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 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.     

Pig blastocyst development in vitro is affected by amino acids

Pig embryos were removed 5 d after onset of estrus and cultured in modified Krebs-Ringer bicarbonate (mKRB) medium containing bovine serum albumin (BSA; 1 mg/ml). Media in Exp. 1 and 2, but not in Exp. 3, contained 10% heat-inactivated lamb serum (LS). In Exp. 1, embryos were cultured in 1) mKRB + LS or mKRB + LS supplemented with 2) glutamine (2 mM); 3) phenylalanine (.1 mM), methionine (.05 mM) and isoleucine (.2 mM); or 4) phenylalanine, methionine, isoleucine and glutamine. Embryos cultured in media with supplemental phenylalanine, methionine and isoleucine attained larger (P less than .05) volumes during culture and initiated hatching at an increased (P less than .05) frequency compared with embryos cultured without additional amino acids. In contrast, adding glutamine depressed (P less than .05) the maximum volumes observed during culture. In Exp. 2 mKRB + LS was compared with mKRB + LS plus phenylalanine (.1 mM), methionine (.05 mM) and isoleucine (.2 mM); methionine and isoleucine; phenylalanine and isoleucine, or phenylalanine and methionine. Embryos cultured in media supplemented with phenylalanine and methionine attained larger (P less than .05) volumes than embryos cultured in either media without added methionine. Fewer (P less than .05) embryos cultured without methionine initiated hatching (56%) compared with embryos provided methionine (89%). In Exp. 3, we evaluated the addition of glutamine (1 mM) with or without phenylalanine (.1 mM), methionine (.05 mM) and isoleucine (.2 mM) to serum-free mKRB. Adding glutamine alone, but not in combination, increased (P less than .05) blastocyst volumes on d 3 and maximum volume attained during culture compared with embryos cultured in mKRB alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

生物频谱对家兔胚胎发育影响的初步研究

本文首次研究了周林生物频谱照射对家兔胚胎体外发育的影响，初步探讨了周林生物频谱在胚胎工程中应用的可能性。周林生物频谱分别照射家兔２－细胞胚胎３、５、７、１０、１５、２０、２５分钟，结果照射１０、１５、２０、２５分钟，可使７２小时（从注射ＨＣＧ后算起，即照射后４８小时）桑椹胚发育率显著增加，照射２０和２５分钟可提高７２、８４、９６和１０８小时囊胚发育率，照射１０分钟和１５分钟可提高１０８小时囊胚发育率，照射７、１０、１５、２０和２５分钟可提高９６小时扩张囊胚发育率及１０８小时孵化囊胚发育率。周林生物频谱照射家兔１６－细胞胚胎２０分钟，其６０小时（从注射ＨＣＧ后起，即照射后１２小时）致密桑椹胚发育率与对照比较差异极显著（Ｐ＜０．０１），囊胚、扩张囊胚和孵化囊胚发育率与对照比较无显著差异（Ｐ＞０．０５）     

Suppressed development of cultured mouse and swine embryos by diethylstilbestrol

Effects of prolonged exposure to the synthetic estrogen diethylstilbestrol (DES) on in vitro development of early mouse and swine embryos were investigated. Two-cell mouse embryos cultured in Whitten's medium (WM) for 192 h were exposed to 10(-4), 10(-7) or 10(-10) M DES dissolved in 1, 10(-3) or 10(-6)% ethanol, respectively. One-cell to eight-cell swine embryos were cultured in WM for 192 h containing 10(-4) or 10(-7) M DES dissolved in 1 and 10(-3)% ethanol, respectively. Embryos cultured in WM containing 1 (0 DES1), 10(-3) (0 DES2) or 10(-6)% ethanol (0 DES3) served as controls. Hatching was inhibited (P less than .05) in mouse embryos cultured in 10(-4) M DES (3.0 +/- 2.1% vs 0 DES1, 25.1 +/- 3.7%). Similar (P greater than .10) percentages of mouse embryos hatched in 10(-7) M DES (36.4 +/- 5.4% vs 0 DES2, 29.1 +/- 5.7%) and 10(-10) M DES (44.4 +/- 4.4% vs 0 DES3, 38.9 +/- 5.3%). Diethylstilbestrol at a concentration of 10(-4) M failed to affect the development of one- to eight-cell swine embryos into blastocysts. However, compared with 0 DES2, 10(-7) M DES reduced (P less than .05) the number of swine blastocysts developing from one- to two-cell (36 vs 78%) and three- to four-cell embryos (50 vs 84%). No significant effects of 10(-7) M DES were detected on the ability of six- to eight-cell swine embryos to develop into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

A Low Oxygen Atmosphere during IVF Accelerates the Kinetic of Formation of In Vitro Produced Ovine Blastocysts

Among the factors that affect in vitro embryo development, oxygen atmosphere is considered to be of great influence. In this study, we evaluated the influence of two different oxygen atmospheres during in vitro fertilization (IVF) of ovine oocytes on their developmental capacity and quality assessed by cryotolerance. Cumulus oocyte complexes derived from ovaries of slaughtered sheep were matured in vitro and subsequently fertilized under low (5%) or high (20%) oxygen atmospheres, and cultured in SOF + aa + 0.4% BSA in 5% CO2 and 5% O2 up to blastocyst stage. The cleavage rates obtained in the fertilization system at 20% O2 were significantly higher than those obtained in the 5% O2 fertilization system (61.2% vs 50.8%; p < 0.01). The distribution of cleaved oocytes at 22, 26 and 40 h of culture intervals was not different in the low or high O2 atmosphere (31.4%, 26.4% and 42.1% vs 28.0%, 29.3% and 42.7% respectively). Blastocysts output on the 6th day post-fertilization (dpf) was significantly higher when oocytes were fertilized under 5% O2 concentration (63.04% in 5% O2 vs 47.36% in 20% O2), while on the 7th dpf the higher number of blastocysts was obtained in the 20% O2 system (35.10%.in 20% O2 vs 26.09% in 5% O2). After vitrification no differences were observed between low or high oxygen atmosphere in the viability rates of blastocysts obtained on day 6 (93.6% vs 96.5%), on day 7 (46.3% vs 41.7%) and on day 8 (11.1% vs 6.6%). After differential staining, no significant differences were observed in the total cell number and inner cell mass and trophoblastic cells ratio of blastocysts produced on 6 dpf (189.6 +/- 51.3 and 0.260 +/- 0.07 vs 223.3 +/- 45.6 and 0.277 +/- 0.09), on 7 dpf (168.3 +/- 25.1 and 0.316 +/- 0.06 vs 172.1 +/- 33,6 and 0.320 +/- 0.06) and on 8 dpf (121.2 +/- 23,8 and 0.302 +/- 0.03 vs 117.0 +/- 35.1 and 0.313 +/- 0.04) under low or high oxygen atmosphere respectively). In conclusion, our data suggest that low oxygen atmosphere during IVF affects positively the production of high quality ovine blastocysts.     

The Effects of Blastomere Biopsy and Oxygen Tension on Bovine Embryo Development, Rate of Apoptosis and Interferon-τ Secretion

A series of experiments was performed to examine the effects of blastomere biopsies on subsequent development of IVF-derived bovine embryos. The first experiment was designed to assess the optimal time for blastomere removal. One blastomere was removed either 48 or 72 h after IVF. Biopsy at 48 h resulted in 17.2% of embryos proceeding to the blastocyst stage, which was lower than when biopsies were performed at 72 h (37.5%, p < 0.05). In the second experiment, embryos were cultured either under atmospheric or 5% O(2) following blastomere removal. Biopsies had no effect on rate of blastocyst formation with 36% of controls and 33.7% of biopsied embryos proceeding to that stage. However, culture under 5% O(2) significantly increased the number of blastocysts from 29.9% to 40.3% (p < 0.05). This effect was significant in both biopsied and control embryos. In the final experiment, biopsied embryos were again cultured under different oxygen tension. Blastocysts were collected and cultured individually for 48 h in medium droplets in their respective O(2) concentration after which time the medium was assayed for concentration of interferon-tau (IFN-tau). Reduced O(2) concentration again significantly increased blastocyst formation from 24.9% to 41.9% (p < 0.05). IFN-tau secretion was not affected by biopsies, but culture under atmospheric O(2) resulted in significantly increased IFN-tau concentration in medium droplets (12274.0 +/- 2825.9 pM vs 5046.5 +/- 2562.2 pM; p < 0.05).     

小鼠2-细胞胚胎细管法和OPS法玻璃化冷冻保存技术的研究

本试验在室温 (2 0℃和 2 5℃ )条件下 ,利用不同浓度的玻璃化溶液 (EFS和EDFS) ,对小鼠 2 细胞胚胎进行细管法和OPS法玻璃化冷冻保存。在 2 0℃室温条件下 ,用EFS4 0平衡 1min细管一步法冷冻 ,解冻后囊胚发育率仅为35 .0 % ,和新鲜 2 细胞体外培养的对照组 (6 5 .0 % )的差异极显著 (P <0 .0 1)。当 2 细胞胚胎在 10 %EG +10 %D溶液中预处理 5min ,再移入EDFS中平衡 30s二步法冷冻保存 ,解冻后囊胚发育率达 4 7.8%～ 4 8.8% ;当室温升至2 5℃时 ,二步法冷冻保存后 2 细胞的囊胚发育率达到 5 2 .2 % ,与对照组无显著差异 (P >0 .0 5 )。改用OPS二步法EFS30冷冻组保存后的 2 细胞胚胎的囊胚发育率高达 6 2 .2 % ,为试验中的最佳组。用最佳细管法和OPS法冷冻组解冻后培养至囊胚移植给受体母鼠均获得产仔     

Improved survival of vitrified in vivo-derived porcine embryos

An efficient cryopreservation protocol for porcine morulae was investigated with three types of vitrification having different cooling rates (Exp. 1). Survival of embryos vitrified after removal of cytoplasmic lipid droplets was also examined by means of the minimum volume cooling (MVC) method (Exp. 2). In Exp. 1, the morula stage embryos were vitrified with a 0.25 ml plastic straw (ST-method), gel loading tip (GLT-method) and the MVC-method, respectively, and stored in liquid nitrogen after which they were warmed in sucrose solutions with cryoprotectants being subsequently removed in a stepwise manner. In Exp. 2, morulae were centrifuged with 7.5 microg/ml cytocharasin B at 12000 x g for 20 min to polarize the cytoplasmic lipid droplets that were then removed from the embryos by micromanipulation (delipation). Both those delipated at the morula stage and the intact embryos at the morula to blastocyst stages were vitrified by the MVC-method. In vitro survival of the vitrified embryos was assessed in both experiments by culturing in NCSU-23 + 10% FCS for 48 h. In vitro developments of vitrified embryos after warming to blastocysts were 20% (6/30) for the ST-method, 39% (18/46) for the GLT-method, and 60% (26/43) for the MVC-method. Embryo survival was further improved by vitrification after delipation (95%, 35/37) compared to intact vitrified morulae (24/42, 57%, P<0.001) and blastocysts (23/31, 74%, P<0.05). Moreover, the number of cells in blastocysts (92 +/- 25) derived from the delipated-vitrified morulae was comparable to those derived from intact control non-vitrified embryos (103 +/- 31). Our results demonstrate that vitrified porcine morulae have the highest survival when using the MVC-method in conjunction with delipation.     

Cryopreservation of bovine somatic cell nuclear-transferred blastocysts: effect of developmental stage

The effect of developmental stage on the survival of bovine somatic cell nuclear-transferred blastocysts after freezing and thawing was evaluated. We also investigated how freezing affects nuclear-transferred (NT) embryos and in vitro fertilized (IVF) bovine embryos. Advanced-stage bovine NT blastocysts survived freezing better than early-stage NT blastocysts (86 vs 14%). The trend was similar with IVF embryos (87 vs 30%). At the stages tested, there was no significant difference in the survivability of NT and IVF embryos from advanced (86 vs 87%) or early-stage blastocysts (14 vs 30%). The average survival rate did not differ between NT and IVF bovine embryos (50 vs 51%). The higher survival rate of advanced-stage blastocysts compared to early-stage blastocysts in NT and IVF bovine embryos might be due to their higher cell number. In NT (128 +/- 25 vs 53 +/- 20) and IVF (128 +/- 29 vs 75 +/- 22) groups, advanced-stage blastocysts contained a significantly higher total cell number than early-stage blastocysts. There was no difference in total cell number between advanced-stage NT and IVF blastocysts (128 +/- 25 vs 128 +/- 29), however, early-stage NT and IVF blastocysts (53 +/- 20 vs 75 +/- 22) differed significantly.     

Effects of delipidation and oxygen concentration on in vitro development of porcine embryos

The effects of delipidation and the oxygen (O(2)) concentration in the atmosphere during culture on in vitro development and H(2)O(2) content were investigated in porcine in vivo fertilized embryos and embryos after in vitro maturation and in vitro fertilization (IVM/IVF embryos). There was no significant difference in the developmental rates to the blastocyst stage between the intact and delipidated IVM/IVF embryos. However, the mean number of cells in blastocysts derived from delipidated IVM/IVF embryos (19.8 +/- 0.8 cells) was significantly smaller than that from intact embryos (24.2 +/- 1.2 cells). Although there were no significant differences in the developmental rates to the blastocyst stage of intact and delipidated IVM/IVF embryos between the cultures under 5% O(2) and 20% O(2), the developmental rate of intact IVM/IVF embryos cultured under 5% O(2) (27.1%) was significantly higher than that of the delipidated embryos cultured under 20% O(2) (19.3%). On the other hand, there was no difference in the developmental rate to the blastocyst stage between in vivo fertilized embryos cultured under 5% O(2) and 20% O(2). Hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species (ROS), is thought to cause damage to embryos. The H(2)O(2) content per embryo derived from oocytes cultured under 5% O(2) (in vivo fertilized, 58.0 +/- 2.5 pixels; IVM/IVF, 79.6 +/- 3.2 pixels) was significantly lower than that (in vivo fertilized, 100.2 +/- 3.8 pixels; IVM/IVF, 103.9 +/- 3.2 pixels) under 20% O(2). Furthermore, the level of H(2)O(2) in delipidated IVM/IVF embryos (94.7 +/- 3.9 pixels) was significantly lower than that in intact embryos (103.9 +/- 3.2 pixels) cultured under 20% O(2). The present results indicate that the delipidation of porcine IVM/IVF embryos and reduction of the O(2) concentration decreased the H(2)O(2) level rather than the in vitro developmental rate to the blastocyst stage.     

In Vitro and in Vivo Developmental Competence of Dromedary (Camelus dromedarius) Embryos Produced in Vitro Using Two Culture Systems (mKSOMaa and Oviductal Cells)

Development competence and pregnancy rate of in vitro-produced (IVP) dromedary embryos were studied in two culture systems: (i) semi-defined modified medium (mKSOMaa) and (ii) co-culture using camel epithelial oviducal cells. Five hundred and three cumulus-oocytes complexes (COCs) were selected, allowed to mature, fertilized and cultured in vitro (38.5 degrees C; 5% CO2, maximum humidity > 95%, with concentration of oxygen of 5% for semi-defined medium and 20% for co-culture cells). Maturation was accomplished by incubation in TCM-199 medium supplemented with 10% heat-treated foetal calf serum (FCS), 10 ng/ml epidermal growth factor, 1 microg/ml follicle-stimulating hormone, 1 microg/ml oestradiol and 500 microM cysteamine for 30 h. In vitro fertilization (IVF) was performed using fresh semen (0.5 x 10(6) spermatozoa/ml in modified TALP solution). Fertilized COCs were denuded by vortexing, then cultured in either mKSOMaa (10% heat-treated FCS was added 24 h post-IVF), under 5% O2 and 90% N2 (group 1; n = 249) or with dromedary epithelial oviducal cell monolayers in TCM-199 with 10% heat-treated FCS under 20% O2 (group 2; n = 254). The rate of cleavage was significant higher (p < 0.05) for group 1 (63%, 156/249) than for group 2 (51%, 130/254). No significant difference was found between the two groups in the rate of development to blastocyst (21% vs 16.5%) and their hatchability (21% vs 14%). Pregnancy rates were similar for the first 60 days. However, all pregnancies were lost after 60 days with the exception of two of six (33%) from recipients of hatched blastocysts from group 1. We conclude that both systems support in vitro production of dromedary embryos by in vitro maturation (IVM)/IVF of oocytes. However, embryos obtained by culture in the semi-defined medium (mKSOMaa) appear to have a better in vivo development ability.     

Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos

This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.     

Comparison of in vitro development of embryos collected from the same gilts at first and third estrus

In vitro development of embryos collected from the same gilts mated at first and third estrus was compared. Embryos from one to eight cells were collected from gilts 36 to 48 h after detection of estrus. Embryos were cultured for 8 d in Whitten's medium in a humidified atmosphere of 5% CO2 in air at 37 degrees C and were observed daily. No differences were detected among percentages of one- to eight-cell embryos developing into morulae from gilts in first or third estrus (P greater than .05). Similar percentages of one- to two-cell embryos from gilts mated at first and third estrus developed into blastocysts (45.8 and 55.2%, respectively), expanded blastocysts (10.4 and 24.1%, respectively) and hatching blastocysts (4.2 and 3.4%, respectively; P greater than .05). Fewer three- to eight-cell embryos from gilts in first estrus than from gilts in third estrus developed into blastocysts (63.4 and 91.1%) and expanded blastocysts (14.6 and 55.6%; P less than .01). Similar percentages of embryos with abnormal morphology were observed among morulae developing from one- to eight-cell embryos collected from gilts mated at first and third estrus (14.9 and 9.9%, respectively; P greater than .05). In contrast, more morphologically abnormal embryos were observed among blastocysts developing from gilts mated at first estrus than at third estrus (31.2% and 14.0%, respectively; P less than .05). The results suggest that the reduced in vitro development of embryos collected from gilts mated at first estrus may be due to an aberration in blastocoel formation and expansion.     

Toxicity potential of absorbed-retained ethylene oxide residues in culture dishes on embryo development in vitro

Four hundred eight-cell mouse embryos were cultured in vitro in either polystyrene (plastic) dishes (Exp. 1) or watch glasses (Exp. 2) to analyze the toxicity potential of absorbed-retained ethylene oxide (EtO). Culture dishes were gas-sterilized with Anprolene equipment and allowed to aerate (21 C) for varying durations. Post-sterilization EtO residues, as determined by weight measurement, were eluted from polystyrene dishes at an exponential rate. After 1 wk of aeration, .625 mg EtO was retained/g of polystyrene product. To determine the effect of EtO residue on in vitro culture, embryos were collected from superovulated donor mice (C57BL/6N), pooled in a modified Dulbecco's phosphate buffered saline (PBI) medium and then randomly allotted to dishes subjected to various aeration durations. Embryos were cultured in Whitten's medium +3 mg/ml bovine serum albumin (BSA) under a humidified 5% CO2 in air atmosphere at 37 C. Gross assessments of embryo development were made using standard morphology and quality grading systems and a fluorescein diacetate staining assay. In Exp. 1, embryo development was retarded at the 8- to 16-cell stage when less than or equal to 12 h aeration of polystyrene dishes was permitted. Aeration for 24 and 36 h resulted in suboptimal embryo development with fewer (P less than .01) blastocysts and greater degeneration rates at 48 h of in vitro culture compared with the control (manufacturer-packaged dishes, no EtO) and 1-wk aeration treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parthenogenetic activation and subsequent development of porcine oocytes activated by a combined electric pulse and butyrolactone I treatment

This study was designed to evaluate the parthenogenetic activation of porcine oocytes matured in vitro for a varied period after combined electric pulse (EP; 1500 V/cm, 100 microsec) and Butyrolactone I (BL I). After 36 h of maturation culture, the rates of activated oocytes and oocytes with two pronuclei were significantly lower than those of oocytes cultured for 42 and 48 h after EP. However, when treated by a combined EP and BL I (150 microM), these rates increased to the same level as 42 and 48 h oocytes. When oocytes cultured for 48 h and activated by a combined EP and BL I treatment were subsequently cultured in mNCSU37 medium, the rates of embryos cleaved and developed to the blastocyst stage were significantly higher than those in Whitten's medium. In contrast, when activated oocytes were cultured in mNCSU37 medium under two oxygen environments (5% vs 20% O(2)), there was no difference in the rates of cleavage, blastocyst formation and nuclear numbers per blastocyst. Our results demonstrated that the combined EP and BL I treatment of porcine oocytes matured in vitro is capable of producing high rates of good quality blastocysts when cultured in a suitable in vitro condition.     

Developmental Competence of Frozen-thawed Blastocysts from Fair-quality Bovine Embryos Cultured with β-Mercaptoethanol

Two-hundred-and-thirty-one fair-quality embryos at the compacted morula stage collected from 89 superovulated cows were cultured in TCM199 or Brinster's BMOC-3 medium with or without 100 microM beta-mercaptoethanol (beta-ME). After 24 h culture, a total of 142 fair-quality embryos developed to the blastocyst stage, of which 106 were subsequently frozen with 1.8 M ethylene glycol. The mean cell number and development rates of frozen-thawed blastocysts from the fair-quality embryos cultured in TCM199 containing beta-ME were higher than those of the fair-quality embryos directly frozen without culture. The pregnancy rates obtained with frozen blastocysts from fair-quality embryos tended to be lower than those of non-cultured fresh fair-quality embryos and cultured fresh blastocysts. These results indicate that the inclusion of beta-ME in pre-freezing culture media improve the development of frozen-thawed blastocysts from fair-quality embryos, but not the pregnancy rate.     

Evaluation of Cheetah and Leopard Spermatozoa Developmental Capability after Interspecific ICSI with Domestic Cat Oocytes

The ICSI procedure is potentially of great value for felids, and it has not been extensively studied in these species. The objectives of this work were to determine the best conditions for ICSI in the domestic cat (DC) to generate interspecific embryos by injecting cheetah (Ch) and leopard (Leo) spermatozoa. Firstly, DC oocytes were matured with insulin–transferrin–selenium (ITS) or without it (MM) and cultured using atmospheric (21%) or low (5%) oxygen tension after ICSI. The group ITS‐5%O₂ showed the highest blastocyst rate (p < 0.05), 20.9% vs 8.7%, 7% and 6.5%, for MM‐21%O₂, MM‐5%O₂ and ITS‐21%O₂, respectively. The best conditions were used to generate the interspecific embryos, together with ionomycin activation (Io) after ICSI. Interspecific embryos resulted in high rates of blastocysts that were not positively affected by Io activation: 32.6% vs 21% for Ch and Ch‐Io, 9.8% vs 21% for Leo and Leo‐Io, and 20% vs 17.4% for DC and DC‐Io. We also evaluated DNA‐fragmented nuclei of experiment 1 and 2 blastocysts, using TUNEL assay. The fragmented nucleus proportion was higher in the ITS‐5%O₂ group, 67.6%. Surprisingly, interspecific blastocysts showed the lowest fragmented nucleus proportion: 27% and 29.9% for Ch and Leo, respectively. We concluded that ITS and 5%O₂ improve blastocyst formation in DC, although with a concomitant increase in DNA fragmentation. Most importantly, cheetah and leopard spermatozoa were able to generate blastocysts without artificial activation, which suggests that developmental capacity of wild felid spermatozoa can be evaluated by interspecific ICSI. This technique should be used to assist wild felid reproduction.     

水牛徒手克隆胚胎培养及冷冻条件的优化

本试验利用微滴、微穴和平板培养系统对徒手克隆(hand-made clone,HMC)重组胚进行体外培养;采用了40% EG(ethylene glycol,EG)、25% EG＋25% DMSO(dimethylsulphoxide,DMSO) 和20% EG＋20% DMSO＋0.5 mol/L蔗糖作为玻璃化冷冻液对HMC囊胚进行了超低温冷冻;并且比较了HMC与传统核移植的胚胎生产效率及囊胚冷冻存活率。结果表明,微穴系统的卵裂率要显著高于平板系统(P<0.05),极显著高于微滴系统(P<0.01);且微穴系统的囊胚率(40.0%)极显著高于平板(19.8%)和微滴系统(8.3%)(P<0.01)。采用20% EG＋20% DMSO＋0.5 mol/L蔗糖作为冷冻保护剂时HMC囊胚存活率极显著高于40% EG(P<0.01);HMC重组胚的融合率和囊胚率均高于传统核移植法(P<0.05;P<0.01),而HMC囊胚的冷冻存活率与传统核移植生产的囊胚没有显著差异。以上结果说明水牛HMC可以替代传统核移植法生产克隆胚胎,微穴体系最适合水牛HMC胚胎的体外培养,且采用20% EG＋20% DMSO＋0.5 mol/L蔗糖对HMC囊胚进行玻璃化冷冻可以取得良好的冷冻效果。