The Animal Sciences Academic Quadrathlon: history, current status, and recommendations.

The Animal Sciences Academic Quadrathlon (AQ) provides opportunities for teams of undergraduate animal and dairy science students to participate in regional American Society of Animal Science (ASAS)/American Dairy Science Association (ADSA) meetings and to collectively exhibit their knowledge and talents competitively in 4 categories: 1) solving practical, hands-on, laboratory-type problems; 2) providing written answers to essay-type questions about principles and concepts; 3) preparing and communicating orally and extemporaneously topics of current animal science interest; and 4) quickly responding to short-answer questions provided in the form of double-elimination quiz bowls. Each team is selected by winning the local AQ at their university. Overall and individual category winning teams are recognized, but team rankings are not emphasized. The ASAS/ADSA members provide leadership for organizing and conducting the AQ, and ASAS and each university provide travel expenses for students. The ultimate purpose is to stimulate academic excellence among undergraduate students and for the students to attend ASAS/ADSA regional scientific meetings to meet faculty and students and to attend scientific research presentations. The purpose of this document was to provide a history of the event and to make recommendations for its improvement. The AQ was conceived in 1967. During the next 10 yr, an ASAS committee developed procedures for a trial AQ held in 1980 at the ASAS Midwestern Section, Kansas State University-Manhattan, and in the next year the first official AQ was held at the ASAS Midwestern Section at the University of Nebraska-Lincoln. Starting in 1985, AQ programs were initiated at the other 3 ASAS sectional meetings, and an estimated 50,000 students representing 60 universities have participated in AQ programs since that time. If the AQ is to continue its improvement over time, it will greatly depend on sustained ASAS/ADSA faculty interest and support, as well as greater adherence to the original AQ procedures.

Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and suppression of apoptosis in vitro.

The object of this study was to investigate the role of epidermal growth factor (EGF) and IGF-I in the regulation of preantral follicular growth, antrum formation, and granulosal cell proliferation/ apoptosis. Porcine preantral follicles were manually dissected and cultured for up to 8 d in Waymouth's (Exp. 1) or alpha-minimum Eagle's essential medium (Exp. 2 and 3) supplemented with 10 microg/mL of transferrin, 100 microg/mL of L-ascorbic acid, and 2 mU/mL of ovine FSH, in the presence (Exp. 1 and 3) or absence (Exp. 2) of 7.5% fetal calf serum. According to the experimental protocol, IGF-I (0, 1, 10, or 100 ng/mL; Exp. 1), or IGF-I (50 ng/mL), EGF (10 ng/mL) and EGF+IGF-I (Exp. 2 and 3) were added to the culture media. In Exp. 1, follicles exhibited a concentration-dependent response (P < 0.05) to IGF-I, with the highest rates of granulosal cell proliferation, follicular integrity, and recovery rate of cumulus cell-oocyte complexes and lowest incidence of apoptosis occurring at the highest IGF-I dose. In Exp. 2 serum-free medium, granulosal cell proliferation was low (1 to 5%), irrespective of whether EGF and/or IGF-I were present and cellular apoptosis was increased (P < 0.05) on d 4 and 8 in the EGF+IGF-I group compared with the addition of either factor alone. In Exp. 3, granulosal cell proliferation was high in all follicles cultured in serum-containing medium for the first 3 d, but fell sharply (P < 0.05) on d 4, except in media containing IGF-I. Collectively, EGF and IGF-I increased granulosal cell proliferation, decreased apoptosis, and promoted follicular antrum formation. These results may provide useful information for developing a preantral follicular culture system in which the oocytes are capable of fertilization and embryonic development.

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.

Minimum number of spermatozoa required for normal fertility after deep intrauterine insemination in non-sedated sows.

A fibreoptic endoscope procedure for non-surgical deep intrauterine insemination in non-sedated sows has been reported. However, the endoscope is an expensive and fragile instrument, and is unsuitable for use under field conditions. The aim of this study was to determine the minimum number of spermatozoa required to maintain optimal fertility using a flexible catheter (1.8 m in length, 4 mm in diameter) for deep intrauterine insemination in 2-6 parity non-sedated sows. Crossbred sows were treated with eCG 24 h after weaning and with hCG 72 h later to induce oestrus. Deep intrauterine insemination was performed 36 h after hCG treatment in 117, 126, 60 and 69 sows with 15.0, 5.0, 2.5 or 1.0 x 10(7) spermatozoa in 10 ml, respectively. Weaned sows (n = 147) not treated with hormones and used for standard artificial insemination (AI) (two inseminations per oestrus with 3 x 10(9) spermatozoa in 100 ml) served as controls. The flexible catheter was passed successfully through the cervix into one uterine horn in 95.4% of the sows in an average of 3.7 +/- 0.09 min. Farrowing rates after deep intrauterine insemination with 15 or 5 x 10(7) spermatozoa did not differ from those of the control group (82.9, 76.2 and 83.0%, respectively), but a significant decrease (P < 0.001) was observed in sows inseminated with 2.5 or 1.0 x 10(7) spermatozoa (46.7 and 39.1%, respectively). In contrast, the number of spermatozoa inseminated did not affect prolificacy. Laparotomy revealed that the tip of the flexible catheter reached approximately the anterior third of the uterine horn. Although deep intrauterine insemination was performed in only one uterine horn, the percentages of embryos collected from the tip of both uterine horns 2 days after deep insemination were not significantly different. The results show that in comparison with standard AI, a 20-60-fold reduction in the number of spermatozoa inseminated and an 8-10-fold reduction in the dose volume can be achieved without decreasing fertility when semen is deposited non-surgically into the upper first third of one uterine horn.

Embryo development and establishment of pregnancy after embryo transfer in pigs: coping with limitations in the availability of viable embryos.

Embryo transfer and pregnancy maintenance strategies in pigs were evaluated with reference to situations in which limited numbers of viable embryos or micromanipulated embryos are available, such as pig cloning. Development of embryos with compromised zona pellucida was compared with development of embryos with intact zona pellucida. Micromanipulation had no effect on blastocyst production rates after development in vivo or in vitro, but development in vivo improved the number of embryos reaching the blastocyst stage. Transfer of embryos with compromised zona pellucida resulted in live piglets. Several hormone treatments to maintain pregnancy were tested in a model in which three embryos were transferred into unmated recipient gilts, compared with transfer of three embryos into mated recipients. None of the hormonal treatments resulted in pregnancy rates of more than 25% at term and no more than 9% of transferred embryos survived, in comparison with 50% of the mated recipients successfully carrying 25% of transferred embryos. Lastly, the developmental potential of parthenogenetic embryos was assessed and 62% of transferred embryos resulted in pregnancies, none of which continued beyond day 55 of gestation. After co-transfer of three fertilized embryos with 55-60 parthenogenetic embryos into each of six recipients, two live piglets were delivered. The results from the present study indicate that transfer of zona pellucida compromised embryos can yield litters of normal piglets. In addition, it was demonstrated in a model system involving the transfer of three fertilized embryos into mature gilts that hormonal pregnancy maintenance strategies support a low proportion of embryos to term. Lastly, the present study shows for the first time a comparably effective but novel alternative for pregnancy maintenance in the pig involving the co-transfer of parthenote embryos.

Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos.

The present study aimed to determine the influence of exogenous epidermal growth factor (EGF) on in vitro preimplantation porcine embryo development and its mRNA expression for EGF receptor (EGFR). Oocytes were aspirated from abattoir ovaries, selected and cultured in defined, protein-free media for 44 hr before in vitro fertilization (IVF). Thirty-six hours after IVF, two-cell stage embryos were selected and treated or cultured until embryo treatment. In experiment 1, compact morulae were selected on day 4 after IVF and randomly allocated into 5 groups: NCSU 23 with PVA as group 1; NCSU 23 with PVA and 0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml EGF as group 2, 3, 4, respectively; NSCU 23 with 0.4% BSA as group 5. In experiment 2, treatment groups were the same as in experiment 1 except that 0.1% crystallized BSA was added to both washing media and all treatment groups instead of PVA. In experiments 3 and 4, two-cell stage embryos were treated and cultured in the same experimental design as experiments 1 and 2, respectively. RT-PCR was used to detect the mRNA expression of EGF receptor in compact morulae and blastocysts. The PCR products were subjected to direct DNA sequencing. There was no significant improvement in the development rate of embryos from compact morulae to blastocysts in the presence of various EGF concentrations (0.1, 1.0, 10.0 ng/ml) versus without EGF addition. They were all significantly lower than those embryos cultured in the continuous presence of 0.4% BSA. However, when a reduced concentration (0.1%) of crystallized BSA was added to all the treatment groups, a significantly lower rate of embryo development was observed in control media (NCSU23 with 0.1% crystallized BSA) compared with those developed in culture media with 0.4% BSA. With the addition of EGF at 10 ng/ml (with 0.1% BSA), embryo development rates were significantly improved over the control group (P < 0.05) and were as good as those rates in 0.4% BSA culture group. When embryos were selected and treated from the 2-cell stage, they did not develop to blastocyst stages after five more days' culture without any protein (BSA) or growth factor addition. When 0.1% BSA was included in the media, blastocyst formation rates were significantly improved by EGF addition at the concentration of both 1.0 or 10 ng/ml (P < 0.05) as compared to 0.0 or 0.1 ng/ml. EGFR mRNA was detected in both compact morulae and blastocyst stages of porcine embryos and confirmed by direct DNA sequencing. Our results indicate that IVM-IVF porcine embryo developmental rates could be improved by the addition of EGF in the culture media with the presence of a reduced amount of defined BSA (>97% albumin). However, EGF alone was not able to elicit any stimulatory effects on embryo development in the absence of protein supplementation. Further studies are needed to investigate the potential synergistic factors in embryo culture media to eventually define the porcine embryo culture media.

Microtubule assembly after treatment of pig oocytes with taxol: correlation with chromosomes, gamma-tubulin, and MAP kinase.

In this study, taxol was used as a tool to study the correlation of microtubule assembly with chromosomes, gamma-tubulin and phosphorylated mitogen-activated protein (MAP) kinase in pig oocytes at different maturational stages. Taxol treatment did not affect meiotic resumption and chromosome condensation but inhibited/disrupted chromosome alignment at the metaphase plate and bipolar spindle formation and thus meiotic progression. Microtubules were co-localized with chromosomes and were found to emanate from the chromosomes in taxol-treated oocytes, suggesting that chromosomes may serve as a source of microtubule organization. In addition, the concentric emanation of microtubules within the chromosome-surrounded area in taxol-treated oocytes suggests that microtubule emanation from the chromosomes may be directed by other microtubule-organizing material. The formation of one large spindle or >/=2 spindles in oocytes after taxol removal shows that minus end microtubule-organizing material can be normally located on both sides of chromosomes only when the chromosomes are aligned on the metaphase plate. The co-localization of gamma-tubulin and phosphorylated MAP kinase with microtubule assembly in both control and taxol-treated oocytes suggests that these two proteins are associated microtubule-nucleating material in pig oocytes. However, Western blot analysis showed that neither cytoplasmic microtubule aster formation nor extensive microtubule assembly in the chromosome region induced by taxol was caused by super-activation of MAP kinase. Taxol also induced microtubule assembly depending on chromosome distribution in the first polar body. The results suggest that chromosomes are always co-localized with microtubules and that emanation of microtubules from the chromosomes may be regulated/directed by microtubule-organizing material including gamma-tubulin and phosphorylated MAP kinase in pig oocytes.

Developmental potential of porcine nuclear transfer embryos derived from transgenic fetal fibroblasts infected with the gene for the green fluorescent protein: comparison of different fusion/activation conditions.

The in vitro developmental potential of porcine nuclear transfer (NT) embryos was evaluated. Oocytes were matured for 42-44 h, and metaphase II-oocytes were enucleated. Fetal fibroblasts infected with the enhanced green fluorescent protein (EGFP) gene were serum-starved for 3-5 days. A single cell was injected into the perivitelline space of the enucleated oocytes. The reconstructed oocytes were allocated to different fusion and activation conditions. In experiment 1, two different fusion/activation conditions were compared: two pulses of 1.2 kV/cm for 30 microsec (group A), or one pulse of 1.6 kV/cm for 30 microsec followed in 30 min by one pulse of 1.2 kV/cm for 30 microsec (group B). Parthenogenetic controls were created by using the group A parameter. The fusion rate in group A (mean +/- SEM, 68.4% +/- 3.9%) was higher (P < 0.05) than in group B (59.4% +/- 2.3%). The rates of cleavage (50.1% +/- 4.6% to 62.8% +/- 5.5%) were not different among control and treatment groups. However, the rate of parthenogenetic control embryos developing to the blastocyst stage (18.1% +/- 3.1%) was higher (P < 0.05) than the rate of NT embryos (5.9% +/- 1.7% and 4.9% +/- 2.5%). In experiment 2, we compared two pulses of 1.2 kV/cm (group C) versus two pulses of 1.3 kV/cm (group D). For two control groups, the same pulses as those given to group C or D, respectively, were supplied. The fusion rate in group D (70.6% +/- 4.2%) was higher (P < 0.05) than in group C (58.9% +/- 2.7%). The cleavage rates were not different among control and treatment groups (58.1% +/- 8.1% to 73.6% +/- 6.0%). However, the rate of embryos developing to the blastocyst stage in group D (3.5% +/- 1.7%) was lower (P < 0.05) than in controls and group C (11.4% +/- 2.0% to 16.4% +/- 1.1%). In experiment 3, we examined whether the presence of cytochalasin B (CB) during donor cell injection affects the development of NT embryos. The fusion rate of oocytes in the group with CB (78.4% +/- 1.4%) was higher (P < 0.05) than in the group without CB (70.9% +/- 0.2%). The cleavage rate of the control group (85.5% +/- 4.9%) was higher (P < 0.05) than those of the treatment groups (61.6% +/- 2.7% and 63.9% +/- 4.3%). However, the rates of embryos developing to the blastocyst stage (8.1% +/- 2.5% to 19.1% +/- 6.0%) and the mean cell number of blastocysts (29.4 +/- 5.2 to 45.7 +/- 6.4) were not different among control and treatment groups. Green fluorescence was observed at all stages in NT embryos. These results indicate that two pulses of 1.2 kV/cm are enough for fusion/activation of NT embryos to develop to the blastocyst stage, and that the presence of CB during donor cell injection is not necessary for early development of NT embryos.

Development and expression of the green fluorescent protein in porcine embryos derived from nuclear transfer of transgenic granulosa-derived cells.

Nuclear transfer (NT) techniques have advanced in the last few years, and cloned animals have been produced from somatic cells in several species including pig. In this study we examined the feasibility of using granulosa-derived cells (GCs) as donor cells combined with a microinjection procedure to transfer those nuclei. In vitro matured oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm. Mural GCs infected with an enhanced green fluorescence protein (EGFP) gene were serum-starved (0.5% serum, 7 days), injected directly into cytoplasm of enucleated oocytes and the oocytes were electrically activated. The reconstructed embryos were cultured for 7 days and stained with Hoechst 33342 to determine the number of nuclei. Non-manipulated oocytes were electrically activated and cultured as controls. At 9 h post-activation, the pronuclear formation rates were 78.7+/-3.7% in NT and 97.4+/-4.4% in controls at 9 h post-activation. After 7 days culture, the cleavage rates were 24.5+/-7.2% in NT and 79.3+/-5.6% in controls. The blastocysts formation rates were 4.9+/-2.4% in NT and 26.8+/-3.8% in controls. To examine the effect of activation time on development of NT embryos, oocytes were activated at 0-0.5, 1-2, or 3-4 h post-injection. At 18 h post-activation the pronuclear formation rates were higher (62.5+/-7.3%) in the 3-4 h group as compared to the 0-0.5 h (22.0+/-12.5%) or 1-2h (44.5+/-6.3%) groups (P<0.05). However, the cleavage rates (9.6+/-4.6 to 10.7+/-4.2%) and the blastocysts formation rates (1.2+/-2.4 to 4.9+/-3.7%) were not different among treatments (P>0.05). The mean cell number of blastocysts was 15.7+/-5.7 in NT and 25.3+/-24.7 in controls. Green fluorescence was observed in roughly half of the embryos from the one-cell to the blastocyst stage. These results indicate that granulosa-derived cell nuclei can be remodeled in the cytoplasm of porcine oocytes, and that the reconstructed embryos can develop to the blastocyst stage. In addition, EGFP can be used as a marker for gene expression of donor nuclei.

Feasibility of producing porcine nuclear transfer embryos by using G2/M-stage fetal fibroblasts as donors.

The type of donor cell most suitable for producing cloned animals is one of the topics under debate in the field of nuclear transfer. To provide useful information to answer this question, G2/M- and G0/G1-stage fetal fibroblasts were used as donor cells for nuclear transfer. In vitro-matured oocytes derived from abattoir ovaries were used as recipient cytoplasts. In both groups, nuclear envelope breakdown and premature chromosome condensation were completed within 1-2 h after donor cells were injected into the cytoplasm of oocytes. Microtubules were organized around condensed chromosomes and formed a spindle within 1-1.5 h after activation. Decondensation of chromosomes could be seen within 2-4 h after activation. Reformation of the new nuclear envelope occurred 4-6 h after activation and was followed by nuclear swelling and formation of a pronucleus-like structure (PN) 8-12 h after activation. Most (80.6%) of the reconstructed oocytes derived from G2/M cells extruded polar body-like structures (PB). However, a much lower frequency of PB (21.7%) was observed in the reconstructed oocytes derived from G0/G1 donors. A variety of PN and PB combinations were observed in reconstructed oocytes derived from G2/M-stage donors, including 1PN+0PB, 1PN+1PB, 1PN+2PB, 2PN+0PB, 2PN+1PB, 2PN+2PB, and 3PN+1PB. Chromosomes of most embryos (10/13) derived from G2/M stage were diploid. The percentage of cleavage and blastocysts and the average nuclear number of blastocysts in the G2/M and G0/G1 groups were not different. These results demonstrate that the G2/M stage can be morphologically remodeled by cytoplasm of MII oocytes in pigs. To maintain normal ploidy, the extra chromosomes derived from G2/M-stage cells could be expelled by oocytes as a second polar body. G2/M-stage fibroblast nuclei could direct reconstructed embryos to develop to the blastocyst stage.

Degradation of maternal cdc25c during the maternal to zygotic transition is dependent upon embryonic transcription.

To gain a better understanding of the molecular differences that may contribute to cleavage arrest and the poorer development associated with laboratory produced embryos, a series of experiments were conducted to quantitate the message levels of the cell cycle controller cdc25c, over the maternal to zygotic transition (MZT) in 4-cell in vivo- and in vitro-derived porcine embryos. The experiments were designed to measure both maternal and embryonic derived cdc25c transcripts by quantitative reverse transcription-competitive polymerase chain reaction (RT-cPCR), determine the point of the transition to zygotic genome activation, and study the interaction between initial embryonic transcription and maternal cdc25c degradation. Analysis of in vivo- and in vitro-derived embryos revealed no difference in cdc25c message level for any of the times P4CC (P > 0.05). Comparison of control embryos from 5- to 33-hr P4CC revealed a reduction in transcript quantities in the 10-hr P4CC group that was maintained at later time points (P < 0.05). Embryos cultured in the RNA polymerase inhibitor, alpha-amanitin, from cleavage to 5-, 10-, 18-, 25-, or 33-hr P4CC displayed no difference in cdc25c levels when compared to controls at similar time points (P > 0.05). However, if embryos were first exposed to alpha-amanitin after 18-hr P4CC with this treatment continuing to 33 hr, the levels of cdc25c transcript were reduced (P < 0.04) when compared to those embryos that were first exposed to the inhibitor at either 5- or 10-hr P4CC. This finding and the comparison of these same embryos to the 0-33-hr alpha-amanitin and control groups allowed us to conclude that cdc25c transcription began between 10- and 18-hr P4CC, with the degradation of maternal cdc25c message dependent on transcriptional initiation.

Expression of pregnancy-associated glycoprotein 1 and 2 genes in in vivo, in vitro and parthenogenetically derived preimplantation pig embryos.

The objective of this study was to determine whether porcine PAG (poPAG) genes are expressed in embryos as they develop from the 1-cell stage to expanded blastocysts, and whether expression differed according to how embryos had been derived. Embryos at various preimplantation stages were assayed after in vivo fertilisation, after in vitro fertilisation of in vitro-matured oocytes, or following parthenogenetic activation of in vitro-matured oocytes. The presence of PAG transcripts was determined at the 1-, 2-, and 4-cell, compact morula and blastocyst stages by reverse transcription-PCR procedures with PAG 1- and PAG 2-specific primers, followed by Southern blotting. The mRNAs for poPAG 1 and 2 were detected in in vitro-derived, in vivo-derived and parthenogenetically derived blastocyst stage embryos. In some replications poPAG 1 could be detected as early as the compact morula stage and poPAG 2 could be detected as early as the 4-cell stage. Our study revealed that poPAG 1 and 2 genes are expressed as early as the compact morula stage and 4-cell stage, respectively, in normal embryos and in parthenogenetically derived blastocysts. Thus it appears that the poPAGs are not maternally imprinted and they may be useful as potential candidates for markers of developmental competence.

Successful non-surgical deep intrauterine insemination with small numbers of spermatozoa in sows.

A 100-fold reduction of the standard dose for artificial insemination in pigs (3 x 10(9) spermatozoa in 80-100 ml fluid) can be used when spermatozoa are deposited surgically next to the uterotubal junction. The present study was performed to develop a technique for non-surgical deep intrauterine insemination in pigs without sedation of the animal. In Expt 1, sows were weaned, treated to induce oestrus and used to evaluate the difficulties involved in the insertion of a flexible fibre optic endoscope through the cervix and along the uterine horn. Deep uterine catheterizations were performed on each sow at 30-40 h after hCG treatment in the crate in which the animal was housed. The endoscope was inserted through an artificial insemination spirette, moved through the cervical canal and propelled forward along one uterine horn until the entire endoscope was inserted. In 30 sows (90.9%) no or minor difficulties were observed during insertion and in these animals the procedure was completed in 4.1 +/- 0.26 min. Insertion of the endoscope through the cervical canal was not possible in only one sow (3.03%). In Expt 2, endoscopic deep intrauterine insemination at 36 h after hCG treatment was performed in 15, 18 and 13 sows with 100, 20 or 5 x 10(7) spermatozoa, respectively, resulting in farrowing rates of 86.6%, 88.9% and 92.3%, respectively; there were no significant differences among groups. Farrowing rates after deep intrauterine inseminations were also not different from those achieved after standard intracervical insemination with 3 x 10(9) spermatozoa (control group: n = 48; 87.5%). Mean litter size (9.41 +/- 0.38 to 10.02 +/- 0.25) was also similar among the different experimental and control groups. In conclusion, endoscopic non-surgical deep intrauterine inseminations can be performed quickly in sows, and normal farrowing rates and litter sizes can be obtained after insemination with a small number of spermatozoa.

Translocation of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro.

The distribution of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro was revealed by using MitoTracker Green staining and confocal laser scanning microscopy. The regulation of mitochondrial translocation by microfilaments and microtubules was also studied. In oocytes collected from small follicles, strong staining of active mitochondria was observed in the cell cortex. Accumulation of active mitochondria in the peripheral cytoplasm and around the germinal vesicles was characteristic of fully grown oocytes collected from large follicles. Mitochondria accumulated in the perinuclear area during meiotic progression from germinal vesicle breakdown (GVBD) to anaphase I. Larger mitochondrial foci were formed and moved to the inner cytoplasm in mature oocytes. Compared with the oocytes matured in vivo, in which large mitochondrial foci were distributed throughout the cytoplasm, mitochondria were not observed in the central cytoplasm in most of the oocytes matured in vitro. Strong staining of mitochondria was observed in the first polar bodies in metaphase II oocytes. In fertilized eggs, active mitochondria aggregated in the pronuclear region. Perinuclear clustering and a cortical ring were the most marked features of early cleavage. Active mitochondria were distributed in both inner cell mass cells and trophectoderm cells of the blastocysts. Disassembly of microtubules with nocodazole inhibited both mitochondrial aggregations to the germinal vesicle area and their inward movement to the inner cytoplasm during oocyte maturation, as well as the translocation of mitochondria to the peri-pronuclear region during fertilization, whereas disruption of microfilaments by cytochalasin B had no effects. These data indicate that: (i) oocyte maturation, fertilization and early embryo development in pigs are associated with changes in active mitochondrial distribution; (ii) mitochondrial translocation is mediated by microtubules, but not by microfilaments; and (iii) in vitro maturation conditions may cause incomplete movement of mitochondria to the inner cytoplasm and thus affect cytoplasmic maturation.

Inhibitors of mitochondrial ATP production at the time of compaction improve development of in vitro produced porcine embryos.

The relationship between partial inhibition of mitochondrial ATP production during the peri-compaction stage and porcine embryonic development was studied. In vitro produced porcine compact morulae were cultured for two days under conditions that should inhibit ATP production via oxidative phosphorylation. The culture conditions included supplementation of the culture medium with sodium azide (NaN3), an oxidative phosphorylation inhibitor; incubation in the presence of 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation; or incubation under 5% O2 concentration. NaN3 (10-20 microM) increased the average nuclear number found in the resulting blastocysts (P<0.05). The embryos developed in the presence of 100 microM DNP formed blastocysts at a significantly higher incidence than the control embryos (P<0.001); the average nuclear number found in these blastocysts was also higher (P<0.005). When these treatments were applied from the 1-cell stage they proved to be detrimental. Elevations in the frequency of blastocyst formation (P<0.05), and in the average nuclear number per blastocyst (P<0.001) were also measured when compact morulae were incubated in an atmosphere containing 5% vs. 20% O2. NaN3 or DNP did not have negative effects on long term development: the treated embryos were able to form viable conceptuses by day 30 after being transferred into recipients. The data indicate that transient inhibition of mitochondrial ATP production is advantageous for porcine embryonic development in vitro.

Production of nuclear transfer-derived swine that express the enhanced green fluorescent protein.

The ability to add or delete specific genes in swine will likely provide considerable benefits not just to agriculture but also to medicine, where pigs have potential as models for human disease and as organ donors. Here we have transferred nuclei from a genetically modified fibroblast cell line to porcine oocytes, matured in vitro under defined culture conditions, to create piglets expressing enhanced green fluorescent protein. The nuclear transfer-derived piglets were of normal size, although some mild symptoms of "large offspring syndrome" were evident. These experiments represent a next step towards creating swine with more useful genetic modifications.

Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector.

Here we report the production of transgenic pigs that express enhanced green fluorescent protein (eGFP). Porcine oocytes were matured in vitro in a serum-free, chemically defined maturation medium, subsequently infected with a replication deficient pseudotyped retrovirus, fertilized and cultured in vitro before being transferred to a recipient female. Two litters were born from these embryo transfers; one pig from each litter was identified as transgenic and both expressed eGFP. From a tool in basic research to direct applications in production agriculture, domestic livestock capable of expressing foreign genes have many scientific applications.

Effect of incubation temperature on in vitro maturation of porcine oocytes: nuclear maturation, fertilisation and developmental competence.

The present study examined the effect of low culture temperature during in vitro maturation (IVM) of pig oocytes on their nuclear maturation, fertilisation and subsequent embryo development. In experiment 1, oocytes were cultured at 35 or 39 degrees C for 44 h in modified tissue culture medium 199 supplemented with 10 ng/ml epidermal growth factor, 0.57 mM cysteine, 75 microg/ml potassium penicillin G, 50 microg/ml streptomycin sulphate, 0.5 microg/ml LH and 0.5 microg/ml FSH to examine the nuclear maturation status. In experiment 2, oocytes were cultured at 35 degrees C for 44 or 68 h and nuclear maturation was examined. In experiment 3, oocytes matured for 44 or 68 h at 39 degrees C and for 68 h at 35 degrees C were co-incubated with frozen-thawed spermatozoa for 5-6 h. Putative embryos were transferred into North Carolina State University (NCSU) 23 medium containing 0.4% bovine serum albumin. At 12 h after insemination, some oocytes were fixed to examine the fertilisation rate and the remaining embryos were examined at 48 and 144 h for cleavage and blastocyst formation rate, respectively. Compared with 39 degrees C, culture of oocytes at 35 degrees C for 44 h significantly (p < 0.05) reduced the metaphase II (M II) rate (79% vs 12%). However, extension of culture time to 68 h at 35 degrees C significantly increased (p < 0.05) the M II rate (7% vs 58%). In experiment 3, compared with other groups, fewer (p < 0.05) oocytes reached M II when cultured at 35 degrees C for 68 h (69-81% vs 49%). Extension of culture duration to 68 h at 39 degrees C stimulated spontaneous activation (28%) of oocytes. No difference in cleavage rates was observed among different groups. Compared with oocytes matured for 44 h at 39 degrees C (31%), the proportion of blastocysts obtained was low (p < 0.05) for oocytes matured at 35 degrees C (13%) or 39 degrees C (3%) for 68 h. The results indicate that lower culture temperature can delay nuclear maturation of pig oocytes. However, extension of culture time can stimulate nuclear maturation and these oocytes are capable of fertilisation and development to the blastocyst stage at moderate rates.

Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes.

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8+/-17.3% vs 28.5+/-3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0+/-7.0% vs 63.3+/-12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0+/-11.6% vs 4.6+/-4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.

Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor.

This study examined the ability of epidermal growth factor (EGF) to improve the developmental competence of pig oocytes matured in a protein-free (PF) in vitro maturation (IVM) system. Oocyte maturation was done in one of three media: 1. PF-TCM: tissue culture medium (TCM) 199 + 0.1% polyvinylalcohol (PVA); 2. PF-TCM+EGF: PF-TCM + 10 ng/ml EGF; and 3. +ve CONT: North Carolina State University (NCSU) 23 medium + 10% porcine follicular fluid. All media contained 0.57 mM cysteine. Hormonal supplements, 0.5 microg/mL LH and 0.5 microg/mL FSH, were present only for the first half (20 to 22 h) of the culture period. After maturation, oocytes were co-incubated with frozen-thawed spermatozoa for 5 to 6 h and transferred to embryo culture medium, NCSU 23 containing 0.4% BSA, for 144 h. In Experiment 1, differences in cumulus expansion were observed for oocytes matured in +ve CONT (Category 4), PF-TCM (Category 2) and PF-TCM+EGF (Category 3). However, no significant differences in nuclear maturation to metaphase II stage were observed. In Experiment 2, no differences in fertilization parameters were observed. Significant (P < 0.01) differences in cleavage rates were observed among the three media for a proportion of the oocytes matured (52, 60 and 69% in PF-TCM, PF-TCM+EGF, and +ve CONT, respectively). Oocytes matured in PF-TCM showed the lowest (P < 0.01) blastocyst development (22%). However, the same rate of blastocyst development was obtained for +ve CONT (37%) and PF-TCM+EGF (37%). Blastocyst cell numbers were significantly higher when oocytes were matured in the presence of EGF (26 vs. 37 to 41). In Experiment 3, oocytes matured in PF-TCM+EGF had a significantly (P < 0.05) higher intracellular glutathione (GSH) concentration (5.9 vs. 11.4 pmol/oocyte) compared with PF-TCM. Twenty-two of 25 embryo transfer recipients became pregnant (Experiment 4). Four animals returned to estrus in within 60 days. Six pregnant animals slaughtered at 26 to 45 days had 43 fetuses (range: 4 to 12) and the remaining 12 animals farrowed 82 piglets (range: 3 to 12). These results indicate that EGF enhances the developmental competence of pig oocytes matured in a protein-free culture medium which is correlated with higher GSH level in oocytes. Birth of piglets indicate that embryos derived from oocytes matured in the presence of EGF are viable.