Transfer of porcine embryos after 3 days of in vitro culture

Two experiments were conducted to determine the viability of porcine embryos transferred after long-term in vitro culture. In Exp. 1, four-cell embryos were kept in culture for 120 h. Embryos that were exposed to fresh culture medium every 12 h survived better than embryos kept in the same medium throughout the culture period. In Exp. 2, four- and eight-cell embryos were cultured in vitro for 72 h before transfer to estrus-induced recipient gilts. Each gilt received, on average, 19 embryos. If recipients were synchronous with donors 3/32 (9%) recipients remained pregnant with an average of 4.0 +/- .6 viable young. If the sexual cycle of the recipients was 24 h behind that of the donors the pregnancy rate was 18/34 (53%) with 4.4 +/- .5 viable young. Average embryo survival rate for the two groups was 1.8 and 12.5%, respectively. A 24-hourly medium replacement during the in vitro culture period had no significant effect on transfer results. When transferring freshly collected blastocysts, pregnancy rate, number of viable young and survival rate of embryos were 6/10 (60%), 7.8 +/- 1.4, and 23.9% for synchronous recipients and 7/10 (70%), 9.3 +/- 1.8, and 32.9% for asynchronous recipients, respectively. Recipients with very high plasma progesterone levels or numerous follicular cysts at the time of transfer were less likely to remain pregnant than others.     

Development of porcine embryos in vitro: effects of culture medium and donor age.

We compared development of porcine embryos in three media and evaluated the effect of age of the donor on embryo development in vitro. In Exp. 1, embryos were collected from 35 postpubertal females on d 2 or 3 after onset of estrus. Embryos were cultured 144 h in Whitten's Medium (WM), North Carolina State University Medium-23 (NCSU-23), or Beltsville Embryo Culture Medium-3 (BECM-3) in 95% air: 5% CO2 at 39 degrees C. More (P < 0.01) embryos that were initially one cell or two cells developed to blastocysts when cultured in NCSU-23 (56%) and BECM-3 (43%) rather than in WM (7.5%). More (P < 0.01) embryos that were four cells at recovery developed to blastocysts in NCSU-23 (97%) than in BECM-3 (69%) or WM (69%). Blastocysts that developed from four-cell embryos cultured in BECM-3 had more (P < 0.01) nuclei than blastocysts that developed from four-cell embryos in the other two media. In Exp. 2, ovarian responses, fertilization rates, and in vitro embryo development in NCSU-23 and BECM-3 were compared for postpubertal (approximately 170-d-old) gilts vs gilts given exogenous gonadotropins at 102 d of age. Ovulation rate (P < 0.01), number of eggs recovered, and number of eggs fertilized per gilt (P < 0.001) were greater in the older gilts. The percentage of eggs fertilized, the number of unfertilized eggs, and the number of unclassifiable eggs were similar (P > 0.10) for both age groups. More (P < 0.10) blastocysts developed from embryos recovered from 170-d-old than from 102-d-old gilts, and more (P < 0.05) blastocysts developed in NCSU-23 than in BECM-3. Zona thicknesses and number of nuclei per embryo were similar (P > 0.10) for both ages. We conclude that embryos from prepubertal gilts do not have the same in vitro developmental potential as those from cyclic gilts. However, superior development of embryos in NCSU-23 from both 102-d-old and 170-d-old gilts indicates that media composition did not differentially affect embryos produced by younger vs older gilts.     

Effect of heat-stress on bovine embryo development in vitro.

Chronic elevation of uterine temperature has long been known to increase embryo mortality in dairy cattle. Short-term elevation in temperature of mouse embryos to 43 degrees C (acute) has been shown to induce intracellular production of heat-shock proteins. In this study, in vitro development of bovine embryos was assessed during short-term (60 h) coculture with oviduct epithelial cells at 38.6 degrees C (T1), 40 degrees C (T2), 38.6 degrees C after a prior pulse treatment (20 min) at 43 degrees C with 5% CO2 (T3), or 38.6 degrees C after a prior pulse treatment (20 min) at 43 degrees C with 100% CO2 (T4). During incubation, embryos cocultured at 40 degrees C had a greater (P < .05) mean embryo development score at 36 h than embryos cocultured at 38.6 degrees C. At 60 h of incubation, embryo development scores were greater (P < .05) for embryos cultured at 38.6 degrees C than for those cocultured at 40 degrees C. The number of embryos hatched at 60 h was similar after coculture at 38.6 degrees C (T1) or a prior pulse treatment with 5% CO2 and 43 degrees C (T3), but the embryo development score at 60 h was greater (P < .05) for the pulse-treated embryos. Embryos in T4 had greater (P < .05) embryo development scores than did T1 embryos from 36 through 60 h. Pulse treatment (T4) resulted in a greater (P < .05) number of hatched embryos at 60 h than T1, T2, and T3. These results indicate a detrimental effect of a chronic elevation in temperature that was evident shortly after embryo hatching.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-culture of ovine ova with oviductal cells in medium 199

Three experiments were conducted to test the suitability of medium 199 supplemented with 10% fetal calf serum (M199FCS) as a medium for co-culture of one-cell sheep ova with sheep oviductal cells. In Exp. 1, ova were co-cultured for 5 d in 5 ml of M199FCS or in Ham's F10 medium supplemented with 10% fetal calf serum (F10FCS). Co-culture did not increase the number of cleavages at the end of 5 d of culture, but M199FCS supported more cleavages than did F10FCS (P = .016). In Exp. 2, ova were cultured for 1 to 3 d in M199FCS alone or on oviductal, uterine or kidney cell monolayers from ewes 2 d postestrus and transferred to recipients from which they were recovered at 8 d postestrus. Co-culture with oviductal cells improved (P less than .001) the cleavage index of recovered embryos compared with culture in medium alone or co-culture with other cell types. In Exp. 3, monolayers of oviductal cells from ewes 2 d postestrus and from luteal-phase ewes were cultured as in Exp. 2. No difference was observed between the two sources of oviductal cells for their ability to support in vitro development of one-cell sheep eggs for 1 or 2 d. These studies suggest that M199FCS may be a good medium to use in an oviductal cell co-culture system for one-cell sheep ova. Results further suggest that specific secretions of oviductal cells may be important for early embryo development in vivo.     

Vascular endothelial growth factor (VEGF) promotes the early development of bovine embryo in the presence of cumulus cells

Three experiments were conducted to determine the effect of Vascular Endothelial Growth Factor (VEGF) on bovine embryonic development in vitro. Human recombinant VEGF(165) was employed at 5 ng/ml in modified synthetic oviduct fluid. In Exp. 1, bovine cumulus oocyte complexes were matured with or without VEGF for 22 hr, inseminated without VEGF for 6 hr, then cultured with or without VEGF for 42 hr. The cleavage rate and the development rate to 4- to 8-cell were higher (P<0.05) in groups with VEGF during in vitro maturation (IVM, 71.4% and 59.6%), in vitro culture (IVC, 70.3% and 62.3%), and both IVM and IVC (75.9% and 67.8%) than in the group cultured thoroughly without VEGF (49.9% and 38.4%, respectively). In Exp. 2, 4- to 8-cell embryos produced in vitro without VEGF were removed from cumulus cells at 48 hr post-insemination (Pi) and cultured with or without VEGF for 144 hr. The development rates to blastocyst at 96 hr (D6), 120 hr (D7) and 144 hr (D8) were similar (P>0.05) in both groups. In Exp. 3, cumulus cells were removed from presumptive embryos produced by IVM and IVF without VEGF at 10 hr Pi. Denuded embryos were cultured with or without VEGF for 38 hr or 182 hr. The cleavage rate and the development rates to 4- to 8-cell at 48 hr Pi and to blastocyst on D6, D7 and D8 were similar (P>0.05) in all groups. These results suggest that VEGF has a beneficial effect on the initial development of bovine embryo through surrounding cumulus cells.     

Co-culture of in vitro fertilized bovine embryos with different cell monolayers.

The effects of different cell monolayers on in vitro development of early bovine embryos derived from in vitro maturation and fertilization were examined in this study. Early embryos (four to eight cells) were randomly allocated to bovine granulosa cell (GC), oviductal cell (OC), or uterine cell (UC) monolayers in Exp. 1 and to GC, skin cell (SC; from 10-d-old chicken embryos), testicular cell (TC; from 10-d-old mouse), and liver cell (LC; from 10-d-old chicken embryos) monolayers in Exp. 2, and cultured for 6 d at 38.6 degrees C in a humidified atmosphere of 5% CO2 in air. The culture medium was 12.5 mM HEPES TCM 199 supplemented with 1% calf serum and 1 mM sodium pyruvate. In Exp. 1, the percentage of four- to eight-cell embryos that developed to blastocysts on GC, OC, and UC monolayers was 26.9 (28/104), 37.5 (39/104), and 39.2 (40/102), respectively. In Exp. 2, the percentage of four- to eight-cell embryos that developed to blastocysts on GC, SC, TC, and LC monolayers was 53.3 (40/75), 42.9 (33/77), 49.3 (37/75), and 44.3 (35/79), respectively. There were no significant differences in development among groups in either experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Donor and recipient rat strains affect full-term development of one-cell zygotes cultured to morulae/blastocysts

The present study was conducted to examine the developmental potential to offspring of rat embryos cultured from 1-cell to morula/blastocyst stage. Pronuclear zygotes from Wistar x Wistar or (SD x DA) x Wistar strains were cultured in modified rat 1-cell embryo culture medium (mR1ECM) for 96 h in 5% CO(2) in air at 37 C. The proportion of the 3-way cross hybrid zygotes developing into morula/blastocyst stage (74%) was higher than that of the Wistar zygotes (66%). Day-5 morulae/blastocysts developed in vitro were transferred into Day-3 or -4 pseudopregnant recipients of Wistar or SD x DA strain. The transfer of cultured embryos resulted in the birth of offspring at 13-59%, while that of non-cultured control blastocysts showed birth rates of 35-65%. The best offspring rate of cultured embryos (59%) was obtained when the hybrid 1-cell zygotes were cultured in mR1ECM medium and transferred into the 2-days earlier uteri of SD x DA recipients. These results suggest that genetic background of recipients as well as donors is a possible factor affecting full-term development of rat morulae/blastocysts derived from 1-cell stage zygotes cultured in vitro.     

Effect of group culture and embryo-culture conditioned medium on development of bovine embryos

We investigated the effect of group culture on bovine embryo development, and also investigated the effect of embryo-culture conditioned medium on developmental competence of individually cultured bovine embryos. Slaughterhouse-derived bovine oocytes were matured and fertilized in vitro. The presumptive zygotes were cultured individually or cultured in groups of 2 to 5 embryos with a constant culture density (5 mul/embryo). After 7 days of culture, the rates of embryos developed to the blastocyst stage were significantly higher (P < 0.05) in group cultures of more than 3 embryos/drop than for embryo culture of 1 or 2 embryos/drop. These results suggest a beneficial effect of group culture may be exerted by possible growth promoting factors secreted by embryos. In the next experiment, we investigated the effect of timing of fresh medium replacement on the development of embryos cultured in groups. The blastocyst formation rate was lower when culture medium was replaced freshly on days 2-4 after fertilization than on days 5-6. The blastocyst formation rates of single-cultured embryos were significantly (p < 0.05) increased by the addition of conditioned medium derived from multiple-embryo culture. These results indicate that group culture promotes embryo development and that embryo culture-derived conditioned medium is effective for supporting development of single cultured embryos.     

The blastocyst production rate and incidence of chromosomal abnormalities by developmental stage in in vitro produced porcine embryos

The present study was conducted to determine the relationship between embryonic development speed at different stages (the cleaved stage at 52 h and the blastocyst stage at 6 days post insemination) and incidences of chromosome abnormalities in in vitro produced porcine embryos. Porcine oocytes were collected from 3-6-mm ovarian follicles obtained at a slaughterhouse and matured in modified NCSU-37 medium for 44-46 h. Following in vitro fertilization with a final concentration of 1 x 10(5) sperm/ml for 3 h, all oocytes were cultured in vitro for 52 h. Day-2 (52 h after insemination) embryos were classified according to their cleaved stages into 2-cell, 3- to 4-cell, 5- to 8-cell, and >8-cell stages; these were cultured separately for additional 4 days (Day 6). The resultant Day-6 blastocysts were classified according to the morphological diameter into 3 grades: Grade A, expanded blastocysts; Grade B, expanding blastocysts; and Grade C, early blastocysts. They were then analyzed chromosomally. The 3- to 4-cell and 5- to 8-cell embryos had significantly high blastocyst development rates (46.1 and 36.9%, respectively), and these blastocysts contained significantly more cells (40.2 and 42.4 cells, respectively) than those derived from 2-cell embryos and >8-cell embryos (28.6 and 26.5 cells, respectively). The incidence of chromosomal abnormalities was significantly higher in the blastocysts derived from 2-cell and >8-cell stage embryos than in the blastocysts derived from the other stage embryos. Furthermore, the grade A blastocysts had the lowest incidence of chromosomal abnormalities (35.3%) and contained the most cells (48.7 cells). Porcine in vitro production (IVP) yielded a high blastocyst rate and an excellent embryo quality when 3- to 4-cell and 5- to 8-cell stage embryos were selected on Day 2 after insemination. The same criteria yielded a higher quality of expanded blastocysts based on the stage of embryo development and morphology.     

Reduced oxygen tension and EDTA improve bovine zygote development in a chemically defined medium

Bovine zygotes produced by in vitro oocyte maturation and fertilization were cultured for 7.5 d in a chemically defined medium without serum or proteins, except .12 IU/mL of insulin. In Exp. 1, embryos were cultured in approximately 20% oxygen (i.e., 5% CO2 in air) or 5% CO2; 5% O2; 90% N2, with the metal chelators EDTA or diethylenetetraaminopentaacetic acid (DTPA) at 0, 5, 25, or 125 microM. More (P < .01) embryos developed to blastocysts at 5% O2 (17%) than at -20% O2 (7%). Also, embryos grown at 5% O2 averaged more cells than embryos cultured at -20% O2 (38 vs 29 cells for morulae and blastocysts and 15 vs 12 cells including all embryos; P < .05). There were interactions (P < .01) among chelator, concentration of chelator, and oxygen tension. The most efficacious treatments were 5 microM EDTA at 5 or -20% O2 (24 and 20% blastocysts), 5 microM DTPA at 5% O2 (28% blastocysts), and 25 microM EDTA at 5% O2 (25% blastocysts). High concentrations of either chelator were detrimental, especially at -20% O2. In Exp. 2, a smaller range of chelator concentrations was compared (EDTA: 3, 9, 27, or 81 microM, DTPA: 3 or 15 microM) in 5% O2. More embryos developed to blastocysts and expanded blastocysts with 3 microM EDTA than the control without a chelator (20 and 16% vs 7 and 3%, respectively; P < .05). However, in Exp. 3, which concerned embryo development in .33, 1, 3, or 27 microM EDTA and .33, 1, or 3 microM DTPA, no concentration of either chelator was better (P > . 1) than the control.     

活性氧(ROS)对小鼠早期胚胎胚细胞分裂的影响

在体外CZB培养的不同时期添加过氧化氢(2.1μmol/L),发育至囊胚阶段,制作胚胎标本,观察其囊胚发育率、胚细胞数和分裂相数。结果显示:0~12 h组和0~72 h组的胚胎发育率显著低于其他几组(P<0.05),对照组、12~24 h组、24~36 h组、36~48 h组之间均差异不显著(P>0.05);各组之间的胚细胞数和分裂指数均无显著差异。说明胚胎在体外培养时,2细胞期胚胎对外源性H2O2最敏感,也最容易发生发育阻滞。但是经H2O2处理之后,度过2细胞期发育阻滞的胚胎能在以后的发育中发生补偿性生长,其胚细胞数和分裂指数都和体外正常发育的胚胎没有差异。     

猪植入前胚胎体外培养条件的优化

探讨了更换胚胎培养液及添加FBS、高渗透压和不同浓度VE对猪卵母细胞体外受精(IVF)和孤雌激活(PA)胚胎体外发育的影响,进一步优化了猪植入前胚胎体外培养体系。试验一：在第2天、第4天更换新的培养液(换液组),在换液基础上第4天更换为添加10%FBS的培养液(FBS组)。试验二：胚胎分别在0.05 mol/L蔗糖(蔗糖组)和138 mmol/L氯化钠(氯化钠组)的PZM-3(300～320 mOsmol)中培养2 d后移至PZM-3(288 mOsmol)中培养5 d。试验三：在培养液中分别添加50、100和200 μmol/L VE。对照组均在PZM-3(288 mOsmol)中培养7 d。结果表明：试验一,IVF和PA胚胎FBS组囊胚率显著高于对照组和换液组(P<0.05);试验二,IVF胚胎氯化钠组卵裂率、囊胚率均显著高于对照组与蔗糖组(P<0.05);试验三,IVF胚胎添加100 μmol/L VE组囊胚率显著高于对照组(P<0.05)。结果提示,在换液的基础上添加FBS有利于猪IVF和PA胚胎的体外发育;氯化钠调节的高渗透压可以促进猪IVF胚胎的早期发育;添加100 μmol/L VE可以改善猪IVF胚胎的体外发育体系。     

Development of pig embryos after electro-activation and in vitro fertilization in PZM-3 or PZM supplemented with fetal bovine serum

The aim of present study was to optimize culture conditions for pig embryos. Initially, we evaluated three different basic culture conditions. When embryos from electro-activation (parthenotes) or in vitro fertilization (IVF-embryos) were cultured in PZM supplemented with 3 mg/ml bovine serum albumin (PZM-3) in 4-well dishes, in medium covered with oil in 4-well dishes or in droplets under oil, 0%, 33% and 20% of the parthenotes, and 11%, 23% and 20% of the IVF-embryos developed to blastocysts. Subsequently, we examined the development of embryos when they were cultured in 4-well dishes in medium covered with oil continuously for 7 days or cultured under the same conditions but with a change to fresh medium on Days 2 and 4. In this experiment, 23% (no medium change) and 34% (change) of the parthenotes developed to blastocysts, respectively. When IVF-embryos were cultured under similar conditions, 33% and 38% of the embryos developed to blastocysts. Further improvement was achieved when PZM was supplemented with FBS from Day 4. In this experiment, 47% of the parthenotes developed to blastocysts with an average cell number of 57 +/- 7.7. In IVF-embryo group, 49% of the embryos developed to blastocysts with a mean cell number of 60 +/- 6.1. These results indicate that a change to fresh medium and inclusion of FBS in the medium during the late stages of culture can generate a higher proportion of high-quality blastocysts.     

The use of human and bovine commercial media for oocyte maturation and embryo development in the domestic cat (Felis catus)

The aim of this study was to examine the suitability of commercial media designed for humans and cattle for oocyte maturation and embryo culture in the domestic cat. In Exp. I, feline oocytes collected ex vivo were subjected to in vitro maturation in a laboratory‐made culture medium (based on M199) or a commercial medium designed for cattle cells (BO‐IVM^®). In Exp. II, ICSI‐derived feline embryos were cultured for 7 days in a commercial human (Continuous Single Culture^®) or bovine (BO‐EC^®) cell medium. The rates of cleavage, morula and blastocyst formation were evaluated at 24 hr, 6 days and 7 days after ICSI, respectively, and compared between experimental groups. At the end of culture, embryos were assessed for viability and apoptotic changes. In Exp. I, no statistically significant difference in oocyte maturation outcome between laboratory‐made (52.7%) and commercial media (58.9%) was observed. However, the use of a commercial medium prepared for use with bovine cells resulted in a significantly lower variance of the maturation rate. In Exp. II, no statistically significant differences between two commercial media were observed for cleavage (67.5% and 64.5%), morula (39.3% and 47.1%) and blastocyst rates (25.0% and 19.6%), as well as for the percentage of late apoptotic blastomeres. Morulae cultured in medium marketed for humans exhibited significantly more early apoptotic (43.2 ± 31.2% vs. 23.4 ± 23.2%) and necrotic (60.6 ± 47.6% vs. 29.4 ± 22.6%) blastomeres. In conclusion, both commercial media tested are suitable for in vitro oocyte maturation and embryo culture procedures in cats. It is remarkable that a culture medium designed for use in cattle for in vitro maturation of cat oocytes provides more reproducible results.     

牛体外受精胚胎一步脱防冻剂冷冻方法的研究

为了研究适合于牛体外受精胚胎的一步除防冻剂的冷冻方法，特进行两个试验。试验１分别用１．５Ｍ甘油＋０．２５Ｍ蔗糖、１．５Ｍ乙二醇和１．５Ｍ丙二醇作防冻剂冷冻体外受精后第７天，已发育至囊胚阶段的牛胚胎。使胚胎降温至－７℃后，植冰，然后以０．３℃／分的速率降温至－３０℃，立即将胚胎投入液氮中冷冻保存。在３７℃水中解冻胚胎后，使其直接在含１５％胎犊血清（ＦＣＳ）的磷酸缓冲液（ＰＢＳ）中脱去防冻剂。经体外培养７２ｈ后，３组胚胎的孵化率分别为７７．２７％（１０２／１３２）、７３．２４％（１０４／１４２）、４７．９０％（５７／１１９），第１、２组的孵化率极显著高于第３组（Ｐ＜０．０１），说明用１．５Ｍ丙二醇溶液冷冻的胚胎，在解冻后不宜直接在ＰＢＳ中脱防冻剂。试验２比较用不同浓度（１．０Ｍ０１．５Ｍ，２．０Ｍ）的乙二醇和不同投液氮温度（－２５℃，－３０℃，－３５℃）冷冻胚胎的效果。结果表明乙二醇浓度和投液氮温度对胚胎孵化率均无显著影响（Ｐ＞０．０５），各组胚胎的孵化率变动于７４．４４％－８５．４８％之间。     

Effects of Retinol on the in vitro Development of Bos Indicus Embryos to Blastocysts in Two Different Culture Systems

The objective of this study was to evaluate the effect of retinol on the in vitro development of early embryos of cultured Bos indicus (Expt 1) to the blastocyst stage in medium simplex of optimization (KSOM) or sintetic fluid of oviduct (SOF) or co-cultured (Expt 2) with an oviduct cell monolayer (OCM) in KSOM or SOF. A total of 3149 cumulus-oocyte complexes obtained by aspirating follicles (2-5 mm diameter) from ovaries of slaughtered animals were selected for IVM and incubated in TCM 199 supplemented with 25 mM HEPES at 39 degrees C in air with 5% CO(2) and maximum humidity for 24 h. In vitro fertilization (IVF) was performed in modified defined medium (mDM) medium. Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes were randomly allocated to the experimental groups. Zygotes cultured (Expt 1) in KSOM + retinol, KSOM, SOF + retinol and SOF were incubated in maximum humidity at 39 degrees C, 5% CO(2), 5% O(2) and 90% N(2). Zygotes co-cultured (Expt 2) in KSOM + retinol + OCM, KSOM + OCM, SOF + retinol + OCM and SOF + OCM were incubated at 39 degrees C, 5% CO(2). In both experiments media were partially changed 48 h after IVF and unfertilized ova were removed. Afterwards embryos were kept in culture or co-culture for further 9 days. In Expt 1, blastocyst rates (day 7) were 14.6% (KSOM + retinol), 15.8% (KSOM), 16.4% (SOF + retinol) and 15.9% (SOF). In Expt 2, the blastocyst rates (day 7) were 25.4% (KSOM + retinol + OCM) 14.2% (KSOM + OCM), 24.3% (SOF + retinol + OCM) and 15.9% (SOF + OCM). The same influence profile of retinol was observed in the formation of the expanded (day 9) and hatched (day 11) blastocysts. The results obtained in Expt 2 demonstrated that the addition of 0.28 microg/ml retinol to the embryo culture media used in this study had a significant (p < 0.05) positive effect on bovine early embryonic development, under the conditions tested, and can be used to enhance in vitro embryo production.     

Fertilization and blastocyst development in oocytes obtained from prepubertal and adult pigs

Polyspermic fertilization and embryo quality are important issues for the in vitro production of pig embryos. We hypothesized that oocyte donor (prepubertal gilt vs. sow) affects polyspermy and blastocyst development in vitro and that the sexual maturity of the oocyte donor affects the response to sperm concentration in the fertilization medium. In Exp. 1, oocytes of sows and gilts were mounted and stained 12 h after insemination to provide fertilization data. In Exp. 2, putative embryos were developed in vitro to 144 h post-insemination before mounting. In both experiments, cumulus-oocyte complexes (COC) were collected from ovaries of prepubertal gilts and adult sows. Sperm were added after maturation of COC for 40 to 44 h. Sperm from two boars at 0.5 to 5.0 x 10(6) sperm/mL was used for insemination. More (P < 0.01) monospermic fertilizations were observed in oocytes derived from gilts than for oocytes from sows. There were fewer (P < 0.02) penetrated sperm per fertilized oocyte in oocytes from gilts compared with sows. There were effects of semen donor (boar) on the percentage of monospermic (P < 0.01) and polyspermic (P < 0.002) fertilizations, and on the number of penetrated sperm/fertilized oocyte (P < 0.02). In Exp. 2, cleavage and blastocyst formation was evaluated at 2 and 6 d postinsemination, respectively. More (P < 0.001) blastocysts developed from sow-derived oocytes than from gilt-derived oocytes. More (P < 0.05) total cells per blastocyst were observed in embryos from sow-derived oocytes than from gilt-derived oocytes. Semen donor affected the percentage of oocytes cleaving (P < 0.02), and a boar x sperm concentration interaction affected (P < 0.05) the incidence of blastocyt formation. Results indicate that sexual maturity of the donor is not responsible for the high incidence of polyspermy in porcine in vitro fertilization. However, blastocyst development is improved by the use of oocytes from sows rather than from prepubertal gilts.     

Effect of medium change on the development of in vitro matured and fertilized bovine oocytes cultured in medium containing amino acids

Bovine in vitro matured and fertilized oocytes were cultured for 153 hr in groups of 3 or 30 in 30 microl of modified synthetic oviduct fluid medium supplemented with amino acids. The concentration of ammonium in culture medium at 153 hr of culture was significantly decreased by medium change at 72 hr of culture. However, regardless of embryo density, medium change had no beneficial or detrimental effect on the development of bovine embryos. Increase in the development to blastocysts and production of ammonium were observed when embryos were cultured in groups of 30. These results indicated that the ammonium concentration detected in this culture system has a negligible effect on the development of bovine embryos to blastocysts.     

Effect of potassium simplex optimization medium and NCSU23 supplemented with beta-mercaptoethanol and amino acids of in vitro fertilized porcine embryos

The present study was conducted to examine the comparative efficacy of potassium simplex optimization medium (KSOM) and North Carolina State University (NCSU)-23 medium supplemented with beta-mercaptoethanol (beta-ME) and amino acids (AA) on the developmental competence of porcine in vitro fertilized (IVF) embryos. Four experiments were conducted. KSOM and NCSU-23 medium were used to culture porcine parthenogenetic (Exp. 1) and IVF (Exp. 2) embryos. KSOM and NCSU-23 were equally effective in supporting porcine parthenogenetic and IVF embryo development from the 1-cell stage to blastocysts. The NCSU-23 medium (Exp. 3) and KSOM (Exp. 4) were supplemented with amino acid (AA; 5 microl/ml non-essential amino acids + 10 microl/ml essential amino acids) and/or 10 microM beta-mercaptoethanol (beta-ME). The quality of blastocysts from Exp. 3 and 4 was evaluated by counting the number of total cells and determining the ratio of the inner cell mass (ICM) to trophoectoderm (TE) cells. Supplementing with AA and beta-ME or beta-ME alone in NCSU-23 produced significant (p<0.05) differences in terms of rate of cleavage to the 2- to 4- cell (80.8 to 85.4% vs. 73.6%) and blastocyst (30.4 to 30.5 vs. 23.5%) stages and the number of TE (51.4 to 53.8 vs. 35.8) and total cells (67.2 to 71.2 to 48.8) over the control group. On the other hand, supplementing KSOM with AA and/or beta-ME produced significant (p<0.05) differences in terms of rate of cleavage to the 2- to 4-cell (78.8% vs. 67.7%) and morula (57.8% vs. 46.3%) stages and the number of ICM (18.6 to 19.2 vs. 11.6) and total cells (62.8 to 70.6 vs. 42.8) over control group. In conclusion, our study demonstrates that both KSOM and NCSU-23 medium supplemented with AA and beta-ME and/or only beta-ME alone are superior to normal KSOM and NCSU-23 for porcine IVF embryo culture in terms of embryo developmental competence and quality.