Fertilization Of Porcine Oocytes Research Articles

In vitro fertilization of porcine oocytes is affected by spermatic coincubation time

Abstract: The aim was to study the effects of different gamete coincubation times on porcine in vitro fertilization (IVF), and to verify whether efficiency could be improved by reducing oocyte exposure time to spermatozoa during IVF. In groups of 50, a total of 508 immature cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium. The COCs were cultured for 44 hours and then inseminated with in natura semen (2,000 spermatozoa/oocyte). The sperm and oocytes were coincubated according to the following treatments (T): T1 = oocytes exposed to spermatozoa for one hour (173 oocytes), T2 = oocytes exposed to spermatozoa for two hours (170 oocytes), and T3 = oocytes exposed to spermatozoa for three hours (165 oocytes). After these coincubation periods, the oocytes were washed in fertilization medium (TALP medium) to remove spermatozoa not bound to the zona pellucida and cultured in another similar medium (containing no sperm). Eighteen to twenty hours after fertilization, the putative zygotes were stained in Hoechst-33342 to evaluate the IVF results. The penetration rate was higher (P<0.05) after two hours of coincubation time than it was for one or three hours. Furthermore, 68.60% of the ova coincubated with the spermatozoa for two hours were monospermic. The oocytes exposed to spermatozoa for one hour (T1) presented a higher (P<0.01) rate of polyspermy than those in T2 and T3. Fertilization performance (%) did not differ (P>0.05) between oocytes exposed to spermatozoa for one (T1) and three hours (T3). However, optimum (P=0.048) results were obtained after two hours of coincubation, when the rate of fertilization performance was 50.16±8.52%. The number of penetrated sperm per oocyte, as well as male pronucleus formation, did not differ (P>0.05) between the treatments evaluated. Under these assay conditions, especially in relation to the sperm concentration used, gamete coincubation for a period of two hours appears to be optimal for monospermy and fertilization performance. Thus, it is the optimal time period for obtaining a large number of pig embryos capable of normal development.

Fertilization of porcine oocytes following intracytoplasmic injection of flame-dried boar spermatozoa

Fertilization of porcine oocytes following intracytoplasmic injection of flame-dried boar spermatozoa

Effect of acrosome reaction progress in frozen-thawed boar spermatozoa on the efficiency of in vitro oocyte fertilization

A good functional status of cryopreserved boar spermatozoa is very important for successful fertilization of porcine oocytes and in vitro embryo production. The purpose of the study was to evaluate the changes in functional status of boar spermatozoa separated from frozen-thawed semen and capacitated in vitro by caffeine. The effect of acrosome reaction development in spermatozoa on the efficiency of oocyte fertilization has been studied in boars A, B and C. Motile spermatozoa were separated by Percoll gradient, untreated (control) or treated with both 1mM and 2mM caffeine, and capacitated or co-cultured with matured oocytes. The motility, viability, chromatin and acrosome integrity, and fertilizing ability of spermatozoa were assessed. The separation significantly increased (P < 0.05) the percentage of viable spermatozoa in all tested boars and percentages of motile and acrosome intact spermatozoa in boars B and C. The capacitation significantly decreased (P < 0.05) the percentages of viable and motile spermatozoa, but after capacitation, the motility and viability were significantly higher (P < 0.05) for the caffeine-treated spermatozoa than for the untreated controls. A fall in the proportion of acrosome-intact spermatozoa was different for each caffeine concentration and each boar, but in all boars, acrosome reaction progress was faster and, similarly, monospermy and the total efficiency of fertilization were significantly higher (P < 0.01) for the spermatozoa treated with 1mM caffeine than for those treated with 2mM caffeine. It can be concluded that there is a potential relationship between the acrosome reaction progress in frozen-thawed boar spermatozoa and the efficiency of fertilization of porcine oocytes. A faster AR induced in spermatozoa by appropriate caffeine treatment resulted in a higher monospermy rate and total efficiency of fertilization. Thus, it is important to test sires before their semen is used for in vitro embryo production. The faster AR induced by 1mM caffeine was more effective in terms of monospermy and total efficiency of fertilization.

Application of a microfluidic sperm sorter to the in-vitro fertilization of porcine oocytes reduced the incidence of polyspermic penetration

The objective of this study was to use a microfluidic sperm sorter (MFSS), designed to isolate motile human spermatozoa with laminar flows (no centrifugation), for porcine IVF. Boar spermatozoa were diluted at 1 × 10 8 cells/mL with a diluent containing 20% seminal fluid and flowed with modified TCM-199 (mM199, with 5 mM caffeine) to introduce motile sperm into the exit chamber for IVF. In Experiment 1, after flowing for 5 min, sperm concentration varied significantly among specific sites within the MFSS collecting chamber (range, 0.8 ± 0.5 × 10 4 to 575.0 ± 56.3 × 10 4 cells/mL; mean ± SEM). In Experiment 2, when porcine IVM oocytes were placed at three locations in the MFSS exit chamber (where only motile spermatozoa accumulated) and subsequently cultured in caffeine-free mM199 for 8 h, sperm penetration rate was not significantly different among places (86.1 ± 10.5 to 100%), but the monospermic penetration rate was lower (P < 0.05) in oocytes 3.5 mm from the exit position (12.5 ± 4.8%) than those at 7.5 mm (53.1 ± 6.0%) or further (41.9 ± 2.8%) from the exit. In Experiment 3, the normal fertilization index (ratio of monospermic oocytes to number of oocytes examined) 8 h after insemination was higher (P < 0.05) in the MFSS-IVF system (0.375 ± 0.040) than both standard IVF and transient IVF (0.222 ± 0.028 and 0.189 ± 0.027, respectively, with co-culture for 8 h and for 5 min). Developmental competence of fertilized oocytes (blastocyst formation) was higher (P < 0.05) in the MFSS-IVF system (40.9 ± 2.3%) than in either standard or transient IVF (22.6 ± 1.4 and 33.7 ± 3.5%). In conclusion, brief co-culture of porcine oocytes with spermatozoa gradually accumulated in the MFSS chamber improved the efficiency of producing monospermic fertilized embryos and blastocysts. Furthermore, efficiencies were significantly affected by oocyte location within the chamber.

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine ( Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (−) FCs (5.2 × 10 6 cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, −FC/S, +FC/R, and −FC/R) under 5% or 20% O 2. Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O 2 using the −FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both −FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the −FC/S culture system for promoting fertilization.

002. PIG SPERM EGG INTERACTION AND FORMATION OF A ZONA PELLUCIDA BINDING COMPLEX

In order to achieve fertilization sperm cells first need to successfully interact with the zona pellucida. Before reaching the zona pellucida the sperm cell undergoes extensive remodeling both in the male and female genital tract. These changes serve to mediate optimal recognition of the zona pellucida in the oviduct (primary zona pellucida binding). Optimal sperm-zona interactions are crucial for porcine oocyte fertilization: The zona pellucida- attached sperm cell is triggered to undergo the acrosome reaction and will also become hypermotile. Together these two responses allow the sperm cell to drill through the zona pellucida (secondary zona pellucida binding) this coincides with local sperm zona drilling so that a few sperm can reach the perivitellin space. This delaying strategy allows only one sperm cell in a given time-point to bind and fuse with the oocyte (fertilization) and thus minimizes the risk of polyspermy. The polyspermy block is essentially executed by the fertilized oocyte that immediately secretes its cortical granule content into the perivitellin space. This content blocks sperm-oocyte fusion either by sticking to the oolemma or by the induction of a biochemical reaction of the zona pellucida (zona pellucida hardening). The cortical reaction thus blocks sperm-zona pellucida binding and/or sperm-zona pellucida drilling. Note that zona pellcuida interactions under pig IVF conditions relatively frequently result in polyspermy. It is not clear whether this is also the case in vivo after natural mating. More importantly we do not know how other artificial reproductive technologies affect polyspermy rates. This is especially relevant for new sperm treatments and insemination technologies in which sperm are activated by capacitation media essentially mimicking the IVF media. Therefore, better understanding of sperm activation and of the arrangement of proteins involved in zona pellucida interactions are relevant for designing strategies to further improve pig reproduction.

The beneficial effect of fructose and glucose on in vitro maturation and the fertilization of porcine oocytes.

This study was conducted to investigate the effects of supplementing fructose in the culture medium on in vitro maturation (IVM), in vitro fertilization (IVF), and metabolism of porcine oocytes. Porcine oocytes were matured in vitro in modified North Carolina State University 37 medium (NCSU-37) and then supplemented with either glucose (5.5 mM), fructose (5.5 mM), or glucose (2.75 mM) plus fructose (2.75 mM). The maturation and fertilization of oocytes, and the incorporation and oxidation of 14C-glucose, 14C-fructose, and 14C-methionine in oocytes at different stages of development were examined. The supplementation of glucose plus fructose significantly promoted (P < 0.05) oocytes germinal vesicle break down (GVBD), maturation to metaphase II (MII), penetration by spermatozoa, and male pronuclear formation compared with glucose. The incorporation and oxidation of 14C-methionine into the oocyte significantly increased (P < 0.05) with glucose plus fructose supplementation than glucose. A significantly higher (P < 0.05) rate of incorporation and oxidation was achieved with 14C-fructose compared to 14C-glucose. Glucose plus fructose supplementation improved maturation, penetration by spermatozoa, male pronuclear formation, and energy metabolism by porcine oocytes.

A modified swim-up method reduces polyspermy during in vitro fertilization of porcine oocytes

The general method of porcine in vitro fertilization (IVF), involving the co-culture of both gametes in a medium drop, is thought to be the main reason for the high incidence of polyspermy. The aim of this study was to reduce the polyspermic fertilization of porcine embryos during IVF by the modified swim-up method, based on general sperm swim-up technique. Within this design, a 70 μm pore sized cell strainer was used to separate the sperm pellet placed at the bottom of a tube from the mature oocytes placed within the upper region. The separation of gametes using this permeable barrier was to ensure that only motile sperm gained access to the oocytes. It was found that the rate of polyspermy was significantly lowered for the sperm preparations from three boar breeds in modified swim-up method when compared with that of the general microdrop method ( p < 0.05). However, the penetration rates were found to be similar in both methods for two boar breeds. The average occurrence of blastocysts with more total cell number was higher in the modified swim-up method, while no significant difference in blastocyst rates between the two IVF methods was observed. The frequency of normal diploid embryos was also significantly higher in the modified swim-up method and polyploidy was more frequently observed in microdrop method ( p < 0.05). Our results demonstrated that the modified swim-up IVF method could reduce polyspermic penetration, and consequently produce better quality and karyotypically normal embryos in porcine IVF.

Effects of epigallocatechin-3-gallate (EGCG) on in vitro maturation and fertilization of porcine oocytes

The beneficial properties of green tea and especially of its principal active polyphenol, epigallocatechin-3-gallate (EGCG), have led to an increased demand for dietary supplements with highly enriched EGCG concentrations. In order to investigate the possible reproductive-related consequence of EGCG supplementation, the effects of this catechin on in vitro maturation (IVM) and fertilization (IVF) of oocyte, using the pig as experimental model, were examined. In the first series of experiments EGCG, at concentrations ranging from 0 to 25 microg/ml, was added during in vitro maturation of pig oocytes. EGCG had no effect on nuclear maturation of pig oocytes and on fertilization traits considered after IVF at any of the doses tested. By contrast, a significant (p<0.05) decrease in the number of embryos that developed to blastocysts following parthenogenetic activation was recorded when 25 microg/ml EGCG was added to IVM medium; in addition this catechin concentration significantly (p<0.05) inhibited progesterone production by cumulus cells after 48 h of culture. When induction of sperm capacitation was performed in presence of EGCG, a significantly lower percentage of spermatozoa showing a Hsp70-capacitated pattern and a significant reduction of sperm H(2)O(2) production were evident at a concentration of 25 microg/ml EGCG (p<0.05). During gamete coincubation EGCG reduced, in a dose response manner, the number of reacted spermatozoa suspended in fertilization medium and increased the number of sperm bound to ZP. Supplementation of 10 microg/ml EGCG during IVF significantly increased the fertilization rate while higher EGCG concentrations (25 microg/ml) decreased the percentage of fertilized oocytes (p<0.05). In conclusion, our data suggest that high EGCG concentrations could affect in vitro maturation and fertilization in pig; it cannot be totally excluded that excessive EGCG concentrations could induce reproductive-related consequences also in vivo.

Effect of amino acids and dipeptides on accumulation of ammonia in the medium during in vitro maturation and fertilization of porcine oocytes.

Aim: The present study was designed to investigate the effect of amino acids and their dipeptides on the accumulation of ammonia in the medium during in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes. Methods: The IVM and IVF media were modified North Carolina State University-37 solution and modified Tyrode's albumin lactate pyruvate, respectively. Porcine oocytes were matured in IVM medium containing 75-2400µmol ammonia. Amino acids (1.0mmol) or their dipeptides (2.0mmol) related to the urea cycle were added individually to the IVM and IVF media containing 300µmol ammonia. Oocyte maturation and fertilization were assessed using acetic-orcein staining, and the accumulation of ammonia in the media was measured using the indophenol method. Results: Percentages of metaphase II (MII) were adversely affected (P<0.05) by ≥300µmol concentrations of ammonia in the IVM medium. In the presence of 300µmol ammonia in the IVM and IVF media, glutamic acid, l-alanyl-L-glutamine (AlaGln), l-glycyl-L-glutamine (GlyGln) and AlaGln+GlyGln showed the highest rate (P<0.05) of MII, monospermic fertilization, and the lowest rate (P<0.05) of ammonia accumulation in the media. Conclusion: AlaGln and GlyGln in IVM and IVF media were more stable and effective than the individual amino acids in reducing the accumulation of ammonia, and increased the rate of porcine oocyte MII and monospermic fertilization in vitro. (Reprod Med Biol 2007; 6: 165-170).

Antihyaluronidase Action of Ellagic Acid Effectively Prevents Polyspermy As a Result of Suppression of the Acrosome Reaction Induced by Sperm-Zona Interaction During In Vitro Fertilization of Porcine Oocytes

The present study was conducted to examine the effects of three tannin relatives (tannic acid, TA; gallic acid, GA; and ellagic acid, EA) on antihyaluronidase and reactive oxygen species (ROS) scavenging activity, in vitro fertilization (IVF) parameters, and the acrosome reaction (AR) induced by sperm-zona interaction. Among the three tannin relatives, TA and EA showed the strongest potency for blocking the hyaluronidase activity of boar sperm, with concentration-dependent inhibition over the range of 2-10 microg/ml. In contrast, ROSs were effectively scavenged by TA and GA, but not EA. When cumulus-free oocytes were inseminated in IVF medium containing 5 microg/ml of the tannin relatives, polyspermy was significantly reduced by TA and EA (32 and 29%, respectively) compared with oocytes treated with or without GA (51 and 69%, respectively) under conditions that maintained a high sperm penetration rate (P<0.05). Interestingly, induction of the AR by treatment of preincubated sperm with progesterone was blocked by TA and GA as a result of their higher levels of ROS scavenging activity, while EA, which possessed weak ROS scavenging activity, did not disturb induction of the AR with progesterone. However, the incidence of AR induced by sperm-zona interaction was significantly decreased by the strong antihyaluronidase actions of TA and EA compared with that in the absence of these compounds. Treatment with the compounds caused neither a protective proteolytic modification of the zona pellucida matrix before fertilization nor a reduction in acrosomal proteolytic activity or the number of zona-bound sperm. These findings suggest that the antihyaluronidase action of EA effectively prevents polyspermy by suppression of AR functionality induced by sperm-zona interaction and that hyaluronidase intervention is therefore required during porcine IVF.

Inhibition of boar sperm hyaluronidase activity by tannic acid reduces polyspermy duringin vitrofertilization 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.

Effect of Relaxin on In Vitro Fertilization of Porcine Oocytes

Porcine relaxin is a peptide hormone belonging to the insulin super family that has a variety of biological functions. The present experiment was designed to investigate the effects of relaxin on sperm function and on in vitro fertilization (IVF) of porcine oocytes. Porcine spermatozoa were washed, swum-up, and incubated for 1-4 h in mTALP medium supplemented with 0, 20 or 50 ng/ml porcine relaxin. Motility was determined by observing the type of forward movement of the spermatozoa, and acrosome status was evaluated by applying the triple staining technique. Immature oocytes were aspirated from antral follicles and matured in IVM medium (modified NCSU-37). Matured oocytes were co-cultured with spermatozoa in IVF medium (mTALP) supplemented with 0, 5, 10, 15 or 20 ng/ml relaxin. After 6 h of sperm-oocyte co-incubation, putative zygotes were cultured for 18 h in oocyte culture medium NCSU-37 and then assessed for the rates of monospermy, polyspermy, and male pronucleus formation after acetic orcein staining. Relaxin improved (P<0.05) sperm motility and increased the percentage of acrosome-reacted live spermatozoa during 1-4 h of incubation, although viability was not significantly improved. Significantly (P<0.05) the highest percentage of monospermic (31.7%) and lowest percentage of polyspermic (16.5%) fertilization was achieved from the sperm-oocyte co-culture group treated with 20 ng/ml relaxin as compared to other groups. The percentage of male pronucleus formation was significantly (P<0.05) greater in the 20 ng/ml relaxin-treated sperm-oocyte co-culture group than in the other groups. These results indicate that supplementation with relaxin is capable of improving sperm function and fertilization of porcine oocytes in vitro.

In vitro maturation and fertilization of porcine oocytes after a 48 h culture in roscovitine, an inhibitor of p34 cdc2/cyclin B kinase

Maintaining oocytes at the germinal vesicle (GV) stage in vitro may permit enhanced acquisition of the developmental competence. The objective of the current study was to evaluate the nuclear and cytoplasmic maturation in vitro of porcine oocytes after pretreatment with S-roscovitine (ROS). Cumulus oocyte complexes (COC) were treated with 50 μM ROS for 48 h and then matured for various lengths of time in a conventional step-wise in vitro maturation (IVM) system by using dibutyryl cyclic AMP. The COC that were matured in the same system for 44 h without pretreatment with ROS were used as the control group. At various periods after the start of IVM, oocytes were assessed for the meiotic stages and subjected to in vitro fertilization (IVF) with fresh spermatozoa. The ROS treatment inhibited GV breakdown of 94.4% oocytes, with the majority arrested at the GV-I stage (67.4%). Maximum maturation rate to the metaphase-II stage after ROS treatment was achieved by 44 h of IVM (92.1%) and no differences were observed with control oocytes (95.0%). Penetration rate was correlated to the maturation rate. The duration of IVM had no effects on polyspermy and male pronuclear (MPN) formation rates at 8 h post insemination (hpi), whereas both rates increased at 22 hpi. Direct comparison with controls assessed at 22 hpi confirmed a lesser MPN formation in ROS-treated oocytes (73.7% compared with 53.6%). Glutathione (GSH) concentrations were less in oocytes treated with ROS than in control oocytes (5 compared with 7.7 pmol/oocyte) as well as blastocyst rate (22.0% compared with 38.1%, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pretreated with ROS for 48 h did not equal that of control oocytes in the current IVM system.

Reduction of polyspermic penetration using biomimetic microfluidic technology during in vitro fertilization

Efforts to improve the in vitro embryo production process in pigs have included modifying culture medium and number of spermatozoa inseminated in order to reduce the incidence of polyspermy. Polyspermy is a pathological condition which results in aberrant embryonic development. The microchannels are designed to more closely mimic the function of the oviduct and create a flow pattern of spermatozoa past the oocytes similar to the pattern in the oviduct. In vitro fertilization of porcine oocytes in the microchannels has produced a higher incidence of monospermic penetration (p<0.05) as compared to the oocytes fertilized in the traditional microdrop system with comparable penetration and male pronucleus formation rates. Additionally, cleavage rates of the embryos as well as development to the blastocyst stage are similar. Here we demonstrate that the biomimetic microchannel in vitro fertilization system can reduce polyspermy and, therefore, increase the number of potentially viable embryos without reducing the overall in vitro production efficiency.

Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos

After in vitro maturation and fertilization of porcine oocytes, the fertilized embryos were cultured under 5 or 20% oxygen (O 2) for 7 days. In embryos cultured under 5% O 2 versus 20% O 2, development to the blastocyst stage was higher (36.3% versus 22.5%, P<0.05); the hydrogen peroxide (H 2O 2) content as a reactive oxygen species was lower (92 pixels versus 111 pixels, P<0.05); and fragmentation of DNA in 8- to 16-cell stage embryos (estimated by the comet assay) resulted in a shorter ( P<0.05) DNA tail (36 μm versus 141 μm). Antioxidants such as β-mercaptoethanol (β-ME) and Vitamin-E (Vit-E) suppressed oxidative damage in the embryos and improved their developmental ability. For embryos cultured under 20% O 2, there were the following differences ( P<0.05) between embryos exposed to 0 μM versus 50 μM β-ME: 28% versus 57% developed to the blastocyst stage; 125 pixels versus 98 pixels per embryo in H 2O 2 content; and a DNA tail of 209 μm versus 105 μm. In addition, for embryos cultured under 20% O 2, there were also differences ( P<0.05) between those exposed to 0 μM versus 50 μM of Vit-E: 28% versus 40% rate of development to the blastocyst stage; 28.9 cells versus 35.9 cells in the expanded blastocyst; 122 pixels versus 95 pixels per embryo (H 2O 2 content); and 215 μm versus 97 μm length of the DNA tail. Therefore, a low O 2 concentration during in vitro culture of porcine embryos decreased the H 2O 2 content and, as a consequence, reduced DNA fragmentation, and, thereby, improved developmental ability.

Factors affecting fertilization of porcine oocytes following intracytoplasmic injection of sperm

The objective of this study was to optimize intracytoplasmic sperm injection (ICSI), and to assess the effects of membrane-damaged sperm on development of porcine oocytes following ICSI. For optimization of development following the ICSI process, sperm injected oocytes were activated 0.5-1.0 hr after ICSI with 1 x 30 micros pulse of 1.2, 1.7, 2.2, and 2.7 kV/cm DC in experiment 1. After 7-days of culture ICSI oocytes activated with [x1]2.2 kV/cm produced more blastocyts ([x2]34.4%, P < 0.05) than other treatment groups. In experiment 2, oocytes were activated with 1 x 30 micros pulse of 2.2 kV/cm at either 0, 1.5, 3, or 6 hr after ICSI. Oocytes activated 1.5 hr after [x3]ICSI yielded more blastocysts (27.6%)[M4] than in other treatments. In experiment 3, sperm were briefly exposed to 0.1% Triton X-100 to induce membrane damage. Live-dead staining of Percoll-sorted untreated spermatozoa (frozen-thawed) used in this study showed that over 96% were "alive" whereas none were "alive" after Triton X-100 treatment. The rate of development to blastocyst of oocytes injected with Triton X-100 treated sperm combined with electrical activation (EA) at 1 x 30 micros pulse of 2.2 kV/cm (EA, 40.0%) was the best, when compared with those injected with untreated sperm plus EA (P < 0.05). In experiment 4, the development rate of oocytes to the blastocyst stage ([x5]32.1%) following injection of a sperm head only was not significantly different from that of oocytes injected with whole sperm (31.0%). In conclusion, we found that an intact membrane and tail structures of pig spermatozoa are not essential for embryo development by ICSI, and furthermore, dead porcine spermatozoa, at an early stage of necrosis caused by plasma membrane damage, support better embryo development than do live non-damaged sperm.

Maturation and fertilization of porcine oocytes from primordial follicles by a combination of xenografting and in vitro culture.

Our objective was to develop a method of endowing oocytes from porcine primordial follicles with full maturation and fertilizing ability as a model for ovarian xenografting of large mammals. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the capsules of both kidneys of ovariectomized athymic mice. The host mice were treated with 5 IU of equine chorionic gonadotropin (eCG) for 10 days (eCG-10), 30 days (eCG-30), or 60 days (eCG-60) after detection of cornified epithelial cells in their vaginal smears. Cumulus-oocyte complexes, ovarian grafts, and blood samples were obtained 48 h after eCG treatment. Forty-five to 70 days after grafting, the host mice in all groups for the first time showed vaginal cornification, accompanied by the formation of a small number of antral follicles in the grafts. However, we recovered large numbers of full-sized oocytes only from mice in the eCG-60 group; the numbers of full-sized oocytes in the other groups were low. Peripheral levels of total inhibin were highest in the eCG-60 group; this supports our finding that the most enhanced growth of antral follicles occurred in this eCG-60 group. Of 573 oocytes obtained from the eCG-60 group, 98 (17%) were at the metaphase II stage after in vitro culture for maturation. Moreover, 55% of matured oocytes with the first polar body (n = 20) were fertilized in vitro. These results clearly demonstrate that fertilization of oocytes from porcine primordial follicles is achievable by a combination of xenografting and in vitro culture.

Reduced polyspermic fertilization of porcine oocytes utilizing elevated bicarbonate and reduced calcium concentrations in a single-medium system.

The development of efficient systems for in vitro production of porcine embryos has been hampered by a high incidence of polyspermic fertilization. A recently developed single-medium system for porcine in vitro maturation (IVM), IVF and in vitro embryo culture (IVC) (Purdue Porcine Medium; PPM) was modified with elevated bicarbonate (44 mM) and reduced calcium concentrations (1.7 mM) for IVF (PPMfert.2). Oocyte penetration was evaluated after maturation in PPMmat (0.5 mg mL(-1) hyaluronan, 0.6 mM cysteine, 10 ng mL(-1) epidermal growth factor (EGF), 0.1 U mL(-1) porcine LH and FSH, and 1 x Minimal Essential Medium (MEM) vitamins) and fertilization (5 h with 5 x 10(5) sperm mL(-1)) in either PPMfert.2 or mTBM (20 mM Tris, 0.0 mM bicarbonate, 7.5 mM calcium). Embryonic development (cleavage and blastocyst stages) was assessed after culture in PPM1 and PPM2. Although penetration was lower in PPMfert.2 (69.9%) compared with mTBM (83.9%), 48.8% of penetrated oocytes were fertilized normally in PPMfert.2 compared with only 27.8% normal fertilization in mTBM. More oocytes cleaved in PPMfert.2 (77.9% v. 53.7%), but development to the blastocyst stage was not different between treatments (14.1% v. 14.3%). Further work is needed to improve embryonic development, but reduced polyspermic penetration is an important step in the optimization of the PPM system for in vitro porcine embryo production.

Effect of sperm survival and CTC staining pattern on in vitro fertilization of porcine oocytes

Polyspermy in pig oocytes fertilized in vitro remains unacceptably high. In this study, we evaluated the effects of gamete coincubation time, and determined if the proportion of capacitated spermatozoa would be predictive of the fertilizing ability of frozen-thawed semen in vitro. Cumulus–oocyte complexes were collected from slaughterhouse prepubertal gilt ovaries and matured in vitro for 44 h in TCM199, with EGF, FSH, cysteamine and follicular fluid. Fertilization was induced with 2×10 5 frozen-thawed spermatozoa/ml in TBM. Penetration of oocytes as well as polyspermic fertilization occurred 2 h after insemination. A strong correlation between penetration and polyspermic fertilization rates has been demonstrated, but there was no correlation between the proportion of capacitated spermatozoa, as assessed by chlortetracycline staining, at the time of insemination and fertilization rates. We also compared the results of IVF in three IVF media: TBM, m199 and TALP. Penetration and polyspermy were very different in these three media: 71±19% and 25±13% in TBM, 37±11% and 6±2% in m199, 10±2% and 0% in TALP, respectively. Nevertheless, survival of spermatozoa or modifications of the capacitation status were not different in these media after 6 h incubation. We concluded that survival and capacitation characteristics of the semen used for IVF could not be predictive of the IVF results. It seems necessary to act at the oocyte level to control both variability between replicates and the incidence of polyspermy. Improving the spermatozoa penetration blocking system of the oocytes and reducing the number of sperm-binding sites on the zona pellucida (ZP) are our further objectives.