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)     

Relationship between time elapsed after human chorionic gonadotropin administration and developmental stage in porcine embryos collected from prepubertal gilts

We examined the relationship between the time elapsed after human chorionic gonadotropin (hCG) administration and developmental stage of porcine embryos after collection. Prepubertal gilts, 7 to 8 months old, were given 1500 IU equine chorionic gonadotropin (eCG) intramuscularly, followed by 500 IU hCG 72 h later. The treated gilts were inseminated artificially on Day 1 (Day 0=the day of hCG administration) and on Day 2. Embryos were collected surgically on Day 6 (140, 144, and 147 h after hCG administration) or on Day 7 (164, 168, and 171 h), and the developmental stages of the collected embryos were examined. From 75.2% (276/367) of the prepubertal gilts treated with hormones, we collected an average of 20.7 embryos per gilt with normal morphology. At 140 h after hCG administration, morulae (54.4%) could be collected. At 144 h, morulae and early blastocysts (57.7% and 28.9%, respectively) were collected. By 147 h, the proportion of embryos at the blastocyst to expanded blastocyst stages had increased (10.0%). From 164 h to 171 h, expanding or expanded blastocysts of more than 200 microm in diameter and hatched blastocysts could be collected. The proportion of hatched blastocysts increased from 3.2% (164 h) to 41.0% (171 h). These results suggests that although the number of ovulations differed among gilts, porcine embryos at the appropriate stages can be collected efficiently by controlling the time elapsed between hCG administration and embryo collection.     

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.     

Steroids and plasminogen activator concentrations in follicular fluid of gilts at first and third estrus.

An experiment was conducted to determine whether morphological and functional characteristics of follicles differed at a similar stage of pubertal (first) and third estrus in the same gilts. Nine prepubertal gilts were checked three times daily for estrus and laparotomized 6 h after detected first and third estrus. Samples of vena cava and ovarian venous blood were collected, follicle numbers and diameters were recorded, and follicular fluid (FF) was aspirated from all follicles 8 to 12 mm in diameter. Sera and(or) FF were analyzed for progesterone (P4), estradiol-17 beta (E2), testosterone (T), androstenedione (A4), 5 alpha-dihydrotestosterone (DHT), plasminogen activator (PA), and plasmin (PLM). Overall mean number of follicles > or = 8 mm in diameter did not differ between gilts at first and third estrus (P > .05) but gilts at first estrus had more follicles 4 to 8 (P < .05) and 8.1 to 10 mm in diameter (P < .01) and fewer 10.1 to 12 mm in diameter (P < .07) than at third estrus. Mean FF concentrations of E2, T, and A4 at third estrus were significantly greater than at first estrus, whereas FF concentrations of P4, DHT, PA, and PLM were similar at first and third estrus (P > .05). Mean concentrations of E2 in systemic and ovarian venous sera were also greater in gilts at third than at first estrus (both P < .05). Systemic concentrations of P4 in gilts at first and third estrus did not differ (P > .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors contributing to early embryonic mortality in gilts bred at first estrus

Gilts bred at first (n = 18) and third (n = 18) estrus were assigned in replicates of equal numbers to be slaughtered on d 3, 15 and 30 post-mating to assess fertilization rate, embryonic losses and serum concentrations of estrogen (estradiol-17 beta + estrone) and progesterone. Mean number of ovulations was lower among gilts bred at first vs third estrus (12.2 vs 14.5; P less than .05), with no difference in fertilization rate (100 vs 98%). Embryonic survival was lower (P less than .05) among gilts bred at first vs third estrus on d 15 (78.1 vs 95.4%) and 30 (66.7 vs 89.4%) of gestation. Serum estrogen (pg/ml) and progesterone (ng/ml) levels, although lower in gilts bred at first vs third estrus, were not significantly different at the three stages of gestation studied. The ratio of progesterone to estrogen in gilts bred at first estrus was higher than in those bred at third estrus on d 15 (439 +/- 71 vs 210 +/- 17) and 30 (597 +/- 106 vs 179 +/- 50), but was lower on d 3 (187 +/- 37 vs 444 +/- 123; stage of gestation X estrous period interaction, P less than .05). These data suggest that changes in the ratio of systemic levels of estrogen and progesterone may be related to early embryonic mortality in gilts bred at pubertal estrus.     

Effect of unilateral hysterectomy and ovariectomy on puberty, uterine size and embryo development in swine

Eighty crossbred gilts were assigned randomly to treatments: 1) removal of an ovary and ipsilateral uterine horn (UHO) at 130 d of age and removal of the remaining ovary and uterine horn 12 d post-puberty; 2) UHO at 130 d of age, mated and reproductive tracts recovered when slaughtered at 30 d of gestation; 3) UHO 12 d post-puberty, mated and slaughtered at 30 d of gestation and 4) unoperated controls that were mated and slaughtered at 30 d of gestation. Age of puberty was not affected by treatments. Gilts in treatment 1 had a mean ovulation rate at the pubertal estrus comparable to gilts in treatment 3. But, gilts in treatments 2 and 3 had 16% fewer (P less than .01) corpora lutea at 30 d of gestation than control gilts. Length and weight of the remaining uterine horn at 12 d post-puberty for gilts treated at 130 d of age were similar to the averages of gilts left intact. Gilts with one uterine horn had 2.2 fewer live embryos at 30 d of gestation than control gilts (P less than .01). But, the proportion of corpora lutea represented by live embryos did not differ significantly among treatments. Gilts with one uterine horn had 1.1 fewer live embryos (P less than .15) after adjustment for number of corpora lutea, less uterine space occupied by each embryo (P less than .01) and less total placental membrane per embryo (P less than .05) than control gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrus, ovulation, conception and embryo survival in confinement-managed gilts of three weight groups

Puberty, cycling, ovulation, conception and embryo survival were determined for gilts managed by a specific regimen in confinement. At weights of 70 to 80, 91 to 100 and 109 to 116 kg (groups A, B, C, respectively) gilts were (1) moved from rearing pens to pens in a separate broodstock building, (2) housed in groups of eight to 10 with unfamiliar penmates and (3) exposed to boar presence in an adjoining pen. Groups A and B were mated at third estrus; group C was mated at first estrus. For the three groups 76, 79 and 79%, respectively, of allotted gilts mated; of these 21, 13 and 11%, respectively, showed estrus again and were remated. Ten, 10 and 11%, respectively, were not pregnant when slaughtered at 25 to 28 d of postmating. Average days on experiment to first estrus were 14, 15 and 16 for groups A, B and C; mean interval between estrous periods was 21 d. Ovulations (13.3, 12.9 and 12.6) and live embryos (11.6, 11.1 and 10.7, respectively) did not differ significantly among groups. None of the reproduction traits were significantly associated with differences in mean age or weight among the three groups at breeding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of dietary organic and inorganic selenium on antioxidant status, embryo development, and reproductive performance in hyperovulatory first-parity gilts

This project aimed to determine the effect of Se as inorganic Na-selenite (MSe) or organic Se-yeast (OSe) on antioxidant status, hormonal profile, reproductive performance, and embryo development in first-parity gilts. Forty-nine gilts were allocated to 1 of the 3 dietary treatments starting at first pubertal estrus and lasting up to 30 d after AI: control [CONT: basal diet (Se = 0.2 mg/kg) without added Se; n = 16], MSe (CONT + 0.3 mg/kg of MSe; n = 16), and OSe (CONT + 0.3 mg/kg of OSe; n = 17). Blood was collected from all gilts on the day after each onset of estrus and on d 30 after AI. Blood was also collected daily from d -4 to d +4 of the third onset of estrus (d 0) in 8 CONT, 9 MSe, and 8 OSe cannulated gilts. Gilts had received, after d 14 and 15 of their third estrus, a hormonal challenge to induce super-ovulation. At slaughter, embryos and corpora lutea (CL) were weighed and measured. Blood Se was less (P < 0.01) in CONT than in Se gilts and greater in OSe than in MSe (P < 0.01) from the first estrus until d 30 of gestation. At the same time, blood Se-dependent glutathione peroxidase (GSH-Px) decreased for CONT gilts, whereas it increased for both Se groups. The increase was greater in MSe than in OSe gilts (treatment × time, P = 0.02). Plasma 3,3',5-triiodothyronine and thyroxine concentrations for MSe tended to be less than for OSe gilts (P < 0.06). In cannulated gilts, plasma FSH tended to change among treatments (treatment × time, P = 0.06), and plasma estradiol-17β (E(2)) was less (P = 0.01) for MSe than for OSe. There was no treatment effect on mean litter size or embryonic antioxidant status. The Se content of individual embryos was greater for Se-treated than for CONT gilts (P = 0.03), and Se content of individual embryos and total litter was greater for OSe than for MSe gilts (P < 0.01). The length, weight, and protein content of embryos were greater in OSe than in MSe gilts (P < 0.05). There was no treatment effect on weight, length, Se content, and ferric reducing antioxidant power of CL, but GSH-Px in CL was greater for Se than for CONT gilts (P = 0.02). In summary, the Se status response of gilts to dietary Se was affected by both the quantity and the source of Se dietary supplements. Moreover, the uterine transfer of Se to embryos was improved with OSe as compared with MSe, and this was concomitant with an enhanced development of embryos.     

The influence of premating feed intake on the reproductive performance of gilts.

In an attempt to improve the reproductive performance of gilts mated at puberty, 70 Yorkshire x Landrace gilts were allocated at 120 d of age and 60 kg body weight to one of two treatments. Restricted gilts were fed 2.0 kg d-1 of a diet formulated to provide 18% crude protein and 14.5 MJ DE kg-1 from selection until mated at their first estrus (n = 35). Flushed gilts were fed 2.0 kg d-1 of the same diet from 120 to 150 d of age, but then had their feed intake increased to 3.5 kg d-1 until mated at their first estrus (n = 35). An additional group of gilts (control fed; n = 33) were fed 3.0 kg d-1 from selection until they were bred at their third estrus in order to investigate the influence of feed restriction on the onset of puberty. During gestation all gilts were fed 1.8 to 2.2 kg d-1 of a 16.8% crude protein diet having 13.7 MJ DE kg-1. Control fed gilts were younger (p less than 0.05) at puberty (150 d) than restricted (165 d) or flushed gilts (165 d). There was no difference in subsequent litter size between the restricted and flushed gilts (7.7 and 8.0, respectively). It is concluded that the institution of a flushing nutritional regime in the prepubertal period will not enhance piglet production from gilts mated at puberty.     

小鼠胚胎的玻璃化冷冻研究

用不同冷冻载体(玻璃管、塑料管和0.25 mL细管)及不同冷冻方法(程序化冷冻和玻璃化冷冻)对小鼠3.5 d~4 d桑椹胚和囊胚进行冷冻保存,并与不做任何冷冻保存处理直接培养进行对比。结果表明,使用玻璃管、塑料管和0.25 mL细管作为胚胎的承载材料进行玻璃化冷冻,效果差异不显著;采用程序化冷冻与OPS玻璃化冷冻法,对小鼠胚胎进行冷冻保存可以取得较好的结果。从而得出,用不同材质的冷冻载体进行玻璃化冷冻,可以获得与程序化冷冻相同的良好效果。     

绵羊玻璃化冷冻胚胎直接移植试验研究

应用EFS40玻璃化液对6.5～7日龄的绵羊胚胎进行玻璃化冷冻及解冻后直接移植试验.结果:桑椹胚、囊胚冷冻解冻后移植的妊娠率分别为37.50%(3/8)和54.55%(6/11),胚胎存活率分别为33.33%(3/9)和50.00%(6/12),差异均不显著(P＞0.05);胚胎解冻后用0.5 mol/L蔗糖脱防冻剂与直接用胚胎存放液脱除防冻剂的妊娠率分别为44.44%(4/9)和50.00%(5/10),胚胎存活率分别为40.00%(4/10)、45.45%(5/11)差异不显著(P＞0.05);10枚解冻后的胚胎细管内脱防冻剂后,直接装管移植给8只受体,妊娠率为50.00%(4/8),胚胎成活率为40.00%(4/10),与同期常规冷冻解冻组相比无显著差异(P＞0.05).     

Relationship between plasminogen activator production and bovine embryo development in vitro

The relationship of plasminogen activator (PA) production to cell stage, cell number and changes in overall diameter and zona pellucida thickness for bovine embryos developing in vitro was determined. Late morulae to blastocysts (n = 80) were collected nonsurgically from naturally mated, estrous-synchronized, superovulated crossbred beef cows. Embryos were cultured, one embryo per 25-microliters microdrop, for 6 d. At 24-h intervals, embryos were evaluated for stage of development and transferred to fresh microdrops; media were recovered for PA analysis. In addition, embryo diameter and zona pellucida thickness were measured with an ocular micrometer. Plasminogen activator production was determined using a caseinolytic assay with urokinase as the standard. Changes in diameter, zona pellucida thickness and PA production per 24-h interval for each embryo were plotted, and the graphs were cut out and weighed. Sixty-one embryos (76%) completed the hatching process. Total PA production was correlated positively (P less than .005) to embryonic size (r = .40), developmental stage (r = .35) and cell number (r = .35) and negatively, but weakly, correlated to zona pellucida thickness (r = -.13; P = .267). Hatched embryos produced more total PA than embryos that did not hatch (.140 +/- .011 vs .070 +/- .019 g; P less than .01). These results suggest that as embryonic size and cell number increase and development progresses, bovine embryos liberate more PA.     

The effect of confinement on days to puberty in gilts

In the late fall and winter of 1982 to 1983, 112 crossbred gilts were used in a factorially arranged experiment to determine the effect of confinement on the age at which a gilt reaches first estrus (puberty). Two environments (confinement and non-confinement) and three ages at movement to non-confinement (100, 140, and 180 d) were studied. No differences were detected (P greater than .05) between confinement and non-confinement in the proportions of gilts reaching puberty by 210 d of age. Gilts were older at puberty (P less than .05) in confinement than in non-confinement (192.0 vs 187.7 d) and had a longer interval (P less than .05) from first boar contact to first estrus (12.1 vs 7.8 d). Age at puberty (192.1 vs 187.0 vs 190.5 d) and the proportion reaching puberty (56.4 vs 45.7 vs 65.8%) were not different (P greater than .05) between age-of-movement groups. However, a higher (P less than .05) proportion of the non-confinement gilts reached puberty within 10 d after the beginning of boar exposure than confinement (44.6 vs 26.8%). Moving gilts from confinement to non-confinement (pasture) at 180 d appeared to be the most effective method tested for inducing puberty in gilts.     

Endocrine changes associated with a dietary-induced increase in ovulation rate (flushing) in gilts

Effects of an increased level of dietary energy (flushing) on plasma concentrations of FSH, LH, insulin, progesterone and estradiol-17 beta and ovulation rate were studied in 16 gilts. Gilts received 5,400 kcal ME/d for one estrous cycle and the first 7 d of a second. On d 8 of the second estrous cycle, gilts received either 5,400 kcal ME/d (control [C], n = 8) or 11,000 kcal ME/d (flushed [F], n = 8) for the remainder of the estrous cycle. Blood was collected daily at 15-min intervals for 6 h from d 8 through estrus. Gilts were examined by laparotomy 6 d after estrus. Ovulation rate was greater (P less than .05) in F than C gilts (16.0 vs 9.4). Mean daily concentrations of FSH were greater (P less than .05) in F gilts at 5 d, 4 d and 3 d prior to estrus compared with C females. In both C and F gilts, FSH decreased (P less than .05) prior to estrus. Mean daily concentrations of LH and LH pulse amplitude were not different (P greater than .05) between treatments. Mean number of LH pulses/6 h at 4 d, 3 d and 2 d prior to estrus were greater (P less than .05) in F than in C gilts. In both treatments, LH pulse amplitude decreased (P less than .05) and pulse frequency increased (P less than .07) prior to estrus. Mean plasma concentrations of insulin tended to be higher (P less than .07) in F than in C females during the 7-d period before estrus.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

小鼠桑椹胚简易玻璃化冷冻技术再探讨

本试验继小鼠扩张囊胚玻璃化冷冻保存成功后，在室温（２５℃）下利用不同浓度的ＥＦＳ玻璃化溶液，对小鼠的桑椹胚简易玻璃化冷冻技术进行再探讨。结果是胚胎在１０％ＥＧ溶液中预先处理５分钟，再移入事先配置好含有ＥＦＳ３０的０．２５ｍｌ塑料细管中１分钟平衡后直接投入液氮中冷冻，解冻后获得的发育率最高（９４％）。冻胚移植后妊娠率和产仔率分别为５６％（９／１６）及４２％（４９／１１６）。与对照组相比差异不显著（Ｐ＞０．０５）     

Interval to first postweaning estrus and causes for leaving the breeding herd in Large White, Landrace, Yorkshire and Chester White females after three parities

Three hundred and five Large White (LW, 93), Landrace (L, 92), Yorkshire (Y, 69) and Chester White (CW, 51) gilts produced at the Roman L. Hruska U.S. Meat Animal Research Center over 3 yr with 15 to 41 gilts per breed group per year were housed in total confinement and mated to farrow at 1 yr of age. After 28 to 30 d lactation, piglets were weaned and all females were observed for estrus daily for 30 d. During the year that each gilt initially farrowed, she was mated to produce a second and third litter if she returned to estrus within 30 d after piglet removal. Interval from weaning (d 0) to estrus and reason for leaving the breeding herd were recorded for each animal. Interval from weaning to estrus was influenced by breed (P less than .01), parity (P less than .01) and interaction between breed and parity (P less than .01). Intervals from weaning to estrus, in first parity females which exhibited estrus, were 7.8, 6.6, 9.3 and 14.0 d for the LW, L, Y and CW breeds. Intervals for all females after the second litter were 6.8, 4.9, 8.0 and 9.1 d, and after the third litter 6.4, 5.2, 8.3 and 10.1 d for the LW, L, Y and CW breeds. Breed, parity and year (P less than .01) affected the number of females not exhibiting estrus. Percentages of females not exhibiting estrus were 6.5, 18.7, 37.7 and 23.5 for the LW, L, Y and CW breeds and 16.1, 2.3 and 2.0 for sows after the first, second and third parities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Day of embryo collection, quality and pregnancy rates in cattle

In 1300 donor cows, total embryos increased from 8.5 to 15.3, (P less than 0.001), the mean number of transferable embryos from 3.1 to 6.5 (P = 0.067) and pregnancies increased from 1.3 to 3.3 (P = 0.584) as the day of collection increased from six to 7.5. Most of the embryos recovered on day 6, 7.5 and 8 were morulae, early blastocysts and late blastocysts respectively. Morulae formed the majority of the embryos collected on days 6 and 6.5. Pregnancy rates in early and late blastocysts were highest on days 7 (54.4 and 60.2 per cent) and 7.5 (53.6 and 53.1 per cent, P = 0.009 and 0.004). There were significant differences in pregnancy rates between embryo stages on days 7 and 7.5 (P less than 0.001), embryo grades on days 7, 7.5 and 8 (P less than 0.001), and within embryo grades 1 (P = 0.015) and 3 and 4 (pooled, P = 0.009). On all days combined there were significant differences in pregnancy rates between both embryo stages (P = 0.007) and embryo grades (P less than 0.001). It appears that the concept of embryo fitness may be applied to blastocysts but not to morulae.     

Development of one-cell porcine embryos to the blastocyst stage in simple media

Porcine embryos were flushed from mated donors and examined for cleavage stage. One- and two-cell embryos were randomly allotted to one of the five following in vitro treatments: M199 with Earle's salts, a modified Tyrode's medium (TL), TL supplemented with 10 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) (TLH), TLH supplemented with 5.5 mM glucose (TLHG), or TLH supplemented with 5 mM glutamine (TLHGL). The bicarbonate concentration of TLH, TLHG, and TLHGL was 2 mM, compared with the 25 mM concentration in M199 and TL. Embryos in M199 and TL were incubated in 95% air:5% CO2 at 39 degrees C. Those in the remaining three treatments were incubated in air at 39 degrees C. Embryos incubated in TL and M199 did not develop past the four- to eight-cell stage, whereas the proportions of embryos developing to the compact morula or blastocyst stage by d 7 of culture in the other treatments were as follows: TLHG, 49.1%; TLHGL, 59.4%; TLH, 63.5% (P less than .005). These results indicate that porcine embryos can be cultured from the one-cell stage to blastocyst in a simple HEPES-buffered medium in air. The ability of porcine embryos to develop without supplemental CO2 may be an important finding for use in situations in which embryos must be transported for long periods before embryo transfer.     

Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at –80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at –80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at –80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at –80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.