Cryopreservation methods using liquid nitrogen (LN2) for gametes and embryos are prevalent in mammalian artificial reproduction. However, the pregnancy rate from frozen embryos has not improved over the past two decades because freeze–thawing causes significant damage. The strict regulation of transportation of LN2 containers by airlines also limits exchange between breeders. In this article, we introduce a medium that enabled bovine embryos to be held for up to 7 days at 4°C. A pregnancy rate of 75% (24/32) was obtained for embryos held for 7 days in this medium and transferred to primed recipients. Its constituents were medium 199, foetal bovine serum, and HEPES for buffering. This technique will enable LN2-free storage and air transportation of embryos provided transplantation to recipients can be completed within 7 days.

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Intrauterine administration of peripheral blood mononuclear cells (PBMCs) prior to bovine embryo transfer (ET) was previously shown to improve the pregnancy rate. To better understand how PBMCs improve the pregnancy rate, we examined gene expression in the cells from uterine lumen and evaluated the morphology of bovine pre-attachment embryos in utero following intrauterine administration of PBMCs. On day 3 of the estrous cycle (day 0 = estrous), bovine PBMCs were isolated and suspended in RPMI 1640, and were incubated for 24 hr. The cultured PBMCs were administered non-surgically to the uterine horn ipsilateral to the corpus luteum on day 4 of the estrous cycle (PBMC group). On day 9, endometrial-luminal lymphoid cells from uterine lumen ipsilateral to the corpus luteum were collected by uterine flushing. Transcripts for macrophage-colony stimulating factor in the lymphoid cells were more abundant in the PBMC group than in the control group (P < 0.05). On day 7 (of the separate experiments), five blastocysts were each transferred to the luminal area, to which PBMCs had been administered on day 4. These embryos were allowed to develop in utero until day 15 of gestation, when embryos were non-surgically retrieved from the uterus. The average length of trophoblasts recovered from the PBMC group was significantly longer than that of the control group (51.6 ± 7.8 vs. 27.4 ± 6.0 mm, P < 0.05). Our results strongly suggest that intrauterine administration of PBMCs improves endometrial environment, which promotes early development of pre-attachment conceptuses.

Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24–48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP–embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming.

Reduction in oocyte quality is a major factor responsible for declining fertility associated with maternal aging in cows. The objective of the present study was to determine whether intracytoplasmic sperm injection (ICSI) could increase the efficiency of embryo production in older cows. We used cows aged 30 to 50 months or >120 months, which were defined as young or aged, respectively. The distribution of cortical granules in oocytes was affected by age as older cows had lower proportion of oocytes with mature cytoplasm containing evenly dispersed cortical granules compared to young cows. Although fertilization rates did not differ significantly between the two groups after in vitro fertilization (IVF), the rate of abnormal fertilization was higher, and the numbers of total and diploid blastocysts were lower for aged cows compared to young cows. However, in the embryos produced by ICSI, there was no significant difference in these parameters between young and aged cows. Although ICSI did not improve the blastocyst development rate, ICSI increased the proportion of diploid blastocysts in aged cows compared to IVF. In conclusion, maternal aging may negatively affect cytoplasmic maturation of bovine oocytes, which could be associated with abnormal fertilization or low developmental competence of oocytes. Our data also suggests beneficial effects of ICSI on the production of chromosomally normal embryos in aged cows.