Successful Production of Piglets Derived from Expanded Blastocysts Vitrified Using a Micro Volume Air Cooling Method without Direct Exposure to Liquid Nitrogen

This study was conducted to clarify the feasibility of newly developed vitrification techniques for porcine embryos using the micro volume air cooling (MVAC) method without direct contact with liquid nitrogen (LN₂). Expanded blastocysts were vitrified in a solution containing 6 M ethylene glycol, 0.6 M trehalose and 2% (wt/vol) polyethylene glycol in 10% HEPES-buffered PZM-5. The blastocysts were collected from gilts and vitrified using the new device (MVAC) or a Cryotop (CT). Blastocysts were stored in LN₂ for at least 1 month. After warming, cryoprotective agents were removed using a single step. Survival of the embryos was assessed by in vitro culture (Experiment 1) and by embryo transfer to recipients (Experiment 2). In Experiment 1, the embryos vitrified by the MVAC or CT and fresh embryos without vitrification (Control) were used. The survival rates of embryos in the MVAC, CT and Control groups were 88.9% (32/36), 91.7% (33/36) and 100% (34/34), respectively, after 48 h culture, and the hatching rates of embryos after 48 h incubation were 69.4% (25/36), 63.9% (23/36) and 94.1% (32/34), respectively. In Experiment 2, 64 vitrified embryos were transferred to 5 recipient gilts, and 8 healthy piglets were produced from 3 recipients in the MVAC group. Similarly, 66 vitrified embryos were transferred to 5 recipient gilts, and 9 healthy piglets were produced from 2 recipients in the CT group. These results indicated that porcine expanded blastocysts can be cryopreserved using the MVAC method without potential pathogen contamination from LN₂.     

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.     

Non‐surgical transfer of vitrified porcine embryos using a catheter designed for a proximal site of the uterus

This study aimed to compare the efficiency of non‐surgical embryo transfer (ET) using a newly developed catheter, which enables transferring embryos into a proximal site of the uterus (mostly uterine body), and surgical ET of vitrified porcine embryos. In Experiment 1, the catheter was inserted into 12 gilts, with each half of the group allocated to skilled or novice operators. The time required for insertion into the uterus did not differ between skilled and novice operators (4 min 9 s and 4 min 6 s, respectively). In Experiment 2, 12 gilts were used as recipients for non‐surgical and surgical ET with vitrified embryos (n = 6, each). There was no significant difference in the rate of piglet production based on the number of transferred embryos between surgical and non‐surgical ET (25.8% vs. 15.4%, p = .098). The results suggest that non‐surgical ET catheter allowed for easy insertion and transfer of embryos without special training. Although the catheter is effective for deposition of embryos into the proximal site of uterus, the efficiency of piglet production is not enhanced compared with surgical ET. The ET method using this catheter, being labor‐saving and less‐invasive, may contribute to the improvement of ET in pigs.     

Birth of piglets through the non-surgical transfer of blastocysts produced in vitro

In this study, we attempted to produce piglets by non-surgically transferring blastocysts produced in vitro, using a flexible catheter as the transfer instrument. Cumulus-oocyte complexes (COCs) were aspirated from the follicles of ovaries obtained at a local slaughterhouse. They were then matured in modified North Carolina State University (NCSU)-37 medium for 44-46 h and fertilized in porcine gamete medium (PGM). Ten hours after in vitro fertilization (IVF), presumptive zygotes were removed from the cumulus cells and cultured in porcine zygote medium (PZM)-5. Blastocysts were cultured for five days after IVF and, using a catheter for deep intrauterine insemination without sedation, they were transcervically transferred into the uterine horn of six recipients (45-50 blastocysts/recipients) whose estrous cycles were synchronized, at 5 days after human chorionic gonadotropin (hCG) injection. Of the six recipients, one sow became pregnant and farrowed seven piglets (four live piglets) 119 days after hCG injection. The body weight at birth of the newborns ranged from 0.8 to 1.4 kg. These results indicate that it is possible to obtain piglets by transcervically transferring blastocysts produced by IVF and in vitro cultures in chemically defined media.     

Transfer of pig embryos to different uterine sites.

Embryo transfer in pigs normally involves surgery. In connection with the development of nonsurgical or endoscopic transfer techniques, it is important to know whether the uterine site to which embryos are transferred has an effect on the success rate. In the present investigation, prepubertal donor gilts were treated with 1,500 IU of PMSG and, 72 h later, with 500 IU of hCG. Gilts were artificially inseminated 24 and 36 h after hCG injection. Embryos at the expanded blastocyst stage were collected from donor gilts. Recipient gilts were treated synchronous with the donors, using 1,000 IU of PMSG followed, 72 h later, with 500 IU of hCG. After a maximum of 3 h in vitro, embryos (n = 15 to 20, mean = 17.3) were transferred surgically to the middle of the uterine horn, to the caudal quarter of the uterine horn, or to the uterine body. Recipients were slaughtered between 28 and 34 d after transfer. The pregnancy rate of the recipients was low when the embryos were deposited in the uterine body (12%), compared with the middle (88%) or the caudal quarter of the uterine horn (81%) (P < .01). The corresponding average number of viable fetuses per pregnant recipient was 8.2 in the uterine body, 5.6 in the middle, and 4.5 in the caudal quarter. Average survival rate of embryos after transfer to the middle of the uterine horn was 41% vs 29 and 3% after transfer to the caudal quarter or the uterine body, respectively (P < .01). Hence, the uterine body seems to be an unsuitable site for embryo transfer in pigs. These results may explain the unsatisfactory results achieved with nonsurgical embryo transfer in the past.     

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).     

Embryo transfer in pigs: a method for introducing genetic material into primary specific-pathogen-free herds.

Fifty-five embryos were transferred from 4 Duroc donor sows into 4 crossbred recipient gilts. The embryo survival rate was 66%; 53% of eggs ovulated by donors were represented by live pigs at term. Three of the litters were obtained by hysterectomy of the recipients and introduced into a primary specific-pathogen-free herd, thereby saving the donor sows for subsequent use. Embryo transfer provided a method for exploiting the genetic potential of superior females.     

Production of Middle White Piglets after Transfer of Embryos Produced In Vitro

The present study was conducted to examine the feasibility of in vitro embryo production and transfer technologies for producing Middle White piglets. After collection from three retired Middle White sows, a total of 222 oocytes were matured, fertilized and cultured in vitro, and a total of 50 embryos from the 4-cell to blastocyst stage were produced by the 4th or 5th day. These embryos were transferred individually into three recipients along with 5 in vivo-derived Duroc blastocysts. All of the recipients became pregnant, and they farrowed a total of 9 Middle White and 9 Duroc piglets. These results suggest that in vitro embryo production using ovaries from retired sows is useful for reproduction of pigs of pure breeds including the Middle White for breeding activities and conservation/utilization of genetic resources.     

Evaluation of the uterine environment and embryos of prepubertal gilts

A series of three experiments was conducted to test the functional status of the uterus and embryos in prepubertal gilts. In Exp. 1, gilts were induced to ovulate by treating with gonadotropins followed by hCG 72 or 96 h later, and were artificially inseminated 24 h after hCG. Five of the 10 gilts treated at 120 d of age, but none of the gilts treated at 100 of age, maintained pregnancies. We next tested the function of the uterine environment by transferring embryos from postpubertal females into gilts of various ages that had been induced to ovulate but not inseminated (Exp. 2). Pregnancy rate at d 50 of gestation was 44% (4/9) for 100-d-old recipients, 67% (2/3) for 140-d-old recipients, and 60% (3/5) for postpubertal recipients (P > 0.20). Therefore, uteri of 100-d-old gilts are able to maintain pregnancies with conceptuses from postpubertal gilts. In Exp. 3, embryos from 100-d-old and postpubertal gilts were transferred into postpubertal recipients. Uterine horns of recipients were surgically separated before transfer, and embryos from 100-d-old and post-pubertal females were transferred to opposite horns of some recipients (experimental). Other recipients received embryos from postpubertal females in both uterine horns (control). When examined on d 50 to 60 of gestation, three of five control gilts were pregnant and three of seven experimental gilts were pregnant (P > 0.50). In experimental recipients, the survival of embryos from 100-d-old gilts was 38% (8/21) compared to 57% (15/26) for embryos from postpubertal gilts (P > 0.30). Because all uterine horns of pregnant recipients contained fetuses, these results support the hypothesis that embryos from 100-d-old gilts are able to initiate and maintain pregnancies in the uteri of postpubertal gilts. Therefore, the uterine environment of 100-d-old gilts provides an environment that supports development of embryos produced by postpubertal gilts, and the embryos produced by 100-d-old gilts can survive and develop in the uteri of postpubertal gilts. It was only the combination of embryos and uteri of 100-d-old gilts that did not permit pregnancy to be maintained.     

Effects of recloning on the efficiency of production of alpha 1,3-galactosyltransferase knockout pigs

Obtaining sufficient transgenic cells via selective cultivation of genetically manipulated somatic cells is difficult due to the limited number of cell divisions. Additionally, if irreversible mutations in a cell's chromosomes occur during selective cultivation and the cell is used as the nuclear donor, somatic cell nuclear transfer (SCNT) embryos often exhibit abnormal development. On the other hand, a SCNT method in which fetal cells derived from SCNT embryos are used as the nuclear donor (recloning method) is an effective technique for obtaining large quantities of transgenic cells. In this study, we compared the in vivo development rate of SCNT embryos produced from porcine alpha1-3 galactosyltransferase gene knockout (GTKO) cells by a recloning method with that of SCNT embryos produced without recloning from porcine GTKO cells (direct method). In the direct method, 557 and 462 cloned embryos were produced using two types of activation methods, the two-step activation (TA) method and the delayed activation (DA) method, and then transferred into 6 and 4 recipients, respectively, but no piglets were born from these recipients. In the recloning method, 956 and 1038 cloned embryos were produced using the TA and DA methods, respectively, and then transferred to 8 and 7 recipients, respectively. Two piglets were born from one recipient in the TA group and 6 piglets were born from 3 recipients in the DA group. This report indicates that the recloning method improved the developmental capacity of SCNT embryos reconstructed with gene-targeted somatic cells.     

猪微注射基因导入系统影响受体受孕率及产仔率的几个因素

在应用显微注射、胚胎移植系统技术，进行猪ＯＭＴ／ＰＧＨ基因导入的研究中，对移入受体的胚数，ＰＭＳＧ处理受体以及不同的移植方法（自体移植与异体移植）等影响受体受孕率及产仔率的因素，进行了试验分析，结果表明：（１）移入受体的注射胚数分别为１０—１９枚、２０—２９枚、３０枚以上时，其受孕率为４５．５％、６４．７％和７１．４％，产仔率为８．０％、１９．６％和１４．５％，以移入２０—２９枚效率最高；（２）用ＰＭＳＧ对受体母猪做同期发情处理，其受孕率和产仔率比选择自然发情的受体分别下降２５％和８．５％；（３）采用自体移植的方式，在移入胚数基本相同的情况下（１５枚左右），比异体移植的受孕率和产仔率分别提高１４．３％和６．４％。     

The transfer of fresh and frozen embryos in an elite swamp buffalo herd

To investigate the development of fresh and frozen swamp buffalo embryos after transfer to synchronized recipients, 14 fresh embryos and 28 frozen embryos, collected from Thai swamp buffalo cows of an elite herd at the Surin Breeding Center, were transferred nonsurgically to 31 synchronized recipients buffalo cows. One fresh embryo was transferred to each of 14 recipients. Twenty eight frozen embryos were transferred to 17 recipients of which 7 cows received I embryo, 9 cows received 2 embryos and 1 cow received 3 embryos. Pregnancy was diagnosed by real time B-mode ultrasonography one month after transfer and confirmed by rectal palpation one and two months later. The pregnant cows were kept under observation until calving. The results of fresh embryo transfer showed that 5/14 (35.7%) were pregnant after 30 days, 4/14 (28.6%) remained pregnant until the 3rd month and 2/14 (14.3%) calved. With the frozen embryos, only one cow which received three embryos became pregnant and remained so for 3 months although the embryo did not survive to full term. The overall pregnancy rate using frozen embryos was 5.9% (1/17). The study demonstrated the possibility of performing embryo transfer in elite buffalo herds for genetic improvement, however the use of frozen embryos needed further investigation.     

Insemination of recipient sows improves the survival to term of vitrified and warmed porcine expanded blastocysts transferred non‐surgically

This study was performed to evaluate reproductive performance after non‐surgical embryo transfer (Ns‐ET) of 10–15 porcine expanded blastocysts (ExBs) that had been vitrified and warmed (V/W) using the micro volume air cooling (MVAC) method. The effect of asynchrony between the donor and recipient estrous cycle was investigated. Ns‐ET was conducted in recipients whose estrous cycle was asynchronous to that of donors by a delay of 2, 1, or 0 days. In the 2‐day and 1‐day groups, the similar farrowing rates (27.3% and 25.0%) and survival rates to term (13.9% and 15.7%) were obtained after Ns‐ET of V/W ExBs. None of the recipients in 0‐day group farrowed. Artificial insemination (AI) prior to Ns‐ET was then evaluated. Ten–15 V/W ExBs were transferred non‐surgically to 12 recipients whose estrous cycles were asynchronous to that of donors by a 2‐day delay. All of the recipients produced piglets, and all (100.0%) delivered piglets were derived from the transferred V/W ExBs. The survival rate of V/W ExBs to term was 25.2%. These results demonstrate that Ns‐ET of V/W ExBs using MVAC can facilitate piglet production, even if 10–15 embryos are transferred. Moreover, piglets were obtained stably when AI was performed prior to Ns‐ET.     

受体绵羊人工授精后移植胚胎的试验

试验通过使用人工授精后绵羊作为受体进行胚胎移植,以寻找一种更加有效的方法提高绵羊胚胎移植的经济效益。试验中使用FSH对10只无角道赛特绵羊进行超数排卵处理,同时对60只受体小尾寒羊进行同期发情。供体羊在发情配种后4.5～5.0d从子宫角收集胚胎。同时,将胚胎移植到同期发情并进行人工授精的受体羊子宫内。总共有57枚可用胚移植给44只受体小尾寒羊,32只怀孕到分娩,共产下羔羊51只(无角道赛特羔28只,道赛特与小尾寒羊杂种羔23只)。此外,经人工授精但未进行手术移植的7只小尾寒羊产下15只杂种羔羊。移胚植受体妊娠率72.7%(32/44),移胚受体繁殖率118%(51/44),受体利用率88.3%(53/60)。移胚受体总妊娠率和受体利用率均显著高于常规ET组(P<0.01)。与常规胚胎移植相比,受体羊人工授精后移植胚胎不仅提高了无角道赛特母羊的繁殖率,而且提高了受体羊的利用率。     

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 serum-free co-culture and synchrony of recipients on development of cultured sheep embryos to fetuses

The percentage of sheep embryos that continued to develop after collection and immediate transfer on d 2 after estrus was similar when phosphate-buffered saline with 10% fetal calf serum (PBSFCS, 45%), physiological saline (50%), or tissue culture medium 199 supplemented with 10% fetal calf serum (M199FCS, 47%) was used to flush embryos from oviducts. Co-culture of sheep embryos for 3 d with oviductal cells tended (P = .1) to reduce the percentage of embryos that developed to fetuses after transfer compared with those embryos transferred immediately. Tissue culture medium 199 supplemented with .3% BSA (M199BSA) was an adequate substitute for M199FCS for culture of sheep oviductal cells if tissue culture wells were pretreated with fibronectin. Estradiol in concentrations from 10 to 1,000 pg/ml and progesterone at concentrations of 1 or 10 ng/ml in M199BSA failed to stimulate embryo development during 3 d of co-culture beyond that seen in co-culture with M199FCS or M199BSA without added steroid. Transfer of sheep embyros co-cultured for 3 d in M199BSA or M199FCS to recipients synchronized with donors resulted in about 19% of the embryos developing to fetuses, whereas transfer to recipients that were in estrus 24 h after donors resulted in 33% of embryos developing to fetuses. The significant (P less than .05) improvement for delayed recipients may reflect the relatively lesser developmental rate of co-cultured embryos compared with that of embryos in vivo. Embryo development into fetuses was similar after co-culture in M199FCS or M199BSA co-cultures; therefore, serum is not required for the co-culture of sheep embryos.     

Matching of embryo stages and grades with recipient oestrous synchrony in bovine embryo transfer

The efficacy of matching embryos with recipients on the basis of embryo stage and grade and donor-recipient oestrous synchrony was investigated using the records of 13,663 embryos that were collected and transferred at a commercial embryo transfer centre. The selection of early blastocysts for exact oestrous synchrony cows was effective and resulted in the highest pregnancy rates. Selection of early morulae was effective for recipients in oestrus after the donor but not when transferred into exact and negative recipients. The matching of late morulae with recipients in oestrus after the donor was not effective and had no influence on pregnancy rates. The selection of late, hatched and collapsed blastocysts for transfer into recipients in oestrus before the donor was ineffective and pregnancy rates were higher in exact and +12 hour recipients. Pregnancy rates declined 23.6 per cent in quality grades 1 to 4 whereas the range between stages was 13.3 per cent. Higher quality embryos of all stages gave the highest pregnancy rates. Examination of pregnancy rates of grades within stages suggested that the more developed the embryo the more difficult it is to grade. The difference in pregnancy rates between exact and -24 (6.9 per cent) and +24 (4.8 per cent) hour recipients was small and declined a further 4.7 per cent and 9.8 per cent in -36 and +36 hour recipients. Grade 3 and 4 embryos tolerated asynchrony better than grade 1 and 2, and early morulae tolerated asynchrony better than the other stages. It was concluded that the matching of certain embryo stages with the donor-recipient oestrous synchrony is advantageous but not always possible.     

The effect of artificial insemination prior to transfer of a limited number of vitrified and warmed porcine embryos by open pulled straw (OPS) method on their survival ability for farrowing

Embryo transfer (ET) of 20 porcine expanded blastocysts (ExBs) vitrified and warmed (VW) by open pulled straw (OPS) to a recipient allows stable piglet production. The efficiency of artificial insemination (AI) prior to ET of 10 VW ExBs for piglet production was investigated. For one trial, 10–15 VW ExBs from single donor were assigned, 10 were used for ET and the remains were assessed for their in vitro viability. In the non‐AI/ET group, 10 were transferred to each of five recipients. As AI/ET group, 10 were transferred to each of five recipients after AI. In AI/non‐ET group, only AI was performed to seven gilts. In the non‐AI/ET group, the pregnancy rate was 40%, but none of them farrowed. In the AI/ET group, all recipients produced piglets. Four (80.0%) delivered piglets from transferred VW ExBs. The survival rate of VW ExBs to term was 20.0% (10/50). In the AI/non‐ET group, six of the seven gilts farrowed. There was no difference in in vitro viability between the non‐AI/ET and AI/ ET groups (62.5% and 68.3%, respectively). AI prior to ET can be an appropriate way to maintain pregnancy and assist the development of a low number of VW ExBs to term.     

Cloned porcine embryos can maintain developmental ability after cryopreservation at the morula stage

The aim of the present study was to clarify the overall efficiency of porcine somatic cell nuclear transfer (SCNT) by incorporating cryopreservation of the cloned embryos before transfer. The SCNT embryos reconstructed with preadipocytes and in vitro-matured (IVM) oocytes were cultured to harvest morula stage embryos; they were then subjected to delipation (removal of cytoplasmic lipid droplets) and vitrification. After warming and culture, the embryos developing to blastocysts were transferred to recipients to obtain cloned piglets. From 372 reconstructed embryos, 188 (50.5%) reached the morula stage and 117 (31.5%) developed to blastocysts after vitrification. Transfer of 98 (26.3%) morphologically normal blastocysts gave rise to 6 (1.6%) piglets, including 1 stillborn. The efficiency of the cloned piglet production was comparable with that obtained using SCNT embryos without cryopreservation (2.7%, 17/635). Here, we demonstrate that porcine somatic cell cloning can be performed without a significant reduction in efficiency even when the SCNT embryos are cryopreserved before transfer.