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.

Effect of time of ovulation and sperm concentration on fertilization rate in gilts.

In normal production practices, sows and gilts are inseminated at least twice during estrus because the timing of ovulation is variable relative to the onset of estrus. The objective of this study was to determine if a normal fertilization rate could be achieved with a single insemination of low sperm number given at a precise interval relative to ovulation. Gilts (n=59) were randomly assigned to one of three treatment groups: low dose (LD; one insemination, 0.5 x 10(9) spermatozoa), high dose (HD; one insemination, 3 x 10(9) spermatozoa) or multiple dose (MD; two inseminations, 3 x 10(9) spermatozoa per insemination). Twice daily estrus detection (06:00 and 18:00 h) was performed using fenceline boar contact and backpressure testing. Transrectal ultrasonography was performed every 6 h beginning at the detection of the onset of standing estrus and continuing until ovulation. Gilts in the LD and HD groups were inseminated 22 h after detection of estrus; MD gilts received inseminations at 10 and 22 h after detection of estrus. Inseminations were administered by using an insemination catheter and semen was deposited into the cervix. The uterus was flushed on Day 5 after the onset of estrus and the number of corpora lutea, oocytes, and embryos were counted. Time of insemination relative to ovulation was designated as 40 to >24 h, 24 to >12 h, and 12 to 0 h before ovulation and >0 h after ovulation. The LD gilts had fewer embryos (P<0.04), more unfertilized oocytes (P<0.05) and a lower fertilization rate (P<0.07) compared to MD gilts. The effects of time of insemination relative to ovulation and the treatment by time interaction were not significant. We conclude that a cervical insemination with low spermatozoa concentration may not result in acceptable fertility even when precisely timed relative to ovulation.

Effect of recombinant porcine somatotropin on fetal and placental growth in gilts with reduced uterine capacity.

Crowded uterine conditions were induced by unilateral hysterectomy-ovariectomy (UHO) in 42 gilts to determine the effect of recombinant porcine somatotropin on fetal and placental growth. Gilts were randomly assigned across three replicates to one of three treatments: Control (C; n = 14), daily injections of 1 mL saline from d 0 to 64 of gestation, Early (E; n = 12), 5 mg of rpST/d from d 0 to 30, followed by 1 mL saline from d 31 to 64, and Late (L; n = 16), 1 mL saline/d from d 0 to 29, followed by 5 mg of rpST/d from d 30 to 64 of gestation. Blood was collected from each gilt via jugular venipuncture at d 0 and every 15 d thereafter. Gilts were hysterectomized on d 65 of gestation. Length of placental attachment and fetal crown-rump length were measured. Placentas and fetuses were weighed. Placental length, wet weight, and dry weight were recorded. Treatment with rpST (either E or L) increased (P < 0.0001) maternal plasma IGF-I concentrations relative to controls. Treatment with rpST did not affect placental wet weight or placental DNA content. However, E and L treatments increased the percentage of placental protein (P = 0.01) and placental dry matter (P = 0.10) and increased contact area of uterine-placental interface (P = 0.01). Despite changes in placental composition and morphology, weights of fetuses collected from L-treated gilts did not differ from controls, whereas weights of fetuses collected from E-treated gilts tended to be less than controls (P < 0.06). Administration of rpST increased maternal IGF-I concentrations and placental surface area but failed to increase fetal growth in the UHO model. Therefore, mechanisms that are independent of maternal IGF-I or placental contact area may control early fetal growth under crowded uterine conditions.

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.

Growth and the initiation of steroidogenesis in porcine follicles are associated with unique patterns of gene expression for individual componentsof the ovarian insulin-like growth factor system.

Ovarian follicular growth and steroidogenesis are controlled by the interaction of insulin-like growth factors (IGFs) and gonadotropins. The objective was to determine the temporal and spatial relationships for gonadotropin receptor, steroidogenic enzyme, and IGF system gene expression during the development of preovulatory porcine follicles. Sows (n = 18) were weaned and follicles were monitored by transrectal ultrasonography. Ovaries were collected from sows when the mean diameter of the preovulatory follicular cohort was approximately 2, 4, 6, or 8 mm. mRNA were measured by in situ hybridization for individual follicles within the preovulatory cohort (3 to 5 follicles per sow). Patterns of gene expression detected by in situ hybridization were confirmed by RNase protection analyses of pooled RNA samples. The amount of LH receptor mRNA and steroidogenic enzyme mRNA (17alpha-hydroxylase and aromatase) increased as the mean diameter of the follicular cohort increased from 2 to 6 mm, but then decreased abruptly for 8-mm follicles. Estradiol concentrations in follicular fluid closely followed the expression patterns of steroidogenic enzymes and LH receptor mRNA. FSH receptor mRNA was present in cohorts of 2-mm follicles but declined in 4-mm follicles and was undetectable in 6- and 8-mm follicles. The expression of IGF-I and type I IGF receptor mRNA were similar for follicles of 2, 4, 6, and 8 mm. In contrast, IGF-II mRNA progressively increased in follicles collected from 2-, 4-, and 6-mm cohorts, and then decreased slightly at 8 mm. Type II IGF receptor mRNA was greatest in 8-mm follicles. IGF binding protein-2 (BP-2) mRNA decreased as follicles achieved progressively larger sizes during the preovulatory period (2 to 8 mm), whereas the IGFBP-4 mRNA remained relatively low for follicles in 2- to 6-mm cohorts but then increased markedly in 8-mm follicles. In summary, temporal and spatial patterns of gene expression for gonadotropin receptor, steroidogenic enzyme, and IGF system genes were characterized in preovulatory porcine follicles by using in situ hybridization and RNase protection analyses. The unique patterns of gene expression suggest interdependence among specific genes that may be essential for preovulatory follicular development.

Glutathione content and embryo development after in vitro fertilisation of pig oocytes matured in the presence of a thiol compound and various concentrations of cysteine.

The present study examined the effect of different concentrations of cysteine in the presence of a thiol compound, beta-mercaptoethanol (BME), during in vitro maturation (IVM) of pig oocytes on cumulus expansion, nuclear maturation, intracellular glutathione (GSH) level and subsequent embryonic development after in vitro fertilisation (IVF). In experiment 1, oocytes were matured in NCSU 23 medium containing 10% porcine follicular fluid, 25 microM BME, 0.5 microgram/ml LH, 0.5 microgram/ml FSH and 0, 0.1, 0.2 or 0.4 mg/ml cysteine for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, cumulus cells were removed and a proportion of oocytes fixed to examine the rate of nuclear maturation. The remaining oocytes were co-incubated with spermatozoa for 5-6 h and putative zygotes were transferred to NCSU 23 medium containing 0.4% bovine serum albumin for 144 h. A proportion of putative zygotes were fixed 12 h after insemination to examine fertilisation parameters. In experiment 2, oocytes were matured as in experiment 1 and the GSH content was measured by a DTNB-GSSG reductase recycling assay. No mean differences among treatments were observed in nuclear maturation (78-89%). The mean differences in penetration rate (69-77%), polyspermy rate (31-40%), male pronuclear formation rate (93-96%) or mean number of sperm per oocyte (1.5-1.8) were not affected by the presence or absence of cysteine during oocyte maturation. Also no difference was observed in cleavage rates 48 h after insemination. However, compared with no addition (19%), the presence of 0.1-0.4 mg/ml cysteine during IVM increased (p < 0.001) the proportion of blastocysts (32-39%) at 144 h. In comparison with controls (5.6 pmol/oocyte), the GSH content of oocytes matured in the presence of cysteine was significantly (p < 0.001) higher (13-15 pmol/oocyte) with no mean differences among different cysteine concentrations. The results indicate that in the presence of a thiol compound, supplementation of IVM medium with cysteine can increase the GSH level and improve the developmental competence of pig oocytes following fertilisation. Further, no effect on either GSH level or embryo development was observed by increasing the levels of cysteine supplementation from 0.1 to 0.4 mg/ml.

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.

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.

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.

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.

Effects of oocyte maturation media on development of pig embryos produced by in vitro fertilization.

Few embryos derived from pig oocytes matured and inseminated in vitro are able to develop to blastocyts in culture. The present study was conducted to examine the effects of oocyte maturation media on the developmental ability of pig oocytes matured and inseminated in vitro. Follicular oocytes collected from ovaries of prepubertal gilts were cultured in NCSU23 medium, tissue culture medium 199 or a modified Whitten's medium. All of the media were supplemented with 0.57 mmol cysteine l-1 and 10% pig follicular fluid. After maturation, some of the oocytes were used for examination of intracellular glutathione content, nuclear maturation and cortical granule distribution. The other oocytes were inseminated in vitro in a modified Tris-buffered medium with cryopreserved, ejaculated spermatozoa for examination of cortical reaction, sperm penetration, male pronuclear formation and blastocyst development. No differences (P > 0.05) were observed in nuclear maturation, cortical granule distribution, sperm penetration, male pronuclear formation, polyspermy and cleavage in oocytes matured in the three media. However, significant (P < 0.05) differences were observed in glutathione content, cortical granule exocytosis, blastocyst development and number of cells in blastocysts. NCSU23 medium gave the best results of the three media, resulting in 5.8 pmol glutathione per oocyte, 97% of cortical granule exocytosis, 30% blastocyst development and 36.8 +/- 17.0 cells per blastocyst. These results clearly indicate that cytoplasmic maturation of pig oocytes was significantly affected by oocyte maturation media even in the presence of cysteine and pig follicular fluid. In addition, it was demonstrated that a large proportion of pig oocytes can develop to blastocysts under in vitro conditions.

Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization.

The effect of stage of maturation of the germinal vesicle of porcine oocytes at the time of in vitro maturation on subsequent developmental competence was examined. A large variation exists in the germinal vesicle morphology of oocytes at the time of collection of cumulus-oocyte complexes (COCs) and after culture in the absence of dibutyryl cAMP (dbcAMP) for 20 h. However, the morphology of the germinal vesicle was synchronized to a specific stage after culture in the presence of 1 mM dbcAMP for 20 h. There was no difference in germinal vesicle breakdown rate (total mean, 75.0 +/- 5.4%) or in maturation rate (total mean, 82.1 +/- 2.1 %) at 28 and 44 h of culture, respectively. However, differences in meiotic progress of oocytes were observed (p < 0.05) at 36 h of culture when COCs were exposed to dbcAMP for the first 20 h of maturation, as compared to controls. The incidence of embryos that developed to the blastocyst stage after in vitro fertilization was higher (p < 0.05) when COCs were exposed to dbcAMP (21.5 +/- 2.5%) as compared to controls (9.2 +/- 1.6%). After transfer of experimental embryos to four recipient gilts, the three pregnant recipients delivered 19 live piglets. These results indicate that exposure of COCs to dbcAMP for the first 20 h of culture for maturation increases the homogeneity of oocyte nuclear maturation and improves the efficiency of in vitro production of swine embryos.

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.

Presence of organic osmolytes in maturation medium enhances cytoplasmic maturation of porcine oocytes.

The effects of organic osmolytes on cytoplasmic maturation of porcine oocytes were examined in maturation medium (modified Whitten's medium) containing various NaCl concentrations. The presence of organic osmolytes, such as taurine and sorbitol, at 6 and 12 mM in maturation medium containing 68.49 or 92.40 mM NaCl increased oocyte glutathione content. Microfilament organization in oocytes was disrupted in maturation medium containing the higher level of NaCl (92.40 mM). However, supplementation with 12 mM sorbitol to the medium reduced the severity of the abnormality. Early embryonic development in vitro to the blastocyst stage was 8.3 +/- 0.9% for oocytes matured in modified Whitten's medium (68.49 mM NaCl) supplemented with 12 mM sorbitol, and 7.9 +/- 0.8% in modified NCSU23 medium (containing 108.73 mM NaCl, 7 mM taurine, 5 mM hypotaurine, and 1 mM glutamine), compared to 4.7 +/- 0.6% in modified Whitten's medium (68.49 mM Na Cl), which did not contain organic osmolytes. These results indicate that the presence of organic osmolytes, such as sorbitol and taurine, reduces the detrimental effects of high NaCl concentration in media used for the maturation of porcine oocytes. This effect is reflected by oocyte glutathione content and microfilament organization at the end of maturation and early development following in vitro maturation and in vitro fertilization.

Responses of porcine corpora lutea to somatotropin administration during pregnancy.

The effects of somatotropin (ST) on functions of porcine corpora lutea (CL) during pregnancy were investigated. Twenty-four crossbred (Yorkshire/Landrace) gilts from d 30 to 43 of pregnancy were injected daily with 5 mg of recombinant porcine somatotropin (rpST; n = 12) or 1 mL of saline (control, n = 12). Blood was collected on d 30, 37, and 43 for analyses of plasma progesterone. Gilts were killed on d 44 of pregnancy, and mRNA were isolated from CL, ovary, and liver. Messenger RNA expression for LH receptor, FSH receptor, ST receptor, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and cytochrome P450 side-chain cleavage enzyme (SCC) were measured. Liver, CL, and ovary contained a 4.7-kb mRNA of ST receptor, but the liver contained more mRNA for ST receptor than did CL or ovary (.97 +/- .18, .47 +/- .04, and .25 +/- .04 units, respectively). There were two variants of LH receptor mRNA in CL (6.8 and 4.4 kb). The CL also contained a 1.8-kb mRNA of SCC and a 1.7-kb mRNA of 3 beta-HSD. No FSH receptor mRNA was detected in CL of the pig. The rpST treatment did not affect the mRNA level of ST receptor, 3 beta-HSD, SCC, or 4.4-kb mRNA of the LH receptor. The 6.8-kb mRNA for the LH receptor was decreased (P < .05) by rpST (.56 +/- .04 vs .78 +/- .05 units). Furthermore, concentrations of plasma progesterone decreased (P < .001) in gilts treated with rpST. Decreased luteal function was associated with decreased expression of LH receptor in rpST-treated gilts. The luteotropic effects of ST observed in vitro do not necessarily occur in vivo when gilts are administered rpST during pregnancy.

Effects of recombinant porcine somatotropin on placental size, fetal growth, and IGF-I and IGF-II concentrations in pigs.

The objective of this study was to determine the effects of recombinant porcine somatotropin (rpST) on placental size, fetal growth, and maternal and fetal IGF-I and IGF-II concentrations. Twenty-four pregnant gilts received daily injections of either 1 mL of saline (control) (n = 12) or 5 mg of rpST (n = 12) from d 30 to 43 of gestation. Gilts were slaughtered on d 44 of gestation, reproductive tracts were removed, and fetal weight and length, placental weight, and implantation length were recorded. There was no effect of rpST on fetal or implantation length. Placental weight increased with rpST administration (71.20 +/- 3.52 vs 58.35 +/- 3.41 g; P < .02), as did fetal weight (18.06 +/- .55 vs 16.44 +/- .53 g; rpST vs control, respectively; P < .05). Implantation lengths were partitioned into quartiles to determine the effect of rpST on fetuses with different implantation lengths. The effect of rpST of fetal weight was greater in the first quartile ( < 137.5 mm) than in the fourth quartile ( > 240 mm) (16.04 vs 13.86 g compared with 19.47 vs 18.21 g, respectively). Analysis using a modified Brody curve suggests that the effect of rpST treatment on fetal weight is equivalent to the effect of increasing implantation length by 58.8 mm. Administration of rpST numerically raised IGF-I (P = .07) and IGF-II (P = .12) concentrations in fetal serum. Although maternal serum IGF-I concentrations were similar at d 30, treatment with rpST increased these concentrations over time (77.76, 247.75, 267.85 vs 82.59, 79.59, 77.97 ng/mL on d 30, 37, 43, respectively; P < .001, SE = 14.09). Maternal IGF-II concentrations were also similar at d 30 but decreased over time with rpST treatment (265.78, 219.61, 191.05 vs 285.44, 284.72, 283.05 ng/mL; P < .03, SE = 14.03). Increased maternal IGF-I concentrations may exhibit negative feedback on maternal IGF-II concentrations. The more pronounced effect of rpST on growth in fetuses with shorter implantation lengths suggests that rpST may increase uptake or utilization of nutrients by fetuses. In addition, nutrient transfer across placental membranes may be enhanced by rpST.

Pronuclear formation and intracellular glutathione content of in vitro-matured porcine oocytes following in vitro fertilisation and/or electrical activation.

Pronuclear formation and intracellular content of glutathione, containing reduced and oxidised forms, in porcine oocytes matured in vitro were determined following insemination and/or electrical stimulation. After insemination, sperm penetration had occurred as early as 3 h and female pronuclei had formed by 6 h with complete development by 12 h. Male pronuclear formation occurred, primarily, between 9 and 12 h after insemination. Glutathione content of the oocytes decreased following sperm penetration and remained at a depressed level until 12 h. After electrical stimulation, oocyte activation had occurred and female pronuclei had formed by 3 and 6 h, respectively. Oocyte glutathione content did not change as a result of oocyte activation. When oocytes were exposed to an electrical pulse and then spermatozoa, female pronuclear formation was observed by 3 h after stimulation/insemination. Sperm penetration was observed between 3 and 9 h. However, the incidence of male pronuclear formation observed at 12 h was extremely low, although sperm decondensation had occurred in some oocytes. Oocyte glutathione content had not decreased by 6 h following electrical activation. These results demonstrate that the changes in glutathione content in porcine oocytes following fertilisation in vitro differ from those due to electrical activation. Further, the decreased intracellular glutathione content in oocytes activated by sperm penetration appears to be due to the presence of a sperm factor.

Use of low-salt culture medium for in vitro maturation of porcine oocytes is associated with elevated oocyte glutathione levels and enhanced male pronuclear formation after in vitro fertilization.

The effects of sodium chloride (NaCl) in Whitten's medium on intracellular glutathione concentration and on cytoplasmic maturation, as determined by monospermic penetration and male pronuclear formation of porcine oocytes, were examined. Porcine cumulus-oocyte complexes were cultured for 20 h in BSA-free Whitten's medium containing different NaCl concentrations (44.50, 68.49, 92.40, 116.40, or 140.35 mM) and supplemented with 10% porcine follicular fluid and hormonal supplements; the complexes were then cultured without hormonal supplements for an additional 20-h period. The mean width of the perivitelline space of oocytes was increased with decreased concentration of NaCl in the culture medium. Intracellular glutathione concentration was elevated in oocytes cultured in medium with lower NaCl concentrations. After co-culture with spermatozoa for 6 h and culture in modified Whitten's medium for an additional 6 h, there were no differences in maturation and penetration rates among experimental groups. However, the rate of male pronuclear formation was higher in oocytes matured in media with the lower NaCl concentrations. In addition, the rates of monospermic penetration and male pronuclear formation were higher in oocytes matured in medium containing 44.50 mM NaCl (59.3 +/- 8.1 and 70.9 +/- 2.0%, respectively) than in medium containing 68.49 mM NaCl (39.4 +/- 5.5 and 57.1 +/- 4.5%, respectively). These data indicated that decreasing NaCl concentration in maturation medium for porcine oocytes below the customary level improved the quality of the matured oocytes as reflected in higher intracellular glutathione content, wider perivitelline space, higher monospermic penetration rate, and increased frequency of male pronuclear formation.

In vitro development of in vitro-matured porcine oocytes following chemical activation or in vitro fertilization.

Porcine cumulus-oocyte complexes were cultured in BSA-free Whitten's medium or modified Medium 199, each supplemented with porcine follicular fluid (PFF) and hormonal supplements (OMWM and OMM199, respectively) for 20 h; they then were cultured without hormonal supplements for an additional 20 (experiments 1 and 3) or 24 h (experiment 2). At the end of culture (experiment 1), the intracellular glutathione concentration was higher (p < 0.05) in oocytes matured in OMWM vs. OMM199. After activation by Ca2+ ionophore (experiment 2), the incidence of activation in the OMWM group was lower (p < 0.01) than in the OMM199 group. However, the incidence of pronuclear formation was higher (p < 0.01) in the OMWM group than in the OMM199 group at 8 h after activation. The percentage of embryos that developed to the morula stage was higher (p < 0.01) in the group matured in OMWM vs. OMM199 after 5 days of culture. After in vitro fertilization (experiment 3), the incidence of male pronuclear formation and the percentage of monospermic oocytes that formed one male and one female pronuclei were higher (p < 0.05) after maturation in OMWM vs. OMM199. The percentage of cleaved embryos that developed to the 8-cell and morula stages was higher (p < 0.05) in the OMWM group as compared to the OMM199 group. These results indicate that culture in modified Whitten's medium as compared with a standard medium (modified Medium 199) improves cytoplasmic maturation of porcine oocytes as evaluated by intracellular glutathione content, pronuclear formation, and development in vitro after artificial activation or fertilization in vitro.

Developmental ability of porcine oocytes matured and fertilized in vitro.

The developmental abilities of porcine oocytes matured and fertilized in vitro were examined in vivo and in vitro. Cumulus-oocyte complexes were cultured in mM199 supplemented with 10% porcine follicular fluid (PFF) and hormonal supplements (PMSG, hCG and estradiol-17beta) for 20 h and then without hormonal supplements for an additional 20 h. In Experiment 1, oocytes were then co-cultured for 6 h with spermatozoa which had been preincubated with 1% PFF (PFF-treated) or without (control). Oocytes were transferred to oviducts of gilts or cultured in modified Whitten's medium for 5 d. The percentages of oocytes with monospermic penetration (59%, 42 71 ) and with monospermic penetration and male and female pronuclei (32%, 23 71 ) were higher (P < 0.01) in the PFF-treated group than in controls (25%, 18 71 and 8%, 6 71 , respectively). After 5 d, the percentages of oocytes that developed to the morula or blastocyst stages in vitro and in vivo in the PFF-treated group (10%, 28 288 and 13%, 41 318 , respectively) were also higher (P < 0.05) than in controls (2%, 6 284 and 6%, 16 248 , respectively). Whereas some oocytes that were matured and fertilized in vitro developed to the blastocyst stage after 5 d in vivo culture (3%, 9 288 in PFF-treated group and 2%, 6 284 in control), no blastocysts were observed after 5 d when oocytes were cultured in vitro. When the progression of in vitro development of porcine oocytes that were matured and fertilized in vitro was examined in Experiment 2, morulae appeared after 72 h of culture, and 3% (3 100 ) of the oocytes developed to the blastocyst stage after 144 h (6 d) of culture. These results demonstrate that decreasing polyspermic penetration and increasing monospermic male pronuclear formation, as a result of PFF treatment of maturing spermatozoa, improved the developmental ability of porcine oocytes matured and fertilized in vitro. However, development in vitro was delayed by approximately 24 h compared with in vivo development, most of the embryos were blocked at the morula stage.