Adjustments in IVF system for individual boars: value of additives and time of sperm-oocyte co-incubation

In vitro fertilization (IVF) in pigs is still considered sub-optimal, due to the variable occurrence of polyspermy, variability mainly related to sperm differences. The present study was conducted in an attempt to increase the efficiency of the in vitro production of porcine embryos by optimizing the in vitro fertilization (IVF) protocol for individual males, with regard to the composition of the fertilization medium (experiments 1 and 2) and the length of gamete co-incubation time (experiment 3). A total of 5,943 COC's were in vitro matured (IVM) and inseminated with frozen-thawed spermatozoa from 2 boars (A and B). Experiment 1 determined the effect of additives caffeine (2mM), hyaluronic acid (HA; [0.5mg/mL]) and adenosine (10 microM), alone or in combination, to the IVF-medium during sperm-oocyte co-incubation. Experiment 2 tested the addition of various HA (0, 0.5, 1.0 and 1.5mg/ml) and adenosine (0, 10, 20 and 40 microM) concentrations in the fertilization medium; while experiment 3 investigated the effect of two periods of sperm-oocyte co-incubation (10 min or 6h). In the case of 10 min sperm-oocyte co-incubation, oocytes with attaching spermatozoa were further cultured in IVF-medium containing no spermatozoa until the 6h of insemination was completed. Presumptive zygotes were cultured in embryo culture medium for 12-15 h to assess fertilization parameters. In experiment 1, only caffeine significantly influenced the outcome of fertilization, albeit being a clearly boar-dependent effect. In experiment 2, similar boar differences were seen for HA supplementation while presence of exogenous adenosine did not influence fertilization parameters in either boar. The results of experiment 3 demonstrated that a short co-incubation time significantly (P<0.001) increased penetration rate and mean number of spermatozoa per oocyte (74.9+/-3.9% versus 62.7+/-3.9% and 1.5+/-3.2 versus 1.3+/-3.5 for 10 min or 6h, respectively), but reduced mono-spermy (P<0.001, 57.9+/-2.5% versus 70.0+/-2.8%) when boar A was used. However, such effects were not seen with boar B, in which sperm-oocyte co-incubation time did not affect the efficiency of fertilization. In view of the present results, a preliminary screening for each individual male is required to select optimal conditions for IVF.     

Effects of modification of in vitro fertilization techniques on the sex ratio of the resultant bovine embryos

The duration of sperm-oocyte co-incubation has been observed to affect the sex ratio of in vitro produced bovine embryos. The purpose of this study was to investigate some factors that may be responsible for the skewed sex ratio. The factors studied were selected combinations of the duration of co-incubation, the presence or absence of cumulus cells, and the level of hyaluronic acid (HA) in the culture medium. Experiment 1 examined the effect of selected combinations of different factors during the fertilization phase of in vitro oocyte culture. The factors were the nature of the sperm or its treatment, the duration of the sperm-oocyte co-incubation, and the level of hyaluronic acid in the culture medium. In experiment 2, the capacitation of frozen-thawed-Percoll-washed sperm (control), pre-incubated, and non-binding sperm was evaluated by the zona pellucida (ZP) binding assay and the hypo-osmotic swelling test (HOST). The purpose of experiment 3 was to determine the oocyte cleavage rate and sex ratio of the embryos (>5 cells) produced as a consequence of the 10 treatments used in experiment 1. In treatments 1-3 (experiments 1 and 3) COC were co-cultured with sperm for 1, 5 or 18 h. Polyspermic fertilization rose as the co-incubation period increased (1 h 6.5%, 5 h 15.9%, 18 h 41.8%; P<0.05), and the highest rate of normal fertilization was observed for 5h culture (73.4%; P<0.05). The sex ratio was significantly (P<0.05) skewed from the expected 50:50 towards males following 1 h (64.4%) and 5 h (67.3%) co-incubation, but was not affected by 18 h incubation (52.3%). In treatment 4, sperm was pre-incubated for 1h and cultured with COC for 5 h. Relative to control sperm, pre-incubation of sperm increased ZP binding (116 versus 180 per ZP; P<0.05) and decreased the proportion of HOST positive sperm (65.8-48.6%; P<0.05; experiment 2). Pre-incubation did not affect the rates of polyspermy, normal fertilization or the sex ratio of the embryos (experiments 1 and 3). The oocytes used in treatments 5-10 of experiments 1 and 3 were denuded prior to fertilization. Co-incubation of denuded oocytes for 1h (treatment 5) or 5h (treatment 6) resulted in levels of polyspermic fertilization similar to that for treatment 2 with significantly lower levels of normal fertilization (41.7% and 52.6%, respectively; P<0.05), and the 1h co-incubation significantly skewed (P<0.05) the proportion of male embryos to 70.0%. Denuded oocytes were fertilized for 5h with sperm unable to bind to cumulus cells (NB sperm) in treatment 7 or those that bound to cumulus cells (B) in treatment 8. These two treatments had similar rates of polyspermic, normal and non-fertilization. However, the B sperm caused the sex ratio of the embryos to be significantly skewed to males (63.9%; P<0.05). Fertilization of denuded oocytes in medium containing hyaluronic acid (0.1 mg/ml, treatment 9; 1.0 mg/ml treatment 10) significantly (P<0.05) reduced the incidence of polyspermic fertilization relative to treatments 2 and 6, and normal fertilization relative to treatment 2, but did not affect the sex ratio of the embryos. It was concluded that exposure of sperm to cumulus cells, either before fertilization of denuded oocytes or during the process of fertilization of complete COC, increased the proportion of male embryos produced by in vitro culture. It was hypothesized that this may be due to the capacitation state of the sperm, the cumulus-sperm interaction, and/or the ability of the sperm to bind to cumulus cells or oocytes.     

Development of embryos after in vitro fertilization of bovine oocytes with sperm from either yaks (Bos grunniens) or cattle (Bos taurus)

The objectives of this study were to investigate differences in fertilization and development of embryos after in vitro fertilization of Bos taurus (cow) oocytes with sperm from either yaks (Bos grunniens) or Holstein bulls. Frozen-thawed spermatozoa (Holstein n=5 sires; yak n=5 sires) were evaluated for motility (forward progression) and acrosomal status immediately post-thaw and then 1, 2, 3, and 8h later. In vitro-matured cow oocytes (n=1652) were inseminated with either Holstein bull or yak spermatozoa and after an 18-h co-incubation period, a proportion of the oocytes were fixed and examined for sperm penetration, polyspermy, and male pronuclear formation. The remaining oocytes were cultured in vitro and evaluated for cleavage and blastocyst production rates. Overall, there were species differences (P<0.05) and an effect of time (P<0.01) in sperm motility and acrosome integrity. An effect (P<0.01) of a species-by-time interaction was detected for motility, but not for acrosome integrity. The percentage of oocytes penetrated and the formation of two pronuclei when cow oocytes were inseminated with yak spermatozoa (97.4% and 81.6%, respectively) were greater (P<0.01) than that achieved with Holstein bull spermatozoa (77.8% and 65.9%, respectively), but the incidence of polyspermy (>2 pronuclei) was similar (P>0.05; 10.8% vs. 15.8%). The yak male symbolxcow combination gave a higher cleavage rate than the Holstein male symbolxcow combination (P<0.05; 76.3% vs. 63.3%), but there was no difference in the blastocyst rate (17.9% vs. 14.5%). It is concluded that yak spermatozoa could successfully fertilize cattle oocytes and their hybrid embryos had normal competence to develop to blastocysts.     

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.     

A detailed analysis of early events during in-vitro fertilization of bovine follicular oocytes

Bovine follicular oocytes collected at slaughter were matured and fertilized in vitro with in-vitro capacitated spermatozoa. Analysis of 621 penetrated ova fixed at various times after in-vitro insemination led to definition of 6 stages of early development. A time sequence for sperm penetration, sperm head decondensation, male pronucleus formation, the activation of second meiotic division, female chromosome decondensation and pronucleus development was established. First sperm penetration into the ooplasm was recorded 6 h after insemination; 1-2 h was required for the sperm head to decondense and another 4-6 h to develop into the opposing pronucleus stage. Synkaryosis and first cleavage occurred 28 h after fertilization. Examination of the early stages revealed four types of abnormalities, i.e. polyspermy, polygyny, asynchrony between male and female pronucleus development, and preactivation of cytokinesis.     

Quercetin supplementation to the thawing and incubation media of boar sperm improves post-thaw sperm characteristics and the in vitro production of pig embryos

Elevated levels of reactive oxygen species can cause oxidative stress, which could lead to membrane damage, decreased fertility, and spermatozoan morphological deformities. Antioxidants can be supplemented to reduce the impacts of oxidative stress. The objective of this study was to determine the effects of supplementing quercetin (0.25, 0.50, 0.75 mM) during the thawing and incubation of frozen-thawed boar semen on spermatozoan characteristics, IVF kinetics (n = 400) and subsequent embryonic development (n = 1340). Spermatozoa were evaluated for motility, viability, and membrane lipid peroxidation levels at 0, 2, 4, 6, 8, and 10 h after thawing. Embryos were evaluated for IVF kinetics 12 h after IVF (penetration, polyspermy, male pronucleus formation, IVF efficiency) and cleavage and blastocyst formation at 48 h and 144 h after IVF, respectively. Spermatozoa supplemented with 0.25 mM quercetin had significantly higher (P < 0.05) motility (51.67±8.50 %) and percent of viable cells (61.21 ± 2.44 %) compared to all other treatments at 10 h after thawing, in addition to having significantly (P < 0.05) lower levels of hydroperoxide (3.38 ± 0.88 μM/10⁷cells). There were no differences in penetration rates and male pronucleus formation between treatment groups. Supplementation of quercetin significantly decreased (P < 0.05) polyspermy and significantly increased (P < 0.05) the percentage of embryos reaching blastocyst stage of development by 144 h after IVF compared to no supplementation. Results indicated that supplementing frozen-thawed boar semen with 0.25 mM quercetin improves sperm characteristics up to 10 h after thawing and decreases polyspermy while improving early embryonic development in pigs.     

Influence of the duration of in vitro maturation and gamete co-incubation on the efficiency of in vitro embryo development in Italian Mediterranean buffalo (Bubalus bubalis)

The aim of this study was to investigate the effect of the duration of oocyte in vitro maturation (IVM) and gamete co-incubation on the in vitro embryo (IVEP) production efficiency in River buffalo. In Experiment 1, abattoir-derived cumulus oocyte complexes were fixed at 0, 3, 6, 9, 12, 15, 18, 21 and 24 h after the start of in vitro maturation to study the kinetics of nuclear maturation. In Experiment 2, cumulus oocyte complexes were fertilized in vitro following in vitro maturation for 18, 21, 24, 27 or 30 h. After 20 h of gamete co-incubation, presumptive zygotes were denuded and cultured in vitro in synthetic oviduct fluid (SOF) medium. In Experiment 3, following in vitro maturation and fertilization, presumptive zygotes were removed from fertilization drops at 8, 12, 16 and 20 h post-insemination (pi) and placed in culture as described above. Representative samples of oocytes were fixed at 4, 8, 12, 16 and 20 h to evaluate the sperm penetration rate and the incidence of polyspermy at different co-incubation times. The main conclusions of the study are that: (1) the majority of buffalo oocytes accomplish nuclear maturation between 21 and 24 h after the start of in vitro maturation; (2) both cleavage and blastocyst rates linearly decrease with increasing duration of in vitro maturation (from 18 to 30 h); (3) sperm-oocyte incubation for at least 16 h is required for maximum blastocyst yields.     

Effects of the porcine oviduct-specific glycoprotein on fertilization, polyspermy, and embryonic development in vitro

This study evaluated the effects of porcine oviduct-specific glycoprotein (pOSP) on in vitro fertilization (IVF), polyspermy, and development to blastocyst. Experiment 1 evaluated the effects of various concentrations (0-100 microgram/ml) of purified pOSP on fertilization parameters, including penetration, polyspermy, male pronuclear formation, and mean number of sperm penetrated per oocyte. Experiment 2 examined the ability of an anti-pOSP immunoglobulin G to inhibit the observed effects of pOSP on fertilization parameters. Experiments 3 and 4 examined various concentrations of pOSP (0-100 microgram/ml) on zona pellucida solubility and sperm binding, respectively. Lastly, experiment 5 assessed the effects of various concentrations of pOSP (0-100 microgram/ml) on the in vitro embryo cleavage rate and development to blastocyst. Pig oocytes matured and fertilized in vitro were used for all experiments. An effect of treatment (P < 0.05) was detected for pOSP on penetration, polyspermy, and mean number of sperm per oocyte. Concentrations for pOSP of 0-50 microgram/ml had no effect on sperm penetration rates; however, compared with the control, 100 microgram/ml significantly decreased the penetration rate (74% vs. 41%). Addition of 10-100 microgram/ml significantly reduced the polyspermy rate compared with the control (61% vs. 24-29%). The decrease in polyspermy achieved by addition of pOSP during preincubation and IVF was blocked with a specific antibody to pOSP. No effect of treatment was observed on zona digestion time relative to the control; however, the number of sperm bound to the zona pellucida was significantly decreased by treatment (P < 0.05). Compared with the control, all concentrations of pOSP examined reduced the number of sperm bound per oocyte (45 vs. 19-34). A treatment effect (P < 0.05) was observed for pOSP on embryo development to blastocyst but not on cleavage rates. Addition of pOSP during preincubation and fertilization significantly increased postcleavage development to blastocyst, but a synergistic stimulation on development was not detected when pOSP was included during in vitro culture. These results indicate that exposure to pOSP before and during fertilization reduces the incidence of polyspermy in pig oocytes, reduces the number of bound sperm, and increases postcleavage development to blastocyst.     

An environmentally relevant organochlorine mixture impairs sperm function and embryo development in the porcine model

We evaluated the effects of an environmentally relevant mixture of more than 15 organochlorines on the development of pig oocytes and sperm during in vitro fertilization (IVF). Oocytes were cocultured with sperm in IVF medium containing increasing concentrations of an organochlorine mixture, similar to that found in women of highly exposed populations. Exposure to the organochlorine mixture diminished oocyte penetration rates and polyspermy in a linear manner. The mixture did not affect rates of cleavage nor development to multicell embryos. However, rates of development to the blastocyst stage were lower at the highest concentration at which oocyte penetration was observed. The same experiment was performed using oocytes that were preexposed during in vitro maturation. This greater exposure to the mixture also reduced penetration in a dose-response manner and affected polyspermy. Frozen-thawed pig sperm were also cultured in IVF medium containing the same organochlorine concentrations. Sperm motility parameters were immediately reduced in a dose-dependent manner by the organochlorines, followed by diminished viability 2 h later. From these results, it appears that reduced sperm quality would account for decreases in fertilization, polyspermy, and blastocyst formation. These results suggest that exposing porcine oocytes and sperm to an environmentally pertinent organochlorine mixture in vitro disrupts the oocyte block to polyspermy, sperm fertility, and further embryonic development, and supports recent concerns that such pollutants harm reproductive health in humans and other species.     

Optimization of in vitro bovine embryo production: effect of duration of maturation,length of gamete co-incubation,sperm concentration and sire

The aim of these experiments was to investigate the effect of duration of IVM, duration of gamete co-incubation, and of sperm dose on the development of bovine embryos in vitro. In addition, the speed of sperm penetration of six bulls of known differing in vivo and in vitro fertility was examined. In Experiment 1, following IVM for 16, 20, 24, 28 or 32 h, cumulus oocyte complexes (COCs) were inseminated with 1 x 10(6) spermatozoa/ml. After 24 h co-incubation, presumptive zygotes were denuded and placed in droplets of synthetic oviduct fluid (SOF). In Experiment 2, following IVM and IVF, presumptive zygotes were removed from fertilization wells at 1, 5, 10, 15 or 20 h post insemination and placed in culture as described above. In Experiment 3, following IVM, COCs were inseminated with sperm doses ranging from 0.01 x 10(6) to 1 x 10(6) spermatozoa/ml. Following co-incubation for 24 h, presumptive zygotes were placed in culture as described above. In Experiment 4, following IVM, oocytes were inseminated with sperm from six bulls of known differing field fertility. To assess the rate of sperm penetration, oocytes were subsequently fixed every 3 h (up to 18 h) following IVF. Based on the results of Experiment 4, in Experiment 5, following IVM for 12, 18 or 24 h, COCs were inseminated with sperm from two sires with markedly different penetration speeds. After 24 h co-incubation, presumptive zygotes were denuded and placed in culture. The main findings from this study are that (1) the optimal duration of maturation of bovine oocytes in vitro to maximize blastocyst yield is 24 h, (2) sperm-oocyte co-incubation for 10 h is sufficient to ensure maximal blastocyst yields, (3) sperm concentrations of 0.25 x 10(6) and 0.5 x 10(6) spermatozoa/ml yielded significantly more blastocysts than any other concentration within the range of 0.01 x 10(6) 1 x 10(6) spermatozoa/ml, (4) there are marked differences in the kinetics of sperm penetration between sires and this may be a useful predictor of field fertility, and (5) the inferior development associated with slower penetration rates may in part be overcome by carrying out IVF at a time when the actual penetration is most likely to coincide with the completion of maturation.     

Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro

The consequences of interactions between porcine sperm, eggs, and oviduct cells before and during fertilization in vitro (IVF) has been examined with particular reference to the block to polyspermy. The pattern of polypeptides secreted by porcine oviduct epithelial cells has been determined and its effects on sperm both during pre-fertilization co-culture and during fertilization have been examined. In standard IVF procedures with no oviduct cell involvement, high rates of penetration (91%) were accompanied by equally high rates of multiple sperm penetration (91% of penetrated eggs). Fertilization on oviduct cell monolayers or a combination of 1 h co-culture of sperm and oviduct cells before the addition of in vitro matured oocytes did not reduce polyspermy. However, a sperm-oviduct cell co-culture period of 2.5 h followed by IVF on oviduct cells selectively reduced the rate of polyspermy by 40% and 50% in two separate series of trials (United Kingdom and Japan, respectively): Overall fertilization rates after this treatment were high (95% or 84%, respectively). A 3.5 h period of pre-fertilization co-culture further reduced polyspermy to only 14% of penetrated eggs, but this treatment was accompanied by a sharp drop in the fertilization rate from an overall mean of 88% for all other groups to 19% after 3.5 h co-culture.     

Effect of adding reduced glutathione during insemination on the development of porcine embryos in vitro

This study evaluated the effect of adding reduced glutathione (GSH) during sperm washing and insemination on the subsequent fertilization dynamics and development of IVM porcine oocytes. Follicular oocytes were matured in vitro in NCSU 23 medium with porcine follicular fluid, cysteine and hormone supplements for 22 h. They were then matured in the same medium but without hormones for another 22 h. Matured oocytes were stripped of cumulus cells and co-incubated with frozen-thawed spermatozoa for 5 h. Putative embryos were cultured in NCSU 23 with BSA for either 7 h to examine fertilization parameters or 6 d to evaluate cleavage (2 d) and blastocyst rates. In Experiment 1, GSH was added to the insemination medium at 0, 0.125, 0.25 or 0.5 mM. The presence of GSH during insemination did not affect (P>0.05) rates of penetration, polyspermy, male pronuclear formation or cleavage, but did increase (P<0.05) blastocyst formation rates when added at concentrations of 0.125 (36%) and 0.25 mM (34%) compared with that of the control (0 mM; 19%). However, the numbers of inner cell mass and trophectoderm cells of blastocysts were unaffected by GSH treatment (P>0.05). The presence of GSH during insemination was found not to significantly increase intracellular glutathione concentrations of oocytes (P>0.05). In Experiment 2, addition of GSH (0.25 mM) during sperm washing did not affect cleavage or blastocyst formation rates or cell numbers (P>0.05). In conclusion, the presence of GSH during insemination improves the developmental competence of IVM pig oocytes in a dose-dependent manner.     

Effect of superoxide dismutase(SOD) on pronucleus formation of porcine oocytes fertilized in vitro

This study was undertaken to evaluate the effects of superoxide dismutase (SOD) on pronucleus formation in porcine oocytes fertilized in vitro by frozen-thawed spermatozoa. No differences were found in penetration rates when SOD was added to maturation or fertilization medium at any level tested in first and second experiments. Pronucleus formation rates were higher (P < 0.05) when SOD at 10 and 100 units was added to the maturation medium (46 and 53%, respectively) compared with the controls (26%). On the other hand, when the fertilization medium was supplemented with SOD at different concentrations (1, 10 and 100 units/ml), pronucleus formation rates (55, 52 and 50%) were significantly higher (P < 0.05) than in the control group. In third experiment, the oocytes were cultured in medium with (1 unit/ml) or without SOD for 8, 16, 24 and 32 h after insemination. The penetration rates had a tendency to increase as time of sperm-oocyte culture was prolonged. No significant differences, however, were observed in penetration rates between groups with and without SOD. On the other hand, the pronucleus formation rates were higher in medium with than without SOD at 8 (7 vs 0%), 16 (14 vs 3%), 24 (48 vs 16%; P < 0.01) and 32 h (49 vs 22%; P < 0.05). These findings demonstrate the advantage of culture with SOD on pronucleus formation in porcine oocytes penetrated by spermatozoa. However, SOD does not affect penetration rates and polyspermy.     

Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration

This study was designed to examine the reduced incidence of normal fertilization in vitrified ovine oocytes. After in vitro maturation for 24 h, the oocytes were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution (VS) without being plunged into liquid nitrogen (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In experiment 1, the treated and control oocytes were matured for another 2 h, and the oocytes were then in vitro fertilized for 12 h to examine sperm penetration. The percentage of monospermy in toxicity group (29.3%) and vitrification group (28.2%) dramatically decreased compared to the control group (45.0%) (P<0.05). To find the mechanism that the VS decreased the monospermy, some treated and control oocytes were used to test the distribution of CG and the resistance of zona pellucida (ZP) to 0.1% pronase E immediately (IVM 24 h), after another 2 h of maturation (IVM 26 h), and after 12 h of in vitro fertilization (IVF 12 h) respectively. Others were used to examine female pronucleus formation after 12 h of culture in fertilization medium with the absence of sperm. The results showed that the percentage of CG completely release in the oocytes (IVM 24 and 26 h) of toxicity group (41.2% and 39.9%) and vitrification group (41.7% and 51.7%) was significantly higher than that of control group (7.1% and 18.4%) (P<0.05). The ZP digestion duration in the oocytes (IVM 26 h) of the toxicity group (435.6 s) and vitrification group (422.3 s) was longer than that of control group (381.6 s) (P<0.05). The percentage of female pronucleus formation in toxicity group (58.7%) and vitrification group (63.9%) was higher than that (8.2%) of control group (P<0.05). The data above demonstrated that the VS containing DMSO and EG could parthenogenetically activate in vitro matured ovine oocytes, resulting in ZP hardening and decreased sperm penetration.     

Effects of ovarian cortex cell co-culture during in vitro maturation on porcine oocytes maturation, fertilization and embryo development

The objective of the experiments was to evaluate the effects of porcine ovarian cortex cells (pOCCs) during in vitro maturation (IVM) of porcine oocytes on IVM of porcine oocytes, in vitro fertilization (IVF) parameters and subsequent embryo development. The pOCCs was cultured in the 500 microl TCM199 without hormone until the confluence, and then cultured in 500 microl TCM199 supplemented with hormone for 12 h before the oocytes added. Porcine oocytes were co-cultured with the pOCCs monolayers in the co-culture system for 44 h, following fertilized in the mTBM for 6 h. Finally, the presumptive zygotes were cultured for 144 h in the NCSU-23 supplemented with 0.4% BSA. The results showed that matured M II oocytes in the co-culture group were higher than that in the control group (P<0.05). Although penetration did not differ between the co-culture and control groups (P=0.481), polyspermy declined in the co-culture group (P<0.05), whereas male pronucleus (MPN) formation was improved in the co-culture group compared with the control group (P<0.05). More blastocysts developed in the co-culture group than that in the control group (P<0.05); however, the cleavage rates and the mean number cells per blastocyst showed no significant difference between the treated group and the control group (P=0.560 and 0.873, respectively). In conclusion, the presence of the pOCCs monolayers during IVM enhanced the maturation quality of the porcine oocytes, reduced the polyspermy, increased the percentages of MPN formation and blastocyst, but the blastocyst quality was not improved.     

Influence of oviductal cells and conditioned medium on porcine gametes

The aim of this study was to optimise porcine in vitro fertilisation (IVF) with cryopreserved semen with the exploitation of the oviduct secretion. The oocytes were cultured in NCSU37 supplemented with db-cAMP (1 mM), porcine follicular fluid (pFF; 10%), cysteine (0.1 mg/ml) and beta-mercaptoethanol (25 microM) for 44 h (the first 20 h with 10 IU/ml hCG and PMSG). The oviductal epithelial cells (OEC) were cultured in TCM-199 medium (with 10% FCS, 0.2 mM pyruvate and 50 microg/ml gentamicin) for 48 h. To determine the effects of OEC and conditioned medium, oocytes were separated into five groups for the last 3 h of maturation and placed in: fresh maturation medium (controls), OEC-cNCSU with OEC in the maturation medium for 24 h; OEC-fNUSU with fresh OEC in maturation medium; cTCM with TCM-199 conditioned with OEC for 48 h; or fTCM with fresh TCM-199. Results indicate that OEC-cNCSU and OEC-fNCSU increase the number of oocytes reaching the two pronucleus (2PN) stage (p < 0.01) and decrease the polyspermy rate (p < 0.01) compared with controls. The rates are significantly lower than controls when cTCM and fTCM were used (p < 0.01). As regards blastocyst rates, an increase was observed in the OEC-cNCSU and cTCM groups (p < 0.05). For the second experiment, spermatozoa were incubated with OEC in IVF medium (mTBM medium supplemented with 0.1% BSA) without caffeine for 4 h prior to IVF. Results indicate that sperm treatment with OEC increases the 2PN rate (p < 0.01) compared with controls and reduces the polyspermy rate (p < 0.01). In conclusion, our study shows that co-incubation of OEC with both oocytes and sperm before IVF reduces polyspermy rates and improves embryo development.     

Inhibition of boar sperm hyaluronidase activity by tannic acid reduces polyspermy during in vitro fertilization of porcine oocytes

The present study was conducted to examine the effects of three polyphenols (tannic acid, apigenin and quercetin) on hyaluronidase activity and in vitro fertilization (IVF) parameters. Among them, tannic acid showed by far the strongest potency for blocking hyaluronidase activity extracted from preincubated boar sperm, causing a dose-dependent inhibition over the range of 2-10 microg/ml. When cumulus-intact and cumulus-free oocytes were inseminated in IVF medium containing tannic acid, the penetration and the polyspermy rates were significantly decreased in the presence of 10 microg/ml tannic acid compared with those in the absence of tannic acid, and the addition of 5 microg/ml tannic acid significantly reduced the polyspermy rate (p < 0.05) compared with that of the control while maintaining the high penetration rate. However, apigenin and quercetin had no effect on the rate of polyspermy. Interestingly, the incidence of polyspermy was significantly reduced in oocytes inseminated with sperm pretreated with 5 microg/ml tannic acid (p < 0.05), although the pretreatment of oocytes had no effect against the polyspermy after insemination with untreated sperm. Treatment with tannic acid caused neither a protective proteolytic modification of the zona pellucida matrix before fertilization, nor a reduction of the proteolytic activity of acrosomal contents or the number of zona-bound spermatozoa. These data suggest that an appropriate concentration of tannic acid prevents polyspermy through the inhibition of sperm hyaluronidase activity during IVF of porcine oocytes.     

Cumulus contributions during bovine fertilization in vitro

A mandatory step in performing micromanipulation techniques, studying sperm-oocyte interactions and evaluating morphological aspects of oocyte quality is the removal of cumulus cells from oocytes or zygotes at various stages. In cattle, cumulus removal shortly before fertilization in vitro strongly decreases sperm penetration rates. This study was conducted to evaluate the function of the cumulus oophorus during bovine fertilization in vitro. The importance of cumulus secretions during IVF was investigated by inseminating cumulus-denuded oocytes (CDOs) in fertilization medium supplemented with individual cumulus secretions, such as progesterone or hyaluronic acid. None of these substances increased the fertilization rate of CDOs. However, fertilizing CDOs in cumulus-conditioned medium or on a cumulus monolayer partially restored the reduction in fertilization rate (P<0.05). The fertilization rate of CDOs inseminated on a cumulus monolayer further increased when physical contact between the gametes and the monolayer was prevented by fertilizing them inside a culture plate insert placed on the monolayer (P<0.05). Finally, the importance of reactive oxygen species (ROS) generation and O(2) concentration during IVF was studied. Luminol-dependent chemiluminescence revealed a higher ROS load in conditioned medium of cumulus-enclosed oocytes (CEOs) than in that of CDOs after sperm-oocyte co-incubation (P<0.05). Furthermore, lowering the external O(2) concentration from 20 to 5% decreased the fertilization rate of both CEOs and CDOs, but had a higher impact on CEOs (P<0.05). In conclusion, this study provides evidence that the cumulus oophorus benefits the fertilizing ability of penetrating spermatozoa by creating a complex microenvironment of both cumulus secretions and metabolic products around the oocyte. Gap junctional communication between the oocyte and corona cells as well as sperm trapping by the cumulus oophorus seem to be essential factors in supporting fertilization.     

In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium,duration of maturation,and sperm calcium ionophore treatment,and comparison with rates of fertilization in vivo after oviductal transfer

Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.     

Oviduct-specific glycoprotein modulates sperm-zona binding and improves efficiency of porcine fertilization in vitro

Oviduct-specific glycoprotein (OGP) displays estrus-associated regional and temporal differences in expression and localizes to the zona pellucida, perivitelline space, and plasma membrane of oviductal oocytes and embryos, suggesting that it may have a role in regulation of fertilization and/or early embryonic development. The aims of this study were to evaluate the effect of exogenous OGP on in vitro fertilization (IVF) and embryo development in the pig using a defined serum-free culture system. In vitro-matured porcine oocytes were incubated with homologous OGP (0, 1, 10, 20, and 40 microg/ml) for 3 h and then washed prior to IVF. Exposure of oocytes to 10 or 20 microg/ml porcine OGP (pOGP) significantly reduced the incidence of polyspermy compared with the control (P < 0.01) while maintaining high penetration rates. When oocytes, spermatozoa, or both were preincubated with 10 microg/ml pOGP prior to IVF, the incidence of polyspermy was similarly reduced (P < 0.01) by all three treatments without affecting penetration rates. The ability of spermatozoa to undergo calcium ionophore-induced acrosome reaction was similar with or without exposure to pOGP. However, significantly fewer spermatozoa (P < 0.01) bound to the zona pellucida when oocytes were preincubated with pOGP. To evaluate the effect of pOGP on embryo development, embryos were cultured in pOGP-supplemented medium for 48 h or 144 h. Both transient and continuous exposure to pOGP significantly enhanced cleavage and blastocyst formation rate compared with the control (P < 0.01). These data demonstrate that exposure of either in vitro-matured oocytes or spermatozoa to pOGP decreased polyspermy and spermatozoa binding while maintaining high penetration rates of pig oocytes fertilized in vitro. Furthermore, pOGP exerted an embryotrophic effect independent of effects demonstrated on spermatozoa and oocytes at fertilization.