Piglet production by non-surgical transfer of vitrified embryos, transported to commercial swine farms and warmed on site.

We investigated the feasibility of piglet production by non-surgical embryo transfer (Ns-ET) of vitrified porcine blastocysts and expanded blastocysts transported to commercial farms and warmed on site (V/T/W embryos). Ns-ET was performed by depositing 11-20 vitrified and warmed embryos at a proximal site within the uterus via a catheter. In Experiment 1, the effect of donor-recipient estrous cycle asynchrony on the efficiency of Ns-ET of vitrified and ordinary warmed embryos was investigated at the experimental facility. With a 1-day delay recipients relative to that of donor, the farrowing rate was 50.0% and the survival rate to term was 21.1%. In Experiment 2, Ns-ET using recipients with a 1-day delay and vitrified embryos after one-step warming and dilution was evaluated at the experimental facility. Although the resulting farrowing rate was 42.9%, the survival rate was 6.4%. In Experiment 3, Ns-ET was conducted using V/T/W embryos at four commercial farms, where piglets derived from them were produced. When artificial insemination was conducted prior to Ns-ET, the farrowing and survival rates obtained using V/T/W embryos were 75.0%, and 21.3%, respectively. These results show that Ns-ET of V/T/W embryos using this protocol would be feasible for piglet production at farms.

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.

Porcine embryo collection using single subcutaneous administration of follicle-stimulating hormone in a large volume of saline.

The effects of a single subcutaneous or intramuscular injection of follicle-stimulating hormone (FSH) on follicular growth and expression of estrous behavior and its single subcutaneous administration on the number of corpora lutea (CL) and embryos were investigated in pigs. All four sows that were subcutaneously administered 5 AU FSH expressed normal estrus and had no ovarian cysts. Two of the four sows that were administered 5 AU FSH intramuscularly did not exhibit estrus, and another sow had a short estrus period. All four sows had ovarian cysts. The mean numbers of CL, embryos, and blastocysts following the subcutaneous administration of 5 AU FSH (16.8, 16.0, and 13.8, respectively) did not differ significantly from those for the control animals treated intramuscularly with 1000 IU equine chorionic gonadotropin (18.5, 16.5, and 14.3, respectively). In conclusion, embryo recovery was possible using a single subcutaneous administration of FSH.

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.

Production of piglets from in vitro-produced blastocysts by ultrasound-guided ovum pick-up from live donors.

The objective of this research was to develop a system for piglet production by transvaginal ultrasound-guided ovum pick-up (OPU), in vitro production (IVP) of embryos and embryo transfer. First, to establish a culture system for a small number of oocytes or embryos, we evaluated the effect of different incubation volumes and culture densities on fertilizing ability and developmental competence in vitro. Porcine oocytes derived from slaughterhouse ovaries were matured, fertilized and then cultured in vitro in groups as follows: 50 oocytes in 500 µL medium for IVM, 20 oocytes in 100 µL medium for IVF and 20 embryos in 40 µL medium for IVC (Group I); 20 in 100 µL for IVM, 20 in 100 µL for IVF and 20 in 40 µL for IVC (Group II); and 10 in 100 µL for IVM, 10 in 100 µL for IVF and 10 in 40 µL for IVC (Group III). Percentages of sperm penetration, cleavage and blastocyst formation did not differ among the groups. Second, to increase the collection efficiency of porcine oocytes by transvaginal ultrasound-guided OPU, the effects of aspiration pressure on follicular oocyte collection were assessed. Oocytes were aspirated from ovaries of live sows using 80 or 100 mmHg. The recovered oocytes were divided into four categories according to the surrounding cumulus cells and quality of oocytes. The number of oocytes recovered using 100 mmHg pressure was significantly higher than with 80 mmHg pressure. However, there were no significant differences in the population of oocytes grouped by the morphological criteria, number of blastocysts per session and the total cell number in blastocysts between the two vacuum pressures. Finally, 81 oocytes obtained by OPU from five donor sows were subjected to IVP and 47 transferable embryos (9.4 ±â€¯4.0 [mean ±â€¯SD] morulae/blastocysts per session) were obtained at 5 days after IVF. When they were transferred into five recipient gilts (5-16 embryos per recipient), three of five recipients became pregnant and farrowed a total of 12 live piglets. The present results demonstrate that porcine blastocysts can be produced by OPU-IVP and develop to full term after embryo transfer.

Prediction of maturational competence of feline oocytes using supravital staining of cumulus cells by propidium iodide.

We examined the relationship between integrity of cumulus cells and nuclear maturation rate after in vitro culture to determine a non-invasive prediction of the maturational competence of feline oocytes. Feline cumulus-oocyte complexes (COCs) were collected from either small (400-800 µm) or large (≥800 µm) follicles. Immediately after collection, cumulus cells were evaluated morphologically (thickness of cumulus cell layers) and stained with propidium iodide (PI), which penetrates only non-viable cells. Cumulus cells without PI staining were judged as having good membrane integrity. After evaluation, COCs were cultured for 30 h and their nuclear maturation rate was determined. The nuclear maturation rate of oocytes derived from large follicles (89.8%) was higher (p < 0.05) than that from small follicles (60.8%). There was no difference in the maturation rate of oocytes from follicles with the same size regardless of cumulus morphology. In contrast, oocytes that had cumulus cells with good membrane integrity showed a higher maturation rate (93.8%) than oocytes with poor cumulus integrity (76.9%) in large follicles (p < 0.05). We conclude that evaluation of membrane integrity of cumulus cells by propidium iodide staining can be used to predict the maturational competence of oocytes.

The effect of ovarian status and follicular diameter on maturational ability of domestic cat oocytes.

The objective of this study was to clarify the effect of ovarian status and follicular size on morphological normality and maturational ability of cat oocytes. Ovarian status was classified into inactive, follicular, luteal and prepubertal, and follicles were classified into three groups according to their diameter (400-800, 800-1200 and 1200-2000 µm). In each ovarian status, the number of follicles decreased but the percentage of morphologically normal oocytes increased with the growth of follicles (p<0.05). Only a single follicle that was 1200-2000 µm in diameter was observed in two of the five prepubertal cats. In follicles that were 800-1200 µm in diameter, the percentage of normal oocytes and maturation rate were higher in prepubertal cats than in mature cats (p<0.05). Oocyte diameter tended to increase with the growth of follicles. After oocytes were cultured individually in droplets of maturation medium, the oocyte maturation rate increased with the growth of follicles in each ovarian status (p<0.05). In conclusion, oocytes collected from larger follicles possess higher maturational ability in vitro in sexually mature cats. In prepubertal cats, a higher maturation rate can be obtained from oocytes derived from small follicles compared with in mature cats.