Biomedical subjects

B N Day

Publications and source records attributed to B N Day.

At least 55 records · Page 3Linked to original sources

PubMed1999

Effect of myosin light chain kinase, protein kinase A, and protein kinase C inhibition on porcine oocyte activation.

Previous studies have shown that exposure to broad-spectrum protein kinase inhibitors results in parthenogenetic activation of metaphase II arrested porcine oocytes. The objective of this study was to determine the effect of inhibitors of myosin light chain kinase and other protein kinases on pronuclear development, dephosphorylation of a 25-kDa protein, and cortical granule exocytosis. Metaphase II arrested oocytes were obtained by in vitro maturation. Cumulus-free oocytes were cultured with specific inhibitors in modified Whitten's medium for 24 h. Treatment with inhibitors that should inhibit myosin light chain kinase--HA100 (250 microM), Wortmannin (1 microM), and a combination of Wortmannin (1 microM), KT5720 (75 nM), and Iso-H7 (50 microM)--resulted in significantly higher pronuclear development (74.0%, 18.0%, and 35.0%, respectively) than in the negative control, H7 (10 microM; 2.0-12.4% depending upon the replication). Treatment with HA100 (250 microM) resulted in the dephosphorylation of the 25-kDa protein to a 22-kDa protein in 80.0% (n = 10) of oocytes exposed. However, Wortmannin (1 microM; n = 17), KT5720 (75 nM; n = 16), and Iso-H7 (50 microM; n = 19) treatment individually and in combination (n = 19) did not result in significant (p < 0.05; n = 19) dephosphorylation over the negative control, H7 (10 microM; n = 19). HA100 treatment resulted in significant cortical granule exocytosis when evaluated by laser confocal microscopy. In addition, protein kinase assays revealed lower myosin light chain kinase activity in electroactivated oocytes (p < 0.05) and protein kinase inhibitor-treated oocytes (p < 0.05) than in negative controls, nonelectroactivated oocytes, and H7 (10 microM)-treated oocytes. Treatment with HA100 (250 microM) resulted in pronuclear formation, dephosphorylation of the 25-kDa protein, and some release of cortical granules. These observations suggest that inhibition of myosin light chain kinase, protein kinase A, and protein kinase C results in activation of porcine oocytes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1999

A protein tyrosine phosphatase inhibitor, sodium orthovanadate, causes parthenogenetic activation of pig oocytes via an increase in protein tyrosine kinase activity.

This study was conducted to determine whether a protein tyrosine kinase (PTK) activity is involved in the initiation of the events that occur at fertilization in pig oocytes. After maturation for 47 h, a 7-h treatment of oocytes with 1 mM sodium orthovanadate, which is an inhibitor of protein tyrosine phosphatase, caused more than 90% pronuclear formation, cortical granule exocytosis, and a decrease in mitogen-activated protein kinase activity. Immunoblotting with an antibody specific for phosphotyrosine showed at least three proteins whose phosphotyrosine contents were significantly increased upon treatment of oocytes with 1 mM sodium orthovanadate. Preincubation of pig oocytes with 50 microM tyrphostin 47, a specific PTK inhibitor, completely blocked the ability of sodium orthovanadate to trigger activation events. In addition, when oocytes were pretreated with the calcium-chelating agent BAPTA-AM, sodium orthovanadate-stimulated pronuclear formation was significantly (P < 0.01) reduced (94.0% vs. 43.1%). These results suggest that PTK may be involved in pig oocyte activation in a calcium-dependent manner and that the stimulation of tyrosine kinase is able to signal a series of intracellular changes that lead to the activation events associated with fertilization.

Animals↗

PubMed1999

Pronuclear location before the first cell division determines ploidy of polyspermic pig embryos.

Polyspermy occurs frequently in the fertilization of mammalian eggs, but little is known about whether polyspermic eggs have developmental ability in vitro or in vivo. We previously reported that poly-pronuclear (PPN; 3 or more pronuclei) pig eggs developed normally to the blastocyst stage despite having fewer inner cell mass cell numbers as compared to blastocysts derived from two-pronuclear (2PN) eggs. Here it is shown that most PPN pig eggs have abnormal cleavage patterns (having 3 or more cells) in the first cell division and retarded development of pronuclei prior to syngamy as compared to 2PN eggs. Most blastocysts (14 of 18) that developed from PPN eggs showed abnormal ploidy (were haploid, triploid, and tetraploid) whereas 20 of 22 blastocysts derived from 2PN embryos were diploid. The size and morphology of most Day 40 fetuses that developed from PPN eggs appeared to be normal. Of 8 Day 40 fetuses analyzed, 1 was triploid (XXY) and another was a mosaic with both diploid (XX) and tetraploid cells (frequency of less than 10%, XXXX), and the others were diploid. Anomalies of chromosomal composition were not detected in these fetuses. Five live piglets and one dead piglet were born from two recipients of PPN eggs. It is proposed that not all pronuclei of PPN pig eggs participate in syngamy, resulting in diploid cells in the conceptus. Our data suggest that there are two types of pronuclei location in polyspermic pig eggs and that the resulting ploidy is determined at the zygote stage before the first cell division according to pronuclear location.

Actins↗

PubMed1999

Cyclin B1 transcript quantitation over the maternal to zygotic transition in both in vivo- and in vitro-derived 4-cell porcine embryos.

Using reverse transcription-competitive polymerase chain reaction (RT-cPCR), the quantity of cyclin B1 transcript present over the maternal to zygotic transition was determined for both in vivo- and in vitro-derived 4-cell porcine embryos. After poly(A) RNA isolation, RT-cPCR was performed on single embryos using an introduced, truncated cyclin B1 DNA competitor. Visualization of embryonic cyclin B1 cDNA and competitor for each reaction allowed a ratio to be formed for use in transcript quantity calculations when compared to cPCR standards. Analysis of in vivo- and in vitro-derived control embryos revealed a decline in cyclin B1 transcripts from 5 to 33 h post-4-cell cleavage (P4CC). The quantity of cyclin B1 for the in vivo-derived embryos at 5 and 33 h P4CC was 11.26 and 4.54 attomol/embryo, respectively (P < 0.03), while the in vitro-derived embryos had 20.18 and 7.52 attomol/embryo, respectively (P < 0.03). Treatment with alpha-amanitin from 5, 10, 18, or 25 h P4CC to 33 h P4CC resulted in cyclin B1 quantities that did not differ from those in the 33-h control embryos, irrespective of time spent in the inhibitor. These findings suggest that maternal cyclin B1 transcript degradation occurred over the 4-cell stage with no detectable embryonic cyclin B1 transcripts produced.

Amanitins↗

PubMed1998

Presence of beta-mercaptoethanol can increase the glutathione content of pig oocytes matured in vitro and the rate of blastocyst development after in vitro fertilization.

The present study examined the effect of beta-mercaptoethanol (BME) during in vitro maturation (IVM) of pig oocytes on in vitro fertilization (IVF) parameters, intracellular glutathione (GSH) concentration, subsequent embryo development and blastocyst cell numbers. Cumulus-oocyte complexes were cultured in North Carolina State University (NCSU)-23 medium containing porcine follicular fluid, cysteine, hormonal supplements and 0 to 50 microM BME for 20 to 22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20 to 22 h. After culture, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 5 to 6 h. Putative embryos were transferred to NCSU-23 containing 0.4% BSA and cultured for 144 h (Experiment 1). In comparisons between the presence or absence of BME, no differences were observed in fertilization parameters. At 48 h, no mean differences were found in cleavage rates. However, at 144 h, compared with no addition (26%), the presence of 12.5 and 25 microM BME increased (P < 0.05) the proportion of blastocysts in a dose-dependent manner (34 and 41%). Further increase from 25 to 50 microM BME reduced (P < 0.05) the blastocyst development rate. Blastocysts derived from oocytes matured with 25 microM BME had the highest (P < 0.05) trophectoderm (TE) and total cell numbers. No difference was found in inner cell mass (ICM) cells among treatments. In Experiment 2, after IVM, oocytes were fixed to analyze the GSH concentration. Compared to no addition, a higher (P < 0.01) level of GSH was found in oocytes matured with 25 microM BME. Compared with 25 microM BME, GSH was low (P < 0.05) at 50 microM BME. The results show that at certain concentrations BME in IVM medium has beneficial effects on subsequent embryo development, and is correlated with intracellular GSH level in pig oocytes.

Animals↗

PubMed1998

Practical considerations for the in vitro production of pig embryos.

The routine maturation, fertilization, and development of pig embryos in vitro has only recently been achieved. Many of the conditions for in vitro production of embryos have been undefined and thus difficult to replicate. The major problems of in vitro production of pig embryos have included maturation of oocytes, both nuclear and cytoplasmic, and development from one-cell to blastocyst. While these barriers have been at least partially overcome there is still a significant problem with polyspermy. Nevertheless, numerous offspring have been produced from in vitro maturation, in vitro fertilization, followed by a brief culture prior to embryo transfer. Here is provided a review of the literature encompassing the current status of the in vitro production of pig embryos.

Animals↗

PubMed1998

Morphologic comparison of ovulated and in vitro-matured porcine oocytes, with particular reference to polyspermy after in vitro fertilization.

This study was conducted to evaluate morphologic differences in pig oocytes matured in vivo and in vitro, with particular reference to the potential relationship between oocyte morphology and the occurrence of polyspermy after in vitro fertilization (IVF). In vivo-matured oocytes were surgically recovered from the oviducts of gilts with ovulated follicles on day 2 of estrus, and in vitro-matured oocytes were obtained by culturing follicular oocytes in a oocyte maturation system that has resulted previously in production of live offspring following IVF. Comparisons were made of the cytoplasm density, the diameter of oocytes with or without zona pellucida (ZP), the thickness of the ZP, the size of the perivitelline space (PVS), ZP dissolution time, and cortical granule (CG) distribution before IVF, and CG exocytosis and polyspermic penetration after IVF. Oviductal oocytes have clear areas in the cytoplasm cortex, while in vitro-matured oocytes have very dense cortex. The diameter of ovulated oocytes with ZPs was significantly (P < 0.001) greater than that of in vitro-matured oocytes. However, no difference was observed in the diameter of the oocyte proper. Significantly (P < 0.001) thicker ZPs and wider PVSs were observed in the ovulated oocytes. The ZPs of ovulated oocytes were not dissolved by exposure to 0.1% pronase within 2 hr, but the ZPs of in vitro-matured oocytes were dissolved within 131.7 +/- 7.6 sec. The ZPs of ovulated oocytes, but not of in vitro-matured oocytes, were strongly labeled by a lectin from archis hypogaea that is specific for beta-D-Gal(1-3)-D-GalNAc. Polyspermy rate was significantly (P < 0.01) higher for in vitro-matured oocytes (65%) than for ovulated oocytes (28%). CGs of oviductal oocytes appeared more aggregated than those of in vitro-matured oocytes. Most of CGs were released from both groups of oocytes 6 hr after IVF regardless of whether they were polyspermic or monospermic oocytes. These results indicate that in vitro-matured and in vivo-matured pig oocytes possess equal ability to release CGs on sperm penetration. Unknown changes in the extracellular matrix and/or cytoplasm of the oocytes while in the oviduct may play an important role(s) in the establishment of a functional black to polyspermy in pig oocytes.

Animals↗

PubMed1998

Functional analysis of activation of porcine oocytes by spermatozoa, calcium ionophore, and electrical pulse.

The present study was conducted to examine differences in the activation of pig oocytes induced by sperm penetration or the artificial stimulators calcium ionophore A23187 and electrical pulse. Cumulus-oocyte complexes were cultured in NCSU 23 medium supplemented with 10% pig follicular fluid and 0.57 mM cysteine for 44 hr and then freed from cumulus and corona cells prior to activation with A23187 or an electrical pulse or inseminated with frozen-thawed ejaculated semen. Cortical granule (CG) exocytosis, zona reaction, nuclear activation, and developmental ability were examined after treatment. A23187 and electrical pulse induced 75.7% and 76.9% of CGs to be released from oocytes. Sperm penetration induced 86.3% of CGs to be released from the oocytes, which was significantly higher (P < 0.05) than those induced by artificial stimulators. Activation induced by A23187 and sperm penetration resulted in a zona reaction, which prevented sperm penetration after insemination or reinsemination, respectively. Activation induced by electrical pulse, however, did not cause a zona block since the penetration rate (80%) of oocytes was not different (P > 0.05) from that in control oocytes (87%). Electrical pulse induced 87% of the oocytes to form a pronucleus(ei), with 53% failing to release the second polar body. A23187 induced 62% of oocytes to form a pronucleus(ei), and 81% of these oocytes released the second polar body. Sperm penetration induced 98-100% of the oocytes to release the second polar body and form a female pronucleus, and 88-89% of sperm penetrated oocytes formed a male pronucleus(ei). Blastocyst formation of oocytes exposed to spermatozoa, electrical pulse, and A23187 was 27%, 10%, and 4% at Day 6 and 28%, 11%, and 5% at Day 7, respectively (P < 0.05). More nuclei were observed in the blastocysts derived from in vitro fertilization (32.3 +/- 12.9) than artificial stimulators. These results indicate that different and possibly overlapping mechanisms may be involved in the activation of pig oocytes by spermatozoa, electrical pulse, and A23187.

Animals↗

PubMed1998

Presence of epidermal growth factor during in vitro maturation of pig oocytes and embryo culture can modulate blastocyst development after in vitro fertilization.

The present study examined the effect of epidermal growth factor (EGF) during in vitro maturation (IVM) and embryo culture on blastocyst development in the pig. In experiment 1, cumulus oocyte complexes were cultured in North Carolina State University (NCSU) 23 medium containing porcine follicular fluid, cysteine, hormonal supplements, and with or without EGF (0-40 ng/ml) for 20-22 hr. They then were cultured for an additional 20-22 hr without hormones. After maturation, cumulus-free oocytes were co-incubated with frozen-thawed spermatozoa for 5-6 hr. Putative embryos were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 hr. In experiment 2, oocytes were matured in medium containing 10 ng/ml EGF, inseminated, and putative embryos were cultured in the presence of 0-40 ng/ml EGF. In experiment 3, oocytes were cultured in the presence of 0, 10 and 40 ng/ml EGF to examine the kinetics of meiotic maturation. In experiment 4, 2- to 4-cell and 8-cell to morula stage embryos derived from oocytes matured with 10 ng/ml EGF were transferred to the oviduct and uterus, respectively, of each of three recipient gilts (3 and 4 days post-estrus, respectively). The presence or absence of EGF during IVM did not affect cumulus expansion, nuclear maturation, fertilization parameters, or cleavage rate. However, compared to no addition (21%), presence of 1 (33%) and 10 ng/ml EGF (42%) during IVM increased (P < 0.01) the rate of blastocyst development in a concentration-dependent manner. Compared to 10 ng/ml EGF, higher concentrations (20 and 40 ng/ml) reduced (P < 0.01) blastocyst development in a concentration-dependent manner (35% and 24%, respectively). No difference was observed between no addition and 40 ng/ml EGF (22%). Compared to no addition and 10 ng/ml EGF, a significantly (P < 0.001) higher proportion (25% vs. 55%) of oocytes reached metaphase II stage 33 hr after IVM with 40 ng/ml EGF. However, no difference was observed at 44 hr. Transfer of embryos to six recipient gilts resulted in three pregnancies and birth of 18 piglets. The results show that EGF at certain concentrations in IVM medium can influence the developmental competence of oocytes. However, addition of EGF during the culture of pig embryos derived from oocytes matured in the presence of EGF is without effect. Birth of piglets provides evidence that embryos derived from oocytes matured in a medium containing EGF are viable.

Animals↗

PubMed1998

Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry.

The present study examined the ability to establish pregnancies after transfer of pig embryos derived from in vitro fertilization (IVF) of in vitro matured (IVM) oocytes by X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/mL), epidermal growth factor (10 ng/mL), LH (0.5 microgram/mL) and FSH (0.5 microgram/mL) for 22 h, then the oocytes were cultured without hormonal supplements for an additional 22 h. Boar semen was collected and prepared by flow cytometry sorting of X and Y chromosome bearing spermatozoa. After IVM, cumulus-free oocytes were co-incubated with sorted X or Y spermatozoa (2 x 10(4)/mL) for 6 to 7 h in modified Tris-buffered medium containing 2.5 mM caffeine and 0.4% BSA. After IVF, putative embryos were transferred to NCSU-23 medium containing 0.4% BSA for culture. A portion of the oocytes was fixed 12 h after IVF, the remainder were cultured up to 96 h. At 96 h after IVF, 8-cell to morula stage embryos (n = 30 to 35) from each gender were surgically transferred to the uterus of recipient gilts. Insemination of IVM pig oocytes with X- or Y-bearing sperm cells did not influence the rate of penetration (67 vs 80%), polyspermy (40 vs 53%), male pronuclear formation (95 vs 96%), or mean number of spermatozoa per oocyte (1.6 vs 1.6), respectively. Furthermore, no difference was observed between cleavage rates at 48 h after IVF (X, 49 vs Y, 45%). Transfer of embryos derived from X-bearing spermatozoa to 18 recipients resulted in 5 pregnancies and delivery of 23 females and 1 male piglet. Similarly, transfer of embryos derived from Y-bearing sperm cells to 10 recipients resulted in 3 pregnancies, with 9 male piglets delivered. The results show that X- and Y-bearing spermatozoa sorted using USDA sperm sexing technology can be successfully used in an IVM-IVF system to obtain piglets of a predetermined sex.

Animals↗

PubMed1998

Coculture with follicular shell pieces can enhance the developmental competence of pig oocytes after in vitro fertilization: relevance to intracellular glutathione.

The present study examined the effect of follicular shell pieces (FSP) during in vitro maturation (IVM) of porcine oocytes on 1) in vitro fertilization (IVF) parameters, 2) subsequent embryo development, 3) oocyte glutathione (GSH) concentration, and 4) viability after embryo transfer. Cumulus-oocyte complexes were cultured in North Carolina State University (NCSU) 23 medium containing porcine follicular fluid, cysteine, and hormonal supplements and with or without FSP for 20-22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20-22 h. After culture, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 5-6 h. Putative zygotes were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 h. In comparisons between the presence and absence of FSP, no differences were observed in fertilization parameters. At 48 h, no mean differences were found in cleavage rates. However, at 144 h, the proportion of embryos that developed to the blastocyst stage was significantly (p < 0.01) higher (18% vs. 36%) for oocytes cocultured with FSP. A significantly (p < 0.05) higher GSH concentration was found in oocytes matured with FSP as determined by dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Transfer of embryos to 9 recipients resulted in 5 pregnancies with the birth of 18 live piglets. The results provide clear evidence of the beneficial effect of FSP during IVM of pig oocytes cultured in the presence of cysteine on subsequent embryo development to the blastocyst stage. The birth of piglets confirms the viability of IVM-IVF-derived embryos.

Animals↗

PubMed1998

Maturation in vitro of pig oocytes in protein-free culture media: fertilization and subsequent embryo development in vitro.

In the present study, attempts were made to develop a protein-free (PF) in vitro maturation (IVM) system for pig oocytes and to examine subsequent embryo development after in vitro fertilization. In experiment 1, four IVM media were tested: 1) control: North Carolina State University (NCSU) 23+10% porcine follicular fluid; 2) PF-NCSU: NCSU 23+0.1% polyvinyl alcohol (PVA)+1% amino acids; 3) PF-TCM: Tissue culture medium (TCM) 199+PVA; and 4) PF-WM: PF-Waymouth MB 752/1 medium (WM)+PVA. Oocytes were cultured in the respective media containing eCG and hCG (10 IU/ml each) for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined. Some oocytes were coincubated with frozen-thawed spermatozoa for 5-6 h in modified Tris-buffered medium containing caffeine and BSA. In experiment 2, oocytes were matured in control, PF-TCM, and PF-WM, fertilized in vitro, and cultured for 144 h in NSCU 23+BSA. Fewer (p < 0.01) oocytes reached metaphase II stage in PF-NCSU (45% vs. 80-85%) than in the other media. Oocytes matured in control medium showed the most cumulus expansion, followed by those in PF-TCM and PF-WM; those in PF-NCSU showed very slight expansion. A lower (p < 0.05) penetration rate was obtained for oocytes matured in PF-NCSU than in the control medium (59% vs. 81%). In contrast to those in control (96%) and PF-TCM (93%), oocytes in PF-WM (65%) showed a lower male pronuclear formation. Compared to that in the control, a significantly lower (p < 0.05) cleavage rate was also observed for oocytes matured in PF-WM. Similar proportions of embryos developed to the blastocyst stage when oocytes were matured in control (22%) and PF-TCM (13%). These results indicate that pig oocytes can be successfully matured in a protein-free medium with subsequent development to the blastocyst stage.

Animals↗

PubMed1998

Parthenogenetic activation of pig oocytes with calcium ionophore and the block to sperm penetration after activation.

Calcium ionophore A23187 can parthenogenetically activate oocytes in many animals. The present study was designed to analyze functionally the mechanism of A23187 activation of pig oocytes matured in vitro. In experiment 1, effects of the concentration of A23187 on intracellular calcium transients, cortical granule (CG) exocytosis, nuclear activation, and zona reaction, which was determined by zona hardening and sperm penetrability, were examined. Cumulus-free oocytes were exposed to 0-100 microM A23187 for 5 min. It was found that the amplitude of the intracellular calcium transients, percentage of CG exocytosis, and percentage of pronuclear formation were increased in a concentration-dependent manner. The time for dissolution of zona pellucida (ZP) was increased in the oocytes treated with 25-100 microM A23187. Penetration of ZP-intact oocytes by spermatozoa was decreased and only 3-4% of oocytes were penetrated by spermatozoa after 50-100 microM A23187 treatment. In experiment 2, oocytes were treated with 100 microM A23187 for 5 min and then cultured for 10 min or 3.5 h before insemination. No difference in penetration rates was observed between the two groups of oocytes (12.0% vs. 12.2%), but the penetration rates were significantly lower than those in controls (85.2% vs. 82.4%). In experiment 3, treatment of oocytes with 100 microM A23187 for 5 min was followed by removal of the ZP from a portion of the oocytes. ZP-intact and ZP-free oocytes were then inseminated for examination of sperm penetration. One of 65 (2%) oocytes with ZP and 48 of 52 (92%) oocytes without ZP were penetrated by spermatozoa. These results indicate that activation of pig oocytes by A23187 is the result of A23187-induced intracellular calcium increase and that A23187-induced cortical reaction can prevent sperm penetration of ZP-intact oocytes, but not ZP-free oocytes.

Animals↗

PubMed1998

Development of early porcine embryos in vitro and in vivo.

In vitro development of early porcine embryos under different culture conditions was evaluated and compared to in vivo development. First, one- and two-cell embryos were collected and cultured individually in 20- microl drops under 5% CO2 in air for 4 days. Embryos from one oviduct were cultured in NCSU-23, and those from the contralateral oviduct were cultured in KSOM/AA. The embryos developed in NCSU-23 had a higher mean number of inner cell mass (ICM) nuclei compared to those developed in KSOM/AA (p = 0.025). They also had higher trophectoderm (TE) and total nuclear number (p = 0.001), while there was no difference in the average ratio of ICM to TE nuclei (p = 0.731). When the effect of different gas atmospheres was tested, the numbers of TE and total nuclei were higher (p < 0.01 and p < 0.025, respectively) in embryos cultured in an atmosphere with 5% CO2 in air than in those developed under 5% CO2:5% O2:90% N2. Next the development of embryos cultured in NCSU-23 was compared to that of embryos incubated in vivo. By the end of the 4-day incubation, the cultured embryos had higher nuclear numbers and a higher ratio of ICM to TE nuclei than those developed in vivo (p < 0.001). Finally, the embryos that developed in NCSU-23 or in vivo were transferred into recipients. By Day 40 of pregnancy, 37.1 +/- 15.3% of the in vitro- and 53.8 +/- 15.3% of the in vivo-incubated embryos formed conceptuses. These results indicate that despite the lower nuclear numbers caused by in vitro conditions, the cultured embryos were developmentally competent.

Animals↗

PubMed1997

Preincubation of cumulus-oocyte complexes before exposure to gonadotropins improves the developmental competence of porcine embryos matured and fertilized in vitro.

The objectives of the present study were to examine whether delayed exposure of porcine cumulus-oocyte complexes (COCs) to gonadotropins affects the diameter of oocytes, the nuclear morphology of the germinal vesicle, the rate of germinal vesicle breakdown (GVBD), and the embryonic developmental rate of inseminated oocytes following maturation and fertilization in vitro (IVM/IVF). After preincubation (experimental) or no preincubation (control) in BSA-free NCSU23 medium containing 1096 porcine follicular fluid for 12 h, COCs were cultured for maturation in the same medium supplemented with gonadotropins for 20 h and then without those gonadotropins for 20 h. During the preincubation period, the nuclear morphology of the germinal vesicles became more homogeneous. Incidence of GVBD after 20 h of maturation culture was not different between the control and experimental group. When cultured in NCSU23 medium for 7 d following IVF, the incidence of embryos that developed to the blastocyst stage (23.1 +/- 3.1%) was higher in the experimental group than in the control group (8.7 +/- 1.2%). Blastocysts in the experimental group had a larger number of cells than control blastocysts. Following embryo transfer into the oviduct of recipient gilts, IVM/IVF embryos had elongated by Day 12 of gestation. These results indicate that preincubation of porcine COCs, before exposure to gonadotropins to induce the resumption of meiosis, increases the rate of development of IVM/IVF embryos to the blastocyst stage.

Journal Article↗

PubMed1997

In vitro penetration of pig oocytes in a modified Tris-buffered medium: effect of BSA, caffeine and calcium.

The effect of BSA, caffeine and calcium was studied on the penetration of pig oocytes by frozen-thawed spermatozoa in a modified Tris-buffered medium (mTBM) without added bicarbonate. Pig cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG: 10 IU/ml each) for 22 h. The COC were then cultured in the same medium but without hormonal supplements for an additional 22 h. After culture, cumulus cells were removed and oocytes were co-incubated with spermatozoa for 6 h in mTBM containing caffeine (5 mM) and 0.1 or 0.4% BSA (Experiment 1). In Experiment 2, oocytes were inseminated in mTBM containing 0.1% BSA and various concentrations of caffeine (0 to 5 mM). In Experiment 3, insemination was carried out in mTBM containing 0.1% BSA, 1 mM caffeine and various concentrations of Ca(2+) (0.5 to 10 mM). Supplementation of mTBM with either 0.1 or 0.4% BSA resulted a high penetration rate with a high polyspermy rate. However, the mean number of spermatozoa per oocyte was significantly higher at 0.4% than at 0.1% BSA. The penetration rate, polyspermy rate and mean number of spermatozoa per oocyte were all significantly higher when 1 to 5 mM caffeine were added to the medium than in caffeine-free medium. No penetration was observed in the presence of 0.5 mM Ca(2+). The penetration rate was significantly increased from 12 to 92% at 2.5 to 10 mM Ca(2+). The mean number of spermatozoa per oocyte did not differ between 2.5 and 5 mM Ca(2+) but increased significantly at 7.5 and 10 mM. These results show the successful in vitro penetration of pig oocytes in a chemically semidefined medium without added bicarbonate. Although BSA and caffeine can modulate the rate of sperm penetration, calcium seems to be an important regulatory ion.

Journal Article↗

PubMed1997

Effects of oviductal fluid and heparin on fertility and characteristics of porcine spermatozoa.

The objective of this study was to determine the effects of oviductal fluid and heparin on sperm penetration and the characteristics of spermatozoa. The addition of oviductal fluid and heparin to the fertilisation medium decreased sperm penetration and the mean number of spermatozoa in penetrated eggs. The number of spermatozoa firmly bound to zona pellucida was also decreased in the presence of oviductal fluid and heparin. Chlortetracycline (CTC) fluorescence patterns were used to determine the incidence of capacitation and the acrosome reaction. The proportion of capacitated and acrosome-free spermatozoa increased when spermatozoa were exposed for 1.5 and 3 h to oviductal fluid and heparin. In contrast heparin alone did not increase the number of capacitated spermatozoa at these time points. These results suggest that factor(s) in oviductal secretions reduce polyspermic fertilisation and the number of spermatozoa that will penetrate porcine oocytes. The reduction of polyspermic penetration by oviductal secretions may be due to a reduced number of spermatozoa in the fertilisation medium with an intact acrosome.

Animals↗

PubMed1997

Chlortetracycline fluorescence patterns and in vitro fertilisation of frozen-thawed boar spermatozoa incubated under various bicarbonate concentrations.

Porcine oocyte-cumulus complexes were cultured in bovine serum albumin (BSA)-free North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG: 10 IU/ml each) for 22h. They were then cultured in the same medium but without hormonal supplements for an additional 22 h. After culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed ejaculated boar spermatozoa in tissue culture medium (TCM) 199 containing caffeine (5 mM), fetal calf serum (FCS; 10%) and varying concentrations (26-56 mM) of NaHCO3 for 9 h (experiment 1). In experiment 2, chlortetracycline (CTC) was used to assess the functional state of spermatozoa incubated under different NaHCO3 concentrations. Experiment 3 examined the effect of FCS (1% and 10%) and NaHCO3 (26 and 46 mM) on fertilisation parameters. Compared with 26 mM, penetration rate was significantly higher (p < 0.05) at 36-56 mM NaHCO3. Polyspermy showed a similar pattern although no difference was observed between 26 and 36 mM. At 46 mM NaHCO3, the mean number of spermatozoa (MNS) penetrated per oocyte increased significantly (p < 0.05). A significantly higher proportion of spermatozoa were capacitated and acrosome reacted at 46 and 56 mM NaHCO3, respectively. The fertilisation medium containing 46 mM NaHCO3 and 1% FCS showed a higher penetration rate (84%) with a relatively low incidence of polyspermy (39%). The results indicate that NaHCO3 stimulates capacitation and/or the acrosome reaction of boar spermatozoa in a dose-dependent manner and thus affects fertilisation parameters.

Animals↗