells/ml increased polyspermic penetration (61.9%) without affecting sperm penetration (86.9%). Reducing sperm concentration to 1 x 106 cells/ml reduced polyspermic penetration (25.6%), but sperm penetration rate (69.9%) was also reduced. When IVF was conducted in the straws with or without air columns, and in the microdrops, it was found that sperm penetration in the straws with air columns (96.5%) was significantly (p < 0.05) higher than that in the straws without air columns (81.7%) and in the microdrop (72.9%). However, the incidence of polyspermic penetration in the straws with air columns (34.2%) and without air columns (36.6%) was significantly (p < 0.05) lower than that (52.4%) in the microdrops. The number of spermatozoa bound to the oocytes was increased gradually in the straws but not in the microdrops in which more spermatozoa bound to the oocytes soon after insemination. CG exocytosis was more complete and faster in the oocytes inseminated in the straws than in the microdrops. These findings indicate that IVF of porcine oocytes in the straws provides a better condition in which more oocytes are fertilized normally than that in the microdrop IVF.     

Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development

The objectives of these experiments were to characterize separation of frozen-thawed bovine spermatozoa on a Percoll gradient and then to compare sperm separation by either a swim-up or Percoll gradient procedure for the ability of spermatozoa to fertilize oocytes in vitro. The Percoll gradient was a 45 and 90% discontinuous gradient. Initial experiments found that centrifugation of semen on the Percoll gradient for 15 min at 700 g was sufficient to obtain optimal recovery of motile spermatozoa. Most of the nonmotile spermatozoa were recovered at the interface of the 45 and 90% Percoll layers, while the motile spermatozoa were primarily in the sperm pellet at the bottom of the gradient. When frozen-thawed semen from each of 7 bulls was separated by swimup, a mean +/- SEM of 9% +/- 1 of the motile spermatozoa were recovered after the procedure. In contrast, more spermatozoa were recovered after Percoll gradient separation (P < 0.05), with 40% +/- 4 of the motile spermatozoa recovered. The effect of separation procedure on in vitro fertilization found swim-up separated spermatozoa penetrated a mean +/- SEM of 74% +/- 5 of the oocytes, while fewer oocytes were penetrated by Percoll separated spermatozoa at 52% +/- 8 (P < 0.05). There was no effect of the separation procedure on the rates of polyspermy as measured by sperm/penetrated ova, with a mean +/- SEM of 1.25 +/-.09 for swim-up separated spermatozoa and 1.14 +/-.07 for Percoll separated spermatozoa (P>0.05). A carry over of Percoll into the fertilization medium with the Percoll separated spermatozoa was found not the cause for the decreased penetration of oocytes by these spermatozoa. In 2 of 3 bulls tested, the decreased penetration of oocytes by Percoll separated spermatozoa could be overcome by increasing the sperm concentration during fertilization from 1 x 10(6) to 5 x 10(6)/ml. When development of embryos fertilized by either swim-up or Percoll separated spermatozoa was compared for the semen from 2 bulls, a difference in cleavage rate was found in favor of swim-up separated spermatozoa (P < 0.05), but there was no effect of separation procedure on development (Day 7) to the morula + blastocyst or blastocyst stage (P>0.05). The disadvantages of the Percoll procedure could easily be overcome and the procedure was faster and yielded a six-fold greater recovery of motile spermatozoa than the swim-up method.     

Effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes

This study was carried out to investigate the effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes. The concentrations of frozen-thawed sperm were 0.2 x 10(7), 2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml, respectively. Culture media were NCSU-23, HEPES-buffered (25 mM) NCSU-23, PZM-3 and PZM-4, respectively. Increasing the sperm concentration from 0.2 x 10(7) to 2 x 10(7)/ml, significantly increased the penetration rate. Also, increasing the sperm concentration from 20 x 10(7) to 200 x 10(7)/ml increased the penetration rate from 62.1% to 69.9%, respectively, with no differences between these two concentrations. A similar pattern was observed for polyspermic penetration and male pronucleus formation. The mean number of sperm per oocyte significantly increased in the 20 x 10(7)/ml and again in the 200 x 10(7)/ml sperm concentrations. The percentage of blastocysts from cleaved oocytes at the 2 x 10(7)/ml sperm concentration was significantly higher than that at the 0.2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml sperm concentrations. The percentage of blastocysts from cleaved oocytes and the cell numbers per blastocyst were significantly higher in the HEPES-buffered NCSU-23 culture medium than in the NCSU-23, PZM-3 and PZM-4 culture media under a gas atmosphere of 5% CO2 in air.     

Effect of sperm exposure time on in vitro fertilization and embryo development of bovine oocytes matured in vitro

This study was designed to investigate the effect of sperm exposure time on the fertilization rate and subsequent developmental capacity of bovine oocytes matured in vitro. Cumulus oocyte complexes (COCs) obtained from 2 to 6 mm follicles were matured for 24 h in TCM-199 supplemented with fetal bovine serum (FBS) and hormones (FSH, LH and estradiol 17-beta). In vitro fertilization (IVF) was performed by incubating 15 to 20 matured oocytes with 1 x 10(6) percoll separated frozen-thawed spermatozoa in 1 ml of IVF-TL medium for either 4, 8, 12, 16, 20, 24 or 28 h. Following sperm exposure for different periods of times, the presumptive zygotes were co-cultured with Buffalo Rat Liver cells (BRLC) monolayers in CZB medium without glucose, a simple semi-defined medium developed for mouse embryo culture, for 3 d post-insemination and then in M199/FBS (TCM-199-HEPES supplemented with 20% heat-treated FBS and 1 mM sodium pyruvate) for 5 d. The fertilization rates differed significantly among the 7 treatment groups, with higher frequencies obtained by co-incubation of gametes for 20, 24 or 28 h (67 to 76%) than for 4, 8 and 12 h (26 to 54.5%), with 16 h (57%) being intermediate. However, the length of sperm exposure time did not significantly affect subsequent embryo development, although an increasing trend was noted from 4 h to 20 h. The number of fertilized oocytes at 3 d post-insemination cleaving to 2- to 4-cell vs 8-cell stage was not different among treatment groups. Development of 8-cell embryos to morulae and blastocysts did not differ among the treatment groups. These data suggest that the optimum duration of sperm-oocyte incubation is 24 h, and periods shorter than 16 h may result in a reduced fertilization rate.     

Influence of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic development of buffalo (Bubalus bubalis) oocytes matured and fertilized in vitro

The objective of the present study was to investigate the effects of sperm concentration and presence or absence of cumulus cells on fertilization, cleavage rate and subsequent embryonic development upto the blastocyst stage in buffalo. Cumulus-oocyte-complexes (COCs) obtained from slaughterhouse ovaries were matured in vitro in TCM-199 + 10% FBS + 5 micrograms/mL FSH-P for 24 h. After maturation the COCs were either used as such (cumulus-intact) or freed from attached cumulus cells by repeated pipetting (cumulus-free). Frozen-thawed buffalo spermatozoa were treated with 10 micrograms/mL heparin and 2.5 mM caffeine for sperm capacitation. Oocytes were fertilized in vitro with 1 to 2, 4 to 5 or 9 to 10 million sperm/mL and the cleavage rate was recorded 42 to 44 h post insemination. The cleaved embryos were co-cultured with buffalo oviductal epithelial cells for 10 d post insemination, and the uncleaved oocytes were fixed and stained with aceto-orcein for determination of the penetration rate. The cleavage rate and the proportion of cleaved embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) whereas the proportion of degenerated oocytes and those that became arrested at the 2 to 16-cell stage were significantly lower (P < 0.05) with cumulus-intact than with cumulus-free oocytes at the 3 sperm concentrations. Increasing the sperm concentration increased the cleavage rate significantly (P < 0.05) from 1 to 2 million through 9 to 10 million sperm/mL but had no effect on the proportion of cleaved embryos that developed to morula and blastocyst stages. In conclusion, the results of the present study suggest that cumulus cells have a positive influence on fertilization, cleavage and subsequent embryonic development. Increase in sperm concentration increases cleavage rate without affecting subsequent embryonic development.     

In vitro fertilization of horse follicular oocytes matured in vitro

In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 micrograms/ml heparin for 4h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 microM calcium ionophore A 23187 for 5-10 min followed by washing. After a given pretreatment, suspensions were diluted into B2 medium to a concentration of 5 x 10(6) sperm/ml and co-incubated with oocytes for 12 h or 24-48 h. In the ionophore-treated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 11 of 45 (24%) cleaved by 24-48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures.     

In-vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa.

Pig oocytes matured in culture were inseminated with frozen-thawed ejaculated spermatozoa without preincubation in modified tissue culture medium (TCM) 199. High penetration rates (85-89%) and increased incidence of polyspermy were obtained at 25-100 x 10(6) spermatozoa/ml. Wide variation in penetration rates (16-89%) was observed in oocytes inseminated in medium containing 5mM caffeine and at 25-50 x 10(6) spermatozoa/ml obtained from 6 boars, regardless of sperm motility. At 25-50 x 10(6) spermatozoa/ml, penetration rates of oocytes were dependent upon the concentration of caffeine in the medium: there was no penetration without caffeine, but penetration was highest (89%) with 5mM caffeine. None of the oocytes was penetrated in the medium supplemented with heparin at 5-40 micrograms/ml. When heparin was included in the medium with 5mM caffeine, it inhibited the efficacy of caffeine to promote sperm penetration of oocytes.     

Production of dromedary (Camelus dromedarius) embryos by IVM and IVF and co-culture with oviductal or granulosa cells

The general objective of this work was to produce dromedary embryos from cumulus-oocyte complexes (COCs) that were matured, fertilized and co-cultured in vitro. A total of 1598 COCs were recovered from 457 ovaries; 1308 were deemed suitable for IVM and were cultured at 38.5 degrees C, 5% CO2, and >95% humidity for 36 h in TCM-199 supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/ml epidermal growth factor (EGF), 1 microg/ml FSH, and 500 microM cysteamine. Matured COCs (n = 88) were denuded, fixed, and stained to determine nuclear status; 63% (56/88) had reached metaphase II (MII) at 36 h. Overall, 1135 COCs were inseminated with ejaculated fresh semen (0.5 x 10(6)spermatozoa/ml in modified TALP-solution). Inseminated oocytes (n = 155) were examined for evidence of fertilization; 68% (106/155) were penetrated by spermatozoa, including 52% (55/106) with two pronuclei and 34% (36/106) with polyspermy. Inseminated, denuded oocytes (n = 819) were co-cultured with dromedary oviductal epithelial or granulosa cells in TCM-199 supplemented with 10% heat-treated FCS. Although the rate of first cleavage (two to eight cells) was similar for the two co-culture systems (32 versus 33%, respectively), more embryos (two-cell to blastocyst stage) were obtained from oocytes co-cultured with oviductal versus granulosa cells (61 versus 45%; P < 0.05). The proportions of fertilized oocytes developing to the early morula stage were 19% (80/417) and 12% (48/402) for oocytes co-cultured for 7 days with oviductal or granulosa cells, respectively (P > 0.05). However, development to the blastocyst stage (10% of fertilized oocytes) occurred only in oocytes co-cultured with oviductal cells. In conclusion, dromedary embryos were produced in vitro using abattoir-derived oocytes, fresh (ejaculated) semen, and oviductal cell co-culture.     

In vitro maturation and fertilization of buffalo oocytes (Bubalus bubalis ): Effects of media, hormones and sera

Media (TCM-199 and Ham's F-10); sera (fetal calf serum, FCS, and buffalo estrous serum, BES); and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol 1 ug/ml) were tested to determine the efficiency of in vitro maturation and fertilization of buffalo follicular oocytes. Immature good quality cumulus-oocyte complexes (COCs) were randomly assigned to 1 of 4 experiments. Each experiment consisted of 6 treatment groups. Oocytes cultured for 24 hours in medium (TCM-199 or Ham's F-10) containing 10% FCS or BES had a significantly higher maturation rate than those in medium alone (P < 0.05). However, the maturation rate was higher in medium supplemented with 10% FCS than with 10% BES. Addition of hormones alone or in combination with sera further improved the maturation rate, but no significant difference was observed in the maturation rate among the 3 hormone-treated groups. Immature oocytes matured in the various cultures were fertilized with frozen-thawed buffalo spermatozoa. Our findings show that hormone and/or serum supplementation of TCM-199 did not improve the fertilization rate. Supplementation of Ham's F-10 with LH alone or in combination with LH + FSH + E(2) and with FCS significantly improved the fertilization rate of oocytes while medium with FSH, E(2) or no hormones did not (P < 0.05); same media supplemented with BES resulted in lower fertilization rates both in the presence or absence of hormones. The results indicate that the culture medium has a marked effect on the fertilization rate of buffalo oocytes. Ham's F-10 + LH + FSH + E(2) supplemented with FCS was the most efficacious culture system of those studied for the in vitro maturation of buffalo oocytes.     

Effects of in vitro fertilization conditions on preimplantation development and quality of pig embryos

The present study was to investigate the effects of in vitro fertilization conditions on in vitro development and structural integrity of pig embryos. Porcine oocytes matured in vitro were co-incubated with four different spermatozoa concentrations (0.6 x 10(5), 1.2 x 10(5), 2.5 x 10(5) and 5 x 10(5) cells/ml) for 6 h, and at a spermatozoa concentration (1.2 x 10(5) cells/ml) for 2, 4 and 6 h, respectively. Spermatozoa penetration and blastocyst formation were observed at 10 and 144 h post insemination, respectively. The allocation of a blastocyst to inner cell mass (ICM) and trophectoderm (TE) cells was determined by using a differential staining method. Polyspermy frequency increased with increasing spermatozoa concentrations. The spermatozoa-oocyte co-incubation period of 2 h provided for decreased in vitro development rate than 4 and 6 h groups (P < 0.05), although no difference was detected in polyspermy frequency between spermatozoa-oocyte co-incubation periods. Interestingly, blastocysts derived from the groups with greater spermatozoa concentrations (2.5 x 10(5) and 5 x 10(5) cells/ml) had significantly fewer ICM cell nuclei as compared with those groups with lesser spermatozoa concentrations (0.6 x 10(5) and 1.2 x 10(5) cells/ml). There was no difference in the structural integrity of blastocysts among the co-incubation periods. Blastocysts derived from respective experiments were individually classified into three groups (I: <20%; II: 20-40% and III: >40%) based on the ratio of ICM to total cells. Proportion of blastocysts in Group II, with a presumptive normal range of structural integrity, was slightly decreased in the groups with greater spermatozoa concentrations (2.5 x 10(5) and 5 x 10(5) cells/ml). The results indicate that the spermatozoa concentration during in vitro fertilization may be important for developmental competence and quality of pig embryos.     

Comparisons between three different extenders for canine intrauterine insemination with frozen-thawed spermatozoa

Ejaculates from 7 dogs were obtained on the same day and were pooled. This pooled semen was separated into 3 equal fractions and processed simultaneously, the only difference being in the extender used for freezing. The extenders were laiciphos (containing laiciphos, egg yolk, distilled water and glycerol- Group 1); Tes/Tris (containing Tes/Tris, egg yolk, distilled water and glycerol- Group 2); and biociphos (containing biociphos with glycerol in it, egg yolk and distilled water- Group 3). Spermatozoa were conditioned in 0.5ml French straws and presented normal characteristics before freezing and after thawing. The sperm concentration of the pooled was 683 x 10(6) sperm/ml; sperm motility was above 95%, the percentage of live spermatozoa was above 95% and was of good quality and mobility. Characteristics of the spermatozoa after thawing were the same for spermatozoa frozen with laiciphos and Tes/Tris. Mean sperm concentration was 201.5 +/- 4.95 x 10(6) sperm/ml, sperm motility was 65%, the percentage of live spermatozoa was 80% and the quality of motility.was good. Spermtozoa frozen with biociphos had the following post-thaw characteristics: sperm concentration was 201 x 10(6) sperm/ml, sperm motility was 50%, the percentage of live spermatozoa was 78% and the quality of mobility was medium. Abnormalities were less than 15% for all spermatozoa after thawing. Intrauterine artificial inseminations were performed by laparoscopic intrauterine insemination twice at Days 3 and 5 after the estimated LH peak in 15 normally cyclic Beagle bitches (5 per group) presenting normal hormonal profiles. There were no differences between groups. The females were inseminated with 1.0 ml of spermoatozoa (concentration of 200 x 10(6) sperm/ml) diluted with 1.0 ml of extender. A 60% pregnancy rate was obtained in bitches inseminated with frozen-thawed spermatozoa extended with laiciphos or Tes/Tris and 100% in bitches inseminated with spermatozoa extended with biociphos. Females inseminated with laiciphos, Tes/Tris and biociphos had a mean litter size of 5 +/- 2.6, 3 +/- 1 and 3.4 +/- 1.3 pups, respectively. This study demonstrated that post-thaw assessment of sperm characteristics is not the best technique for evaluating sperm fertility after freezing or for assessing different semen extenders.     

Effect of in vivo oocyte aging on sperm chromatin decondensation in the golden hamster

Aged spontaneously activated hamster oocytes recovered from adult females 18 and 24 hours after ovulation were at the pronuclear stage. These oocytes and fresh controls were inseminated in vitro with capacitated hamster spermatozoa and observed with the phase-contrast microscope. The percentage of fertilization in fresh control oocytes was 98%, as compared to 36% and 18% when the oocytes were recovered 18 and 24 hours after ovulation, respectively. The mean number of sperm decondensations per egg in control oocytes was 10, and in the aged ones it was 0.69 and 0.12 when the oocytes were recovered 18 and 24 hours after ovulation, respectively. When similarly treated oocytes were studied with scanning and transmission electron microscopy, it was found that the degree of gamete membrane fusion was greater than that observed with the phase-contrast microscope, but that most of the spermatozoa failed to decondense the chromatin. We suggest that parthenogenetic oocytes at the pronuclear stage are in a similar stage of the cell cycle as in fertilized eggs, in which the cytoplasm does not have the ability to decondense the sperm chromatin.     

In vitro fertilizing characteristics of bovine spermatozoa with multiple nuclear vacuoles: A case study

A study was designed to determine the in vitro fertilizing characteristics of bovine semen with a high percentage of spermatozoa with multiple nuclear vacuoles. In Experiment 1, a total of 620 oocytes was divided into 2 groups and inseminated with spermatozoa from 1 of 2 different bulls at a concentration of 2 × 10⁵/ml. After Percoll washes, 73.5 ± 3.0% of spermatozoa from Bull A contained multiple nuclear vacuoles, while no sperm cells from Bull B contained vacuoles. After 19.5 ± 0.5 h of co-incubation of oocytes with spermatozoa, loosely attached sperm cells were removed by washing, and the oocytes were fixed between 2 poly-l-lysine coated glass slides. Mean (±SD) percentage of fertilization was significantly lower (P < 0.05) in Bull A (19.7 ± 7.0%) than in Bull B (67.6 ± 4.5%). In one-third of the oocytes fertilized by spermatozoa from Bull A, sperm head decondensation was incomplete and normal male pronucleus formation did not occur. All oocytes fertilized by Bull B had normally decondensed sperm heads. Although fewer (P < 0.05) spermatozoa from Bull A were bound to the zona pellucida than from Bull B, the percentage of vacuolated sperm cells bound to the zona pellucida (73.3 ± 7.8%) did not differ from that in the inseminate. The mean number of sperm cells binding to fertilized oocytes was higher than to unfertilized oocytes for both bulls (P < 0.05). In Experiment 2, 748 salt-stored oocytes (zonae) were inseminated with semen from the same 2 bulls to determine the ability of spermatozoa to penetrate the zona pellucida. The percentage of zonae penetrated by spermatozoa from Bull A (69.9 ± 3.5%; a mean of 2.4 ± 2.3 spermatozoa) was lower (P < 0.05) than from Bull B (96.5 ± 14.7%; a mean of 11.3 ± 9.9). Although the proportion of vacuolated sperm cells from Bull A that bound to the zona pellucida did not differ from that in the inseminate, the proportion of those penetrating the zona pellucida (52.7%) was lower (P < 0.05). In summary, vacuolated sperm cells apparently gained access to the oocyte and bound to the zona pellucida, but they penetrated the zona pellucida at a lower rate and apparently did not form normal male pronuclei.