The effect of L-carnitine supplementation during in vitro maturation on oocyte maturation and somatic cloned embryo development.

The quality of the recipient cytoplasm was reported as a crucial factor in maintaining the vitality of SCNT embryos and SCNT efficiency for dairy cows. Compared with oocytes matured in vivo, oocytes matured in vitro showed abnormal accumulation and metabolism of cytoplasmic lipids. L-carnitine treatment was found to control fatty acid transport into the mitochondrial ß-oxidation pathway, which improved the process of lipid metabolism. The results of this study show that 0.5 mg/ml L-carnitine significantly reduced the cytoplasmic lipid content relative to control. No significant difference was observed in the rate of oocyte nuclear maturation, but the in vitro developmental competence of SCNT embryos was improved in terms of increased blastocyst production and lower apoptotic index in the L-carnitine treatment group. In addition, the pregnancy rate with SCNT embryos in the treatment group was significantly higher than in the control group. In conclusion, the present study demonstrated that adding L-carnitine to the maturation culture medium could improve the developmental competence of SCNT embryos both in vitro and in vivo by reducing the lipid content of the recipient cytoplasm.

Maternal dietary supplementation of arginine increases the ratio of total cloned piglets born to total transferred cloned embryos by improving the pregnancy rate of recipient sows.

The extremely low full-term developmental efficiency of cloned pig embryos limits the practical application of pig cloning techniques. Maternal dietary supplementation of the nutritionally important amino acid, arginine, can enhance prenatal developmental rate of in vivo fertilization-derived pig embryos. It was hypothesized that maternal dietary addition of arginine can also improve the developmental capacity of cloned pig embryos. To test this hypothesis, there was a comparison of the reproductive performance between recipient sows fed an L-arginine-supplemented diet (L-Arg group) and those fed the control diet (control group). There was a subsequent comparison of the developmental indexes of cloned piglets farrowed in the L-Arg and control groups of surrogate sows. Dietary supplementation of L-arginine during gestation days 14-75 increased the plasma concentrations of arginine and arginine metabolites, including nitric oxide, spermidine, and putrescine in recipient sows of transferred cloned pig embryos. Although maternal arginine addition did not affect the birth weight and placental development indexes of newborn cloned piglets, it significantly increased the ratio of total cloned piglets born to total transferred cloned pig embryos by increasing the pregnancy rate of recipient sows. The results of this study suggest that nutritional management of recipient sows is an effective approach to improve the developmental rate of cloned pig embryos.

Zinc supplementation during in vitro maturation increases the production efficiency of cloned pigs.

Zinc supplementation (0.8 µg/ml) in in vitro maturation (IVM) medium significantly enhances oocyte quality. In this study, we compared the development of somatic cell nuclear transfer (SCNT) embryos produced from conventional IVM (control) and zinc-supplemented IVM oocytes. A total of 1206 and 890 SCNT embryos were produced using control and zinc-supplemented oocytes, respectively, and then were transferred to 11 and 8 recipients, respectively. Five control recipients and three zinc-supplemented recipients became pregnant. Two live piglets and eight mummies were born from two control recipients, and ten live piglets and six stillborn piglets were born from three zinc-supplemented recipients. The production efficiency significantly increased in the zinc-supplemented group (0.33% vs. 3.02%). This report suggests that zinc supplementation in IVM medium improved the production efficiency of cloned pigs.

Astaxanthin Normalizes Epigenetic Modifications of Bovine Somatic Cell Cloned Embryos and Decreases the Generation of Lipid Peroxidation.

Astaxanthin is an extremely common antioxidant scavenging reactive oxygen species (ROS) and blocking lipid peroxidation. This study was conducted to investigate the effects of astaxanthin supplementation on oocyte maturation, and development of bovine somatic cell nuclear transfer (SCNT) embryos. Cumulus-oocyte complexes were cultured in maturation medium with astaxanthin (0, 0.5, 1.0, or 1.5 mg/l), respectively. We found that 0.5 mg/l astaxanthin supplementation significantly increased the proportion of oocyte maturation. Oocytes cultured in 0.5 mg/l astaxanthin supplementation were used to construct SCNT embryos and further cultured with 0, 0.5, 1.0 or 1.5 mg/l astaxanthin. The results showed that the supplementation of 0.5 mg/l astaxanthin significantly improved the proportions of cleavage and blastulation, as well as the total cell number in blastocysts compared with the control group, yet this influence was not concentration dependent. Chromosomal analyses revealed that more blastomeres showed a normal chromosomal complement in 0.5 mg/l astaxanthin treatment group, which was similar to that in IVF embryos. The methylation levels located on the exon 1 of the imprinted gene H19 and IGF2, pluripotent gene OCT4 were normalized, and global DNA methylation, H3K9 and H4K12 acetylation were also improved significantly, which was comparable to that in vitro fertilization (IVF) embryos. Moreover, we also found that astaxanthin supplementation significantly decreased the level of lipid peroxidation. Our findings showed that the supplementation of 0.5 mg/l astaxanthin to oocyte maturation medium and embryo culture medium improved oocyte maturation, SCNT embryo development, increased chromosomal stability and normalized the epigenetic modifications, as well as inhibited overproduction of lipid peroxidation.

Production of fertile offspring from oocytes grown in vitro by nuclear transfer in cattle.

Because of recent advancements in reproductive technology, oocytes have attained an increasingly enriched value as a unique cell population in the production of offspring. The growing oocytes in the ovary are an immediate potential source that serve this need; however, complete oocyte growth before use is crucial. Our research objective was to create in vitro-grown (IVG) oocytes that would have the ability to perform specialized activities, including nuclear reprogramming, as an alternative to in vivo-grown oocytes. Bovine oocyte-granulosa cell complexes with a mean oocyte diameter of approximately 100 µm were cultured on Millicell membrane inserts, with culture medium supplemented with 4% polyvinylpyrrolidone (molecular weight, 360,000), 20 ng/ml androstenedione, 2 mM hypoxanthine, and 5 ng/ml bone morphogenetic protein 7. Oocyte viability after the 14-day culture period was 95%, and there was a 71% increase in oocyte volume. Upon induction of oocyte maturation, 61% of the IVG oocytes extruded a polar body. Eighty-four percent of the reconstructed IVG oocytes that used cumulus cells as donor cells underwent cleavage, and half of them became blastocysts. DNA methylation analyses of the satellite I and II regions of the blastocysts revealed a similar highly methylated status in the cloned embryos derived from in vivo-grown and IVG oocytes. Finally, one of the nine embryos reconstructed from the IVG oocytes developed into a living calf following embryo transfer. Fertility of the offspring was confirmed. In conclusion, the potential of a proportion of the IVG oocytes was comparable to that of in vivo-grown oocytes.

Defined media optimization for in vitro culture of bovine somatic cell nuclear transfer (SCNT) embryos.

The objective was to establish an efficient defined culture medium for bovine somatic cell nuclear transfer (SCNT) embryos. In this study, modified synthetic oviductal fluid (mSOF) without bovine serum albumin (BSA) was used as the basic culture medium (BCM), whereas the control medium was BCM with BSA. In Experiment 1, adding polyvinyl alcohol (PVA) to BCM supported development of SCNT embryos to blastocyst stage, but blastocyst formation rate and blastocyst cell number were both lower (P < 0.05) compared to the undefined group (6.1 vs. 32.6% and 67.3 ± 3.4 vs. 109.3 ± 4.5, respectively). In Experiment 2, myo-inositol, a combination of insulin, transferrin and selenium (ITS), and epidermal growth factor (EGF) were added separately to PVA-supplemented BCM. The blastocyst formation rate and blastocyst cell number of those three groups were dramatically improved compared with that of PVA-supplemented group in Experiment 1 (18.5, 23.0, 24.1 vs. 6.1% and 82.7 ± 2.0, 84.3 ± 4.2, 95.3 ± 3.8 vs. 67.3 ± 3.4, respectively, P < 0.05), but were still lower compared with that of undefined group (33.7% and 113.8 ± 3.4, P < 0.05). In Experiment 3, when a combination of myo-inositol, ITS and EGF were added to PVA-supplemented BCM, blastocyst formation rate and blastocyst cell number were similar to that of undefined group (30.4 vs. 31.1% and 109.3 ± 4.4 vs. 112.0 ± 3.6, P > 0.05). In Experiment 4, when blastocysts were cryopreserved and subsequently thawed, there were no significant differences between the optimized defined group (Experiment 3) and undefined group in survival rate and 24 and 48 h hatching blastocyst rates. Furthermore, there were no significant differences in expression levels of H19, HSP70 and BAX in blastocysts derived from optimized defined medium and undefined medium, although the relative expression abundance of IGF-2 was significantly decreased in the former. In conclusion, a defined culture medium containing PVA, myo-inositol, ITS, and EGF supported in vitro development of bovine SCNT embryos.

Increasing glucose in KSOMaa basal medium on culture Day 2 improves in vitro development of cloned caprine blastocysts produced via intraspecies and interspecies somatic cell nuclear transfer.

This study was conducted to evaluate the efficiency of potassium simplex optimization medium with amino acids (KSOMaa) as a basal culture medium for caprine intraspecies somatic cell nuclear transfer (SCNT) and caprine-bovine interspecies somatic cell nuclear transfer (iSCNT) embryos. The effect of increased glucose as an energy substrate for late stage development of cloned caprine embryos in vitro was also evaluated. Enucleated caprine and bovine in vitro matured oocytes at metaphase II were reconstructed with caprine ear skin fibroblast cells for the SCNT and iSCNT studies. The cloned caprine and parthenogenetic embryos were cultured in either KSOMaa with 0.2 mM glucose for 8 days (Treatment 1) or KSOMaa for 2 days followed by KSOMaa with additional glucose at a final concentration of 2.78 mM for the last 6 days (Treatment 2). There were no significant differences in the cleavage rates of SCNT (80.7%) and iSCNT (78.0%) embryos cultured in KSOMaa medium. Both Treatment 1 and Treatment 2 could support in vitro development of SCNT and iSCNT embryos to the blastocyst stage. However, the blastocyst development rate of SCNT embryos was significantly higher (P < 0.05) in Treatment 2 compared to Treatment 1. Increasing glucose for later stage embryo development (8-cell stage onwards) during in vitro culture (IVC) in Treatment 2 also improved both caprine SCNT and iSCNT embryo development to the hatched blastocyst stage. In conclusion, this study shows that cloned caprine embryos derived from SCNT and iSCNT could develop to the blastocyst stage in KSOMaa medium supplemented with additional glucose (2.78 mM, final concentration) and this medium also supported hatching of caprine cloned blastocysts.

Effects of mineral supplements on ovulation and maturation of dog oocytes.

The aim of this study was to assess the effects of trace mineral supplements near the time of ovulation on the number of ovulated oocytes, in vivo oocyte maturation and pregnancy for dog cloning. Sixteen oocyte donor dogs were used in each control and mineral supplement group, and 136 and 166 corpora lutea were counted from each group. No significant difference was observed between oocyte recovery rates in the control (91.2 ± 2.7%) and mineral (89.9 ± 2.7) groups. Proportions of mature (86.2 ± 7.2 and 88.4 ± 6.8%) and aged (13.8 ± 7.2 and 11.6 ± 6.8%) oocytes were not different in the control and mineral groups, respectively. Oocytes with fair (91.5 ± 3.6 and 93.6 ± 2.1%) and poor (8.5 ± 3.6 and 6.4 ± 2.1%) quality also showed no difference between the control and mineral groups. The concentrations of manganese and ferrous iron were higher and lower on the day of ovulation, respectively, in both groups, but trace element concentrations in peripheral blood were not affected by mineral treatment. Oocytes were used to make cloned embryos; after embryo transfer, four and two pups were delivered from the control and mineral group, respectively, but there was no difference in the delivery rate (4.6 and 2.7%). In conclusion, intravenous mineral supplements administered once close to the LH surge in oocyte donor dogs and recipients had no effect on the number of ovulated oocytes, in vivo oocyte maturation or pregnancy in dog cloning in this study.

A 12-month feeding study of reproduction/development in rats fed meat/milk powder supplemented diets derived from the progeny of cloned cattle produced by somatic cell nuclear transfer.

The present 12-month feeding study was carried out with rat groups fed a diet supplemented with meat or milk (meat/milk) derived from the progeny of clones produced by somatic cell nuclear transfer (SCNT) technology. It was conducted to obtain data concerning the chronic toxicities of these edible products during the process of development and reproduction in rats fed such products. The rats were subjected to clinical observations for general health condition and examinations such as sensory/reflex function, grip strength, motor activity, body weight, food consumption, ophthalmology and urinalysis. Moreover, sexually matured rats fed the test diets were mated and examined for items such as the reproductive performances of the dams and health of their pups. After the feeding period, factors related to rat health status, based on the findings for hematology, blood biochemistry, necropsy, organ weight and histology, were examined. There were no biologically significant differences in these factors between the rat groups fed meat/milk powder supplemented diets derived from the progeny and those fed meat/milk powder supplemented diets derived from conventionally bred cattle. Therefore, the present chronic toxicity study suggests that meat and milk derived from the progeny of SCNT cattle might be equivalent to those derived from conventionally bred cattle in use as dietary supplements for rats.

Production of a healthy calf by somatic cell nuclear transfer without micromanipulators and carbon dioxide incubators using the Handmade Cloning (HMC) and the Submarine Incubation System (SIS).

The aim of this work was to investigate the minimum technical requirements for production of live offspring with somatic cell nuclear transfer. The experiment was performed in a field type laboratory without micromanipulators and carbon dioxide incubators. All long-term incubations were performed in the Submarine Incubation System (SIS) using various gas mixtures. The somatic cell culture was established from ear biopsy of a 9-year-old Holstein cow. Nuclear transfer was performed using the Handmade Cloning (HMC) technique. Zona-free oocytes were randomly bisected by hand with a disposable blade and a stereomicroscope. Cytoplast were selected using Hoechst staining and a fluorescent microscope. After a two-step fusion embryos were activated with calcium ionophore and dimethylaminopurine. Embryos were cultured in microwells (WOWs) in SOFaaci medium supplemented with 5% cattle serum. In two consecutive experiments, six blastocysts were produced from 52 reconstructed embryos. On Day 7, five blastocysts were transferred into synchronized recipients. All three recipients became pregnant but two pregnancies aborted at 6 and 7 months, respectively. A heifer calf weighing 27 kg was delivered at term by Caesarean section from the third pregnancy. The healthy 6-month-old heifer, the first cloned animal of Africa, is living evidence that nuclear transfer technology may be successfully used under basic laboratory conditions.

Nuclear transfer in cattle using colostrum-derived mammary gland epithelial cells and ear-derived fibroblast cells.

To assess the developmental potential of nuclear transfer embryos in cattle using mammary gland epithelial (MGE) cells derived from the colostrum, we compared the effectiveness of cloning using those cells and fibroblast cells derived from the ear. The fusion rate of the enucleated oocytes with fibroblast cells (75 +/- 4%) was significantly higher than that with MGE cells (56 +/- 7%, P<0.05). There were no significant differences in the cleavage rate (85 +/- 3% vs. 91+/- 2%) or in the developmental rate to the blastocyst stage (35 +/- 6% vs. 35 +/- 5%) using MGE cells vs. fibroblast cells as donor nuclei (P>0.05). After transfer of blastocysts derived from nuclear transfer embryos produced using MGE cells and fibroblast cells, 13% (4/31) and 16% (6/37) of recipient heifers were pregnant on Day 42 as assessed by ultrasonography, respectively. Two of the 4 and 4 of the 6 recipients of embryos with MGE cell- and fibroblast cell-derived nuclei, respectively, aborted within 150 days of pregnancy. Four live female calves were obtained from MGE cells or fibroblast cells. However, one died from internal hemorrhage of the arteria umbilicalis. The other three calves were normal and healthy. There were no differences in the pregnancy rate or calving rate when using MGE cells vs. fibroblast cells. Microsatellite DNA analyses confirmed that the cloned calves were genetically identical to the donor cows and different from the recipient heifers. We conclude that colostrum-derived MGE cells have the developmental potential to term by nuclear transfer, and the efficiency of development of those cloned embryos was the same as that of embryos obtained using fibroblast cells as donor nuclei, although there was a significant difference in the fusion rate. This method using MGE cells derived from colostrum, which is obtained easily and safely from live adult cows, is more advantageous for cloning with somatic cells.