Comparative evaluation of the cost and efficiency of four types of sexing methods for the production of dairy female calves.

This study was conducted to evaluate and compare the economic benefits of different embryo sexing methods, based on the cost per female dairy calf produced. Female calves were produced from four kinds of female embryos: (1) those collected from superstimulated donors at 7-8 days after artificial insemination (AI) with X-sorted semen; (2) those sex-determined by loop-mediated isothermal amplification assay of a biopsy sample of embryos collected from superstimulated donors after AI with conventional unsorted semen; (3) those obtained by invitro embryo production (IVEP), using X-sorted semen and in vitro-matured oocytes collected from donors by ovum pick-up (OPU); and (4) those obtained by IVEP, using X-sorted semen and oocytes collected by OPU after dominant follicle ablation and follicle growth stimulation of the donors. The respective productivities of female calves per technical service and the total production cost per female calf of each sexing method were compared. The production cost per female calf (66,537 JPY), as calculated from the number of female calves per service (1.30), pregnancy rate of transfer (42.9%), rate of female calves obtained (92.9%), and total cost of the method (56,643 JPY plus embryo transfer fee), was less for IVEP with X-sorted semen and follicular growth-stimulated (FGS) oocytes than for the other groups (P < 0.05). The results demonstrate that embryo production with X-sorted semen and FGS oocytes provides a more efficient method for producing female calves than the other embryo sexing methods.

Normal calves produced after transfer of embryos cultured in a chemically defined medium supplemented with epidermal growth factor and insulin-like growth factor I following ovum pick up and in vitro fertilization in Japanese black cows.

The objective of this study was to examine whether high concentrations of epidermal growth factor (EGF) and/or insulin-like growth factor I (IGF-I) would have a beneficial effect on bovine embryo development in vitro and to obtain normal calves by using an ovum pick up method and embryo culture in a chemically defined medium. When compared with controls, EGF (100 or 200 ng/ml) or IGF-I (50 or 100 ng/ml) significantly increased the rate of embryos that developed into blastocysts during an 8-day culture after the in vitro fertilization of oocytes obtained from ovaries from a slaughterhouse. IGF-I induced a dose-dependent increase in cell number in both the inner cell mass and the trophectoderm, whereas EGF stimulated proliferation only in the inner cell mass. A combination of EGF (100 ng/ml) and IGF-I (50 ng/ml) produced an additive effect, and embryos developed into blastocysts at a comparatively high rate (27.9%) compared with controls (12.0%). A similar rate of development was achieved using a combination of EGF and IGF-I in the culture of embryos following ovum pick up by ultrasound-guided transvaginal follicular aspiration and in vitro fertilization, and 5 blastocysts that developed after the culture were transferred into uteri; two embryos implanted, and normal calves were born. These results suggest that the combined use of EGF and IGF-I makes bovine embryo culture in a chemically defined medium a practical and useful procedure for producing blastocysts, and its application to embryo culture following ovum pick up and in vitro fertilization could be useful for producing normal calves.

Production of Japanese Black calves by the transfer of embryos developed from in vitro-fertilized oocytes derived by ovum pick up and matured in culture with the mitogen-activated protein kinase kinase inhibitor U0126.

This study investigated whether treatment with the mitogen-activated protein kinase kinase inhibitor U0126 during in vitro maturation (IVM), which has previously been reported to improve oocyte developmental competence, is practical for use in calf production using ovum pick up (OPU)-derived oocytes. Two Japanese Black cows were repeatedly and simultaneously treated to stimulate follicular growth and were prepared for OPU. Cumulus-oocyte complexes (COCs) were collected from one cow using a collection medium containing 5 µM U0126 and were cultured in medium supplemented with the same concentration of U0126 for the first 2 hr of IVM; COCs from the other cow were used as controls without U0126 treatment. The cows were exchanged between the two groups at every sequential OPU (n=8). The number of oocytes developing to blastocysts in the U0126-treated group (39.1%, 34/87) was significantly higher than that in the control group (22.1%, 19/86). Eight blastocysts produced with U0126 treatment were transferred to recipients, and four normal calves were obtained. The results indicate that embryos develop efficiently from OPU-derived oocytes treated with U0126, and that these embryos may be of practical use in calf production.

Hair cortisol levels of lactating dairy cows in cold- and warm-temperate regions in Japan.

We compared the hair cortisol levels of lactating dairy cows in a cold- and a warm-temperate region out of four climatic zones in Japan. We simultaneously investigated the effects of calving number, lactation period and month of hair sampling. Hair of nine Holstein lactating cows chosen from each region (i.e. 18 cows per sampling) was sampled in March, June, September and December. Number of calvings (1, 2, ≥3) and lactation duration (early: <100, middle: 101-200, and late: >201 days) were balanced between regions. Cortisol was extracted from hair by methanol, and its level was determined with a cortisol immunoassay kit. A multi-way analysis of variance revealed that the effects of month of hair sampling (P < 0.001) and its combination with region (P < 0.05) were significant. In a multiple comparison test, significant differences (P < 0.01) in hair cortisol level (pg/mg of hair) were found between June (13.0 ± 1.0) and the other 3 months, and between September (1.6 ± 0.2) and December (4.5 ± 0.3). The rise in cortisol level from March to June was more intense in the cold-temperate region. These results demonstrate the necessity of considering seasonal variations in each climatic region when we use hair cortisol level as an indicator of stress.

Improvement of preimplantation development of in vitro-fertilized bovine zygotes by glucose supplementation to a chemically defined medium.

The influences of glucose supplementation on early development of bovine embryos in BSA-free synthetic oviduct fluid were examined. Among the groups supplemented with 1.5, 2.0, 4.0 or 5.6 mM glucose either at 0, 72 or 144 hr after fertilization, blastocysts yield significantly increased in the group supplemented with 4.0 mM glucose 144 hr after fertilization compared to the controls without glucose supplementation. The results suggest that appropriate amounts of glucose supplemented to the medium at the specific stage of embryo culture may be useful for the production of bovine blastocysts.

Viability of porcine embryos after vitrification using water-soluble pullulan films.

The efficiency of a porcine embryo vitrification method that uses water-soluble films of pullulan, a naturally-occurring polysaccharide polymer, was compared with two other types of vitrification methods using different devices and solutions for vitrification and warming. Blastocysts collected in vivo and vitrified by the conventional straw (ST), Cryotop((R)) (MVC) or pullulan film vitrification (PFV) methods were stored in liquid nitrogen for a certain period of time, after which the cryoprotective agents were removed by stepwise dilution. Fresh embryos were used as controls for the non-vitrification group. The vitrified-warmed embryos were incubated in TCM199 with 0.1 mM beta-mercaptoethanol and 20% fetal bovine serum for 24 h at 38.5 C in humidified air with 5% CO(2) to evaluate their viability. The survival rate of embryos in the ST group (48.3%) was significantly lower than that of those in the MVC (70.7%), PFV (79.0%) and non-vitrification (94.4%) groups. The oxygen consumption rate after vitrification was significantly lower than that before vitrification in the ST group, but was not significantly different in the MVC and PFV groups. Both the oxygen consumption rates of embryos after warming and the live cell numbers in the ST group were lower than those in the MVC group, while they did not differ significantly between the PFV and MVC groups. There was a correlation between the oxygen consumption rate and the number of live cells in vitrified embryos after warming. Our results demonstrated that in vivo-derived porcine embryos could be vitrified using pullulan films.