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 F2α (PGF2α ) 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 PGF2α 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 PGF2α 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.

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.

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 F2α (PGF2α ) 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 PGF2α 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 PGF2α administration to synchronize estrus for subsequent embryo harvest, is a suitable alternative to the artificial abortion method.

Birth of piglets from in vitro-produced porcine blastocysts vitrified and warmed in a chemically defined medium.

We examined the effect of different embryo developmental stages, culture periods, and media on the cryotolerance of in vitro-produced porcine blastocysts. All media used for in vitro production, vitrification, and warming were chemically defined. When in vitro-produced embryos at the blastocyst and expanded blastocyst stages were vitrified using the Cryotop method on Day 5 or 6 (Day 0 = the day of IVF), the survival rate and hatching rate of expanded blastocysts after warming were higher than those of blastocysts. The viability after vitrification and warming of Day-6 embryos cultured in porcine blastocyst medium from Day 5 were higher than that of embryos cultured in porcine zygote medium 5. On the other hand, there were no significant differences on the cryotolerance between Day-5 embryos cultured in porcine zygote medium 5 and those replaced with porcine blastocyst medium on Day 4. There were no significant differences in viability between the embryonic ages of 5 and 6 days after vitrification and warming. When expanded blastocysts vitrified on Day 5 or 6 were surgically transferred to recipient gilts, all three recipients became pregnant in the Day-5 group, whereas only one out of three recipients became pregnant in the Day-6 group. These results indicate that the cryotolerance of porcine in vitro-produced blastocysts after vitrification appears to depend on the embryonic stage, culture period, and medium.

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.

Effects of the estrous cycle on the efficacy of oocyte collection and in vitro embryo production in Duroc-breed.

Collection efficacy and in vitro embryo developmental ability of oocytes obtained from Duroc-breed ovary donors at different stages of the estrous cycle (days 6, 12 and 16 after estrus) were performed. The numbers of collected oocytes did not differ significantly among the different estrous cycle groups (total 72-90 oocytes per gilt). However, the blastocyst rates of oocytes collected on days 12 and 16 (9.2% and 19.4%, respectively) were significantly higher than those on day 6 (1.1%). More oocytes were obtained on day 16 from small follicles (<2 mm in diameter; 85.3 oocytes per gilt) than from medium-sized (≥2-<6 mm) and large (≥6 mm) follicles (17.5 and 12.8 oocytes, respectively). The blastocyst rates in both the medium-sized and large follicle groups (20.0% and 19.2%, respectively) were significantly higher than that in the small follicle group (6.3%). The blastocyst cell numbers in both the medium-sized and large follicle groups (39.4 and 43.3 cells, respectively) were significantly higher than that in the small follicle group (30.6 cells). The results suggest that oocyte collection from cycling Duroc pigs can be carried out efficiently from the late luteal to follicular stage. Those oocytes collected from medium-sized and large follicles show better embryo development.

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.

In-straw cryoprotectant dilution for bovine embryos vitrified using Cryotop.

The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitro-produced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (P<0.05) than those of embryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (P<0.05) lower than those of embryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment.

Effect of volume of non-surgical embryo transfer medium on ability of porcine embryos to survive to term.

Non-surgical embryo transfer is a promising method for improving efficiency in the pork industry and also for biotechnology applications, such as in vitro embryo production, transgenesis and cloning. Several groups have reported successful piglet production using an artificial insemination catheter or flexible catheter designed for this procedure; however, the efficiency of the technique is still low. The critical points that need to be addressed in order to improve this procedure are (1) the embryo deposition site and (2) volume of transfer medium associated with the embryos; however, the latter has not yet been examined systematically. In the present study, we evaluated the effect of the volume of non-surgical embryo transfer medium on the ability of porcine embryos to survive to term by using a recently produced flexible catheter. The catheter consists of a guide and an injector. Blastocysts 200-230 mum in diameter were collected from donor gilts and transferred to recipient gilts. The time required for the completion of embryo transfer using this catheter was 14.6 +/- 3.9 min. The tip of the injector was determined by laparotomy to be located in a uterine horn 20-30 cm anterior from the branching point of the uterus body. We transferred 17.0-17.3 embryos with different volumes of medium (1.6, 3.2 and 10 ml) into each of 5, 4 and 4 recipients, respectively, and pregnancy was confirmed in 4, 3 and 1 of these recipients, respectively. Three recipients in the 1.6 ml group farrowed a total of 19 piglets (4, 5 and 10 piglets, respectively). These results suggest that successful non-surgical embryo transfer is affected by the volume of transfer medium.

Batchwise assessment of porcine embryos for cryotolerance.

The viability or developmental ability of porcine embryos after slow-freezing and thawing differs depending on the embryonic stage or the batch, which is defined as a group of embryos obtained from one donor at one time. We froze porcine blastocysts in batches and assessed their cryotolerance by using two expanded blastocysts (EBs) as samples to predict the developmental potential of other blastocysts from the same batch at different stages. Two EBs from the same batch that had been separately frozen were thawed and cultured in vitro for 48 h to examine their in vitro ability to develop to the hatched blastocyst stage. Thereafter, each batch was assigned to Grade A, B, or C according to the viability of the two EBs, i.e., 100% viability (2/2: number of hatched blastocysts/number of cultured EBs) was Grade A; 50% (1/2) was Grade B; and 0% (0/2) was Grade C. The viability of EBs after freeze-thawing and in vitro culture varied depending on the batch and was lower (31.0+/-10.2%, mean+/-S.E.M.; P<0.01) than that of unfrozen controls (96.8+/-2.3%). The viability of frozen-thawed hatched blastocysts (HBs) did not differ among the graded batches, but the blastocyst diameter decreased (from 409 to 326 microm) as the batch grade decreased (from A to C). When both EBs and HBs from batches of the same grade were transferred to recipients (average 11.7 EBs and 16.0 HBs per recipient), the rate of pregnancy and farrowing in recipients decreased (from 77.8% to 0%) and the number of piglets obtained decreased (from 15.3 to 0) as the batch grade decreased. However, when not only frozen-thawed EBs from Grade B or C batches, but also four helper embryos at the morula to early blastocyst stage (which were expected to support the pregnancy) were transferred, the number of piglets generated was higher from EBs from Grade B batches (16.0) than from EBs from Grade C batches (0.0). When frozen-thawed HBs and helper embryos were transferred, the number of piglets generated was higher from HBs from Grade B batches (12.7) than that from HBs from Grade C batches (1.9). After slow-freezing of porcine blastocysts, their rate of survival to the piglet stage differs batchwise, and in vitro viability assessment of sample EBs after freezing and thawing may help in assessing the post-freezing and post-thawing developmental potential of other blastocysts at different stages from the same batch.

Successful piglet production by IVF of oocytes matured in vitro using NCSU-37 supplemented with fetal bovine serum.

Recently, piglets have been obtained from in vitro-produced blastocysts by using in vitro maturation systems in which oocytes have been matured in North Carolina State University (NCSU) solution supplemented with porcine follicular fluid (PFF). However, PFF is not available commercially. To prepare PFF from the ovaries required time and effort and there is substantial variation in quality among batches. Furthermore, PFF is considered a potential source of infectious agents. We evaluated another commercially available potential protein source, fetal bovine serum (FBS), for in vitro maturation, to produce embryos and piglets. Cumulus-oocyte complexes were matured in NCSU-37 with PFF or with one of four batches of FBS. The proportions of oocytes with expanded cumulus cells were lower in all FBS batch groups (P < 0.05, 15-41%) than that in the PFF group (74%). The proportions of oocytes that matured were also lower in all FBS batch groups (P < 0.05, 26-41%) than in the PFF group (73%), irrespective of cumulus expansion. However, the proportions of oocytes that underwent germinal vesicle breakdown were almost the same in all groups (76-96%). After in vitro fertilization, the rate of sperm penetration into matured oocytes was higher in the PFF group (P < 0.05, 63%) than in one batch of FBS (22%) and removal of the compacted cumulus cells after maturation did not affect fertilization status (21%). Subsequent in vitro embryo culture of the PFF and FBS groups for 6 day resulted in similar rates of blastocyst formation (17 and 19%, respectively) and similar numbers of cells per blastocyst (43 and 46 cells, respectively). When blastocysts obtained from oocytes matured with FBS were transferred into two recipients, one became pregnant and farrowed seven piglets. Transfer of blastocysts obtained from oocytes matured with PFF into two other recipients resulted in one pregnancy and production of four piglets. These data suggested that porcine in vitro maturation in NCSU-37 supplemented with FBS reduced the maturational ability of oocytes, but once oocytes have matured, they have the same ability to develop to term after in vitro fertilization and embryo transfer as those matured with PFF.