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.     

Effects of BSA and fetal bovine serum in culture medium on development of rat embryos

Rat 1-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (mR1ECM) or in mR1ECM supplemented with BSA (4 mg/ml; mR1ECM-BSA) or fetal bovine serum (FBS; 10%, v:v; mR1ECM-FBS) instead of polyvinylalcohol. There was no difference in percentages of embryos that developed to the 2-cell to blastocyst stages between mR1ECM and mR1ECM-BSA, but in mR1ECM-FBS, no development beyond the 2-cell stage was observed. When embryos were transferred to mR1ECM-FBS from mR1ECM after 24 to 64 h of culture, development of embryos to and beyond the 4-cell stage was inhibited. However, when transferred after 80 h of culture, more embryos developed to blastocysts and hatching or hatched blastocysts than in embryos cultured in mR1ECM. When 8-cell embryos and early morulae obtained after 72 and 80 h of culture in mR1ECM, respectively, were cultured in mR1ECM-FBS, a higher proportion of early morulae developed to the blastocyst stage than did 8-cell embryos. When morulae obtained after culture in mR1ECM or mR1ECM-BSA were transferred to recipient females, there was no difference in proportions of fetuses obtained. However, a higher proportion of blastocysts cultured in mR1ECM-FBS developed to fetuses compared with those obtained in mR1ECM. These results indicate that BSA has neither deleterious nor beneficial effects on development of rat 1-cell embryos. In contrast, FBS has deleterious effects on early cleavage of embryos but it promotes more rapid development of morulae to blastocysts, resulting in better quality blastocysts.     

Induction of short-term pseudopregnancy in gilts by the administration of estradiol benzoate or estradiol dipropionate to achieve ovulatory synchronization for embryo collection

A study was conducted to investigate whether ovulation in gilts could be synchronized for embryo collection by the administration of estradiol benzoate (EB) or estradiol dipropionate (EDP) to induce pseudopregnancy, followed by the treatment with prostaglandin F_2α (PGF_2α) on 10 days after. Ten gilts each received a total of 20 mg of EB or EDP on Day 10 or EB on Day 10 and 14 to induce pseudopregnancy (Day 0 = onset of estrus). Donors received PGF_2α 10 or 15 days (as a control) after the first administration of estrogens and subsequently eCG and hCG, and were then inseminated artificially. The embryos were collected 7 days after the administration of hCG, and assessed for embryo yield and their developmental stages. All protocols resulted in good embryo yield (9.8–13.2 embryos in average), and the embryos showed average ability to develop to the expanded blastocyst stage (3.29–4.03 as developmental scores) without any significant differences among the protocols. These results suggest that the administration of PGF_2α 10 days after the treatment of gilts with EB or EDP would allow synchronization of ovulation and embryo collection, as well as shortening the period from estrus detection to embryo collection, thus improving embryo collection efficiency.     

Successful blastocyst production by intracytoplasmic injection of sperm after in vitro maturation of follicular oocytes obtained from immature female squirrel monkeys (Saimiri boliviensis)

Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.     

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3-100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8-89.5%) and hatched blastocyst rates (61.1-69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3-30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.     

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.     

猪早期胚胎体内、外发育规律研究

系统地掌握猪早期胚胎体内外发育规律是进行猪胚胎冷冻、胚胎分割、鲜胚移值、长途运输引种以及基因转移等生物技术研究和应用的基础。本研究包括２个实验，实验１，利用８９头超排母猪和１９头自然发情母猪，于配种后第２、３、４、５、６、７、８、９和１０天手术采得胚胎１６１２枚，系统地观察猪早期胚胎体内发育规律，即：２日龄胚胎中原核胚占７７．３９％（３９７／５１３），３日龄胚胎中４和６细胞胚占７９．０５％（８３／１０５），４日龄胚胎中４～８细胞占４０％（３５／７６），５日龄胚胎多为桑椹和囊胚８３．１２％（６４／７７），６日龄胚多为膨胀囊胚４２．０９％（１８１／４３０）和孵化囊胚３４．１９％（１４７／４３０），７和８、９、１０日龄胚发育到孵化囊胚分别为７６．３４／ｏｏ（７１／９３）和１００％（７７／７７），而自然发情的５日龄胚胎中桑椹和囊胚仅占３２．５％（１３／４０），６日龄胚胎中膨胀囊胚仅占１１．１１％（１７／１５３），孵化胚仅占１９．６１％（３０／１５３）。各发育阶段的胚胎比例均明显低于５、６日龄超排供体（Ｐ＜０．０５）。实地２，将１３８枚囊胚和膨胀囊胚，分别体外培养２４和１２小时，囊胚发育阶段的胚胎５０％（４８／９６）?     

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。     

Effect of human chorionic gonadotropin on preovulatory luteinizing hormone surge and ovarian hormone secretion in gilts

The influence of varying doses of human chorionic gonadotropin (hCG) on the preovulatory luteinizing hormone (LH) surge, estradiol-17 beta (E2) and progesterone (P4) was studied in synchronized gilts. Altrenogest (AT) was fed (15 mg X head-1 X d-1) to 24 cyclic gilts for 14 d. Pregnant mares serum gonadotropin (PMSG; 750 IU) was given im on the last day of AT feeding. The gilts were then assigned to one of four groups (n = 6): saline (I), 500 IU hCG (II), 1,000 IU hCG (III) and 1,500 IU hCG (IV). Human chorionic gonadotropin or saline was injected im 72 h after PMSG. No differences in ovulation rate or time from last feeding of AT to occurrence of estrus were observed. All gilts in Groups I and II expressed a preovulatory LH surge compared with only four of six and three of six in Groups III and IV, respectively. All groups treated with hCG showed a rapid drop (P less than .01) in plasma levels of E2 11, 17, 23 h after hCG injection when compared with the control group (35 h). The hCG-treated gilts exhibited elevated P4 concentrations 12 h earlier than the control group (3.1 +/- .5, 3.4 +/- .72, 3.1 +/- .10 ng/ml in groups II, III and IV at 60 h post-hCG vs .9 +/- .08 ng/ml in group I; P less than .05). These studies demonstrate that injections of ovulatory doses of hCG (500 to 1,500 IU) had three distinct effects on events concomitant with occurrence of estrus in gilts: decreased secretion of E2 immediately after hCG administration, failure to observe a preovulatory LH surge in some treated animals and earlier production of P4 by newly developed corpora lutea.     

Vitrification of biopsied mouse embryos

Solid surface vitrification (SSV) was compared with in-straw vitrification for cryopreservation of biopsied mouse embryos. Eight-cell stage embryos were zona drilled and one blastomere was removed. Developed morulae or blastocysts were vitrified in microdrop (35% EG + 5% PVP + 0.4 M trehalose) or in straw (7.0 M EG + 0.5 M sucrose). Following recovery, embryos were cultivated in vitro or transferred into recipients. Cryopreservation had an effect not only on the survival of biopsied embryos but also on their subsequent development in vitro. Cryosurvival of biopsied morulae vitrified in straw was significantly inferior to SSV. The post-warm development of biopsied and non-biopsied morulae was delayed on Day 3.5 and 4.5 in both vitrification groups. A delay in development was observed on Day 5.5 among vitrified non-biopsied blastocysts. The percentage of pups born from biopsied morulae or blastocysts following cryopreservation did not differ from that of the control. No significant differences could be detected between methods within and between embryonic stages in terms of birth rate. The birth rate of biopsied embryos vitrified in straw was significantly lower compared to the non-biopsied embryos. The novel cryopreservation protocol of SSV proved to be effective for cryopreservation of morula- and blastocyst-stage biopsied embryos.     

Embryo collection in prepubertal gilts and attempts to develop an improved embryo transfer technique

Prepubertal gilts were treated with 1,500 iu equine chorionic gonadotrophin, followed 72 hours later by 500 iu human chorionic gonadotrophin (hCG), and inseminated 36 and 48 hours later. Embryos were collected at slaughter 168 hours after the hCG treatment. Blastocysts classified as 'good' or 'fair' were transferred to synchronised recipients, either by conventional surgical means or by a 'semi-endoscopic' approach, and the recipients were slaughtered four weeks later. Of 238 donor gilts, 98.4 per cent had responded with a mean (se) 23.5 (1.0) ovulations and 19.1 (1.0) ova or embryos, of which 47 per cent were considered morphologically intact and transferable. The large proportion of non-transferable embryos was not associated with the age or weight of the gilts, the season or with their housing conditions. Conventional surgical transfer of 15 to 20 (mean 17.4) blastocysts to synchronised recipients yielded 88 percent (14 of 16) pregnancies with between seven and 14 (mean 8.2) viable fetuses, and an embryo survival rate of 47 per cent in the pregnant recipients and 41 per cent in all the recipients. The corresponding data for the semi-endoscopic transfers were 16 to 20 (mean 17.7) blastocysts transferred, 47 per cent (eight of 17) pregnancies, four to 12 (mean 7.3) viable fetuses per pregnant recipient and an embryo survival rate of 41 per cent in the pregnant recipients and 19 per cent in all the recipients. Significantly fewer of these recipients became pregnant and a significantly smaller proportion of the embryos survived (P<0.05).     

山羊类ES细胞的分离与克隆

采集山羊交配后6～8d的桑椹胚、囊胚和孵化囊胚,将桑椹胚和囊胚分别放在小鼠原代胎儿成纤维细胞(PMEF)饲养层和同源原代胎儿成纤维细胞(PGEF)饲养层上比较其脱带时间及脱带率。脱带后,将各自一半胚胎切割,把含ICM的半胚分别放在相应饲养层上进行培养,另一半整胚在各自饲养层上继续培养,而孵化囊胚直接于PGEF饲养层上培养。当ICM增殖一定程度时进行传代,以比较其类ES细胞分离与克隆的效果。结果表明,在2种不同饲养层上,囊胚的脱带时间均短于桑椹胚,囊胚的脱带率均高于桑椹胚,而饲养层的种类对胚胎的脱带时间以及脱带率影响不大。脱带切割囊胚不论在PMEF还是在PGEF饲养层上,其贴壁时间均短于脱带整胚及孵化囊胚,而贴壁率高于脱带整胚,与孵化囊胚相似。脱带整胚及脱带切割胚在PMEF饲养层上所获类ES细胞只能维持3代,而在PGEF饲养层上,脱带切割半胚和孵化囊胚所获类ES细胞传至5代。由此认为,对脱带后的胚胎进行切割处理,有利于ICM的贴壁和增殖;应用同源原代胎儿成纤维细胞饲养层培养系统,有利于类ES细胞的分离与克隆。     

Blastocyst production from in vitro-produced day-2 bovine embryos classified by cleavage stage, and cytogenetical evaluation of the resultant day-8 blastocysts

The present study was conducted to determine the criteria for selecting good quality embryos on Day-2 post-insemination and at the blastocyst stage. Bovine oocytes were matured, fertilized and cultured in vitro. First, Day-2 embryos were classified based on the number of blastomeres into 2-cell, 3- to 4-cell, 5- to 8-cell and >8-cell stage embryos; chromosome samples were then prepared. In the second experiment, the Day-2 embryos classified according to the number of blastomeres were cultured separately for an additional 6 days (Day 8). The resultant Day-8 blastocysts from each group of Day-2 embryos were classified into the following 3 grades based on morphology and diameter: Grade A, hatched and hatching blastocysts; Grade B, expanded blastocysts; and Grade C, unexpanded blastocysts. Chromosome samples were then prepared. The 5- to 8-cell stage Day-2 embryos had the lowest incidence of chromosomal abnormalities (13.5%, P<0.05) and the highest development rate to blastocysts (59.2%, P<0.05). Furthermore, the blastocysts derived from the 5- to 8-cell stage embryos had the largest mean number of cells (102.8+/-42.4, P<0.05), largest number of metaphases per blastocyst (9.5+/-4.8, P<0.05) and lowest incidence of chromosomal abnormalities (24.6%, P<0.05). The Grade A blastocysts had the largest mean number of cells (136.6+/-33.4, P<0.05), a large number of metaphases per blastocyst (11.9+/-5.5, P<0.05) and a low incidence of severe chromosomal abnormalities (17.3%). The results showed that, at Day 2, the 5- to 8-cell stage embryos were of better quality since they had the lowest incidence of chromosomal abnormalities and the highest blastocyst rate and the resultant blastocysts had the largest number of cells and lowest incidence of chromosomal abnormalities. In particular, selection of Grade A blastocysts can improve the development rate to term.     

Influence of estrus synchronization of prepubertal gilts on embryo quality

Synchronization and superovulation are commonly used to obtain large numbers of embryos for experimental and practical purposes. This study compared the number, quality, and in vitro development of embryos recovered from gilts following single or double estrus synchronization and superovulation. Prepubertal gilts from the single synchronization group were injected with 1500 I.U. PMSG and 1000 I.U. hCG 72 h later. The double synchronized group of gilts was treated with 750 I.U. PMSG and 500 I.U. hCG 72 h later. After 17 days, 1500 I.U. PMSG followed by 1000 I.U. hCG was administered. Five days after insemination embryos were recovered and cultured for 6 days. Both single and double hormonal stimulation schedules resulted in recovery of elevated numbers of embryos (28.4 and 23.4 vs. 11.3; p<0.01 and p相似文献   

In vitro fertilisation of in vivo matured porcine oocytes obtained from prepuberal gilts at different time intervals after hCG injection

The goal of the present study was to find out the best interval after hCG injection in PMSG primed prepuberal gilts for retrieval of in vivo matured oocytes for in vitro fertilisation (IVF). Altogether 66 gilts were superovulated with 1500 IU PMSG and 500 IU hCG 72 h later. Ovum pick up was performed endoscopically 24, 28, 32 or 36 h after hCG and a total of 869 cumulus-oocyte-complexes (COCs) were aspirated from 1400 follicles. COCs were tested for quality, and an aliquot was immediately fixed and stained to determine meiotic configuration. The remaining COCs were fertilised in vitro using frozen-thawed epididymal semen. Quality and developmental stage of embryos were tested after IVF, and the number of nuclei was counted. At 24 to 32 h after hCG only few oocytes have entered the second meiotic cycle (18 to 25% vs. 58% at 36 h, p < 0.05). The overall cleavage rate was significantly influenced by insufficient maturation rate at the early collection times (14% at 24 h vs. 49% at 36 h). Additionally, when oocytes were collected 24 to 32 h vs. 36 h the cleavage rate based on mature oocytes was lower (26 vs. 62%, p < 0.05). Once embryonic development has been initiated, the further in vitro development to blastocyst stages did not differ between groups. However, the number of cells was lower at collection times 24 to 32 h as compared to 36 h after hCG (12 to 15 cells vs. 22 cells, p < 0.05). The results indicate that the time of COC collection affects the in vitro developmental competence up to the blastocyst stage and should not be performed earlier than 36 h after hCG treatment.     

Embryo collection from pigs post‐pseudopregnancy induced by estradiol dipropionate

We aimed to define whether embryo collection carried out after pseudopregnancy was of similar outcome and quality as after artificial abortion. To induce pseudopregnancy, 30 gilts or sows were given 20 mg intramuscular estradiol dipropionate (EDP) 10–11 days after the onset of estrus. Ten additional pigs were inseminated artificially at natural estrus as a control group. Prostaglandin F_2α (PGF_2α) was administered twice with a 24 hr interval beginning 15, 20, or 25 days after EDP‐treatment (n = 10 per group) or between 23 and 39 days after artificial insemination in control pigs. Following this, all pigs were given 1,000 IU equine chorionic gonadotropin and 500 IU human chorionic gonadotropin (hCG) and then inseminated. Embryos were recovered 6 or 7 days after hCG treatment and outcome was recorded. There was no significant difference in the number of normal embryos collected from the pigs with PGF_2α initiated at different time points or from the control group. Embryonic developmental stages 7 days after hCG treatment also did not differ among groups. These results indicate that the use of EDP to induce pseudopregnancy, followed by PGF_2α administration to synchronize estrus for subsequent embryo harvest, is a suitable alternative to the artificial abortion method.     

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.     

Effect of nerve growth factor (NGF) on the development of preimplantation rabbit embryos <Emphasis Type="Italic">in vitro</Emphasis>

This study aimed to investigate the effect of nerve growth factor (NGF) on the development of preimplantation rabbit embryos in vitro. Zygotes were collected from superovulated New Zealand rabbits 19 h after injection of hCG and immediately mating and cultured in TCM-199 plus fatty-acid free BSA with different concentrations of NGF. Zygotes not treated with NGF served as control. At 24 h, 48 h, 72 h and 96 h of the culture, the numbers of the early cleavage stage, morulae, blastocysts and hatching blastocysts were determined. The intrazonal diameter of the blastocyst and the total cell numbers per blastocyst were measured after 96 h of culture. The results showed: (1) NGF at 100 ng/mL and 1000 ng/mL could improve the numbers of the hatching blastocysts which developed compared to the control treatment (p < 0.05); (2) All concentrations of NGF increased the total cell numbers in the blastocysts compared to the control treatment (p < 0.05); (3) NGF had no significant effect on the blastocyst intrazonal diameter of the blastocysts at 96 h of culture (p = 0.493); (4) The proportion in the early cleavage stage at 24 h of culture (p = 0.635), of morulae at 48 h of culture (p = 0.812) and of blastocysts at 72 h of culture (p = 0.812) in all treatments were not significantly different.     

In vitro and in vivo developmental ability of oocytes derived from porcine primordial follicles xenografted into nude mice

We evaluated the developmental ability of oocytes in porcine primordial follicles xenografted into nude mice. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the kidney capsules of ovariectomized nude mice. Forty-nine to 89 days after grafting (mean +/- SEM, 66.9 +/- 1.9 days; n = 64), the host mice showed the presence of cornified epithelial cells in their vaginal smears for the first time. The mice were then treated with 4 IU of equine chorionic gonadotropin (eCG) 60 days after first detection of vaginal cornification. Oocytes were collected from the host mice 48 h after treatment with eCG, and then matured. The maturation rates, based on the incidence of first polar body, ranged from 25.1% to 42.5%. They were then fertilized in vitro and cultured in vitro for 6 days, or transferred into estrous-synchronized recipients and recovered after 6 days. On Day 6 of culture, 15.4% of the matured oocytes had cleaved to the 2- to 8-cell stage. However, neither the embryos cultured in vitro nor those transferred and recovered developed to advanced embryonic stages, such as morulae or blastocysts. This result suggests that the developmental ability of xenografted oocytes is insufficient, even after in vitro maturation. Further strategies, such as improvement of hormonal treatment for host mice, are required to enable oocytes in xenografted ovarian tissues to acquire the cytoplasmic maturation necessary for embryonic development.