Our previous studies have shown that oocytes collected from prepubertal calves lack developmental competence. The overall objective of this study was

Intracytoplasmic sperm injection (ICSI) has become a useful technique for clinical applications in the horse-breeding industry. However, both ICSI blastocyst and offspring production continues to be limited for most farm and wild species. This article reviews technical differences of ICSI performance among species, possible biological and methodological reasons for the variable efficiency and potential strategies to improve the outcomes. One of the major applications of ICSI in animal production is the reproduction of high-value specimens. Unfortunately, some domestic species like the bovine show low rates of pronuclei formation after sperm injection, which led to the development of various artificial activation protocols and sperm pre-treatments that are discussed in this article. The impact of ICSI technique on equine breeding programs is considered in detail, since in contrast to other species, its use for elite horse reproduction has increased in recent years. ICSI has also been used to produce genetically modified animals; however, despite numerous attempts in several domestic species, only transgenic pigs have been consistently produced. Finally, the ICSI is a promising tool for genetic rescue of endangered and wild species. In conclusion, while ICSI has become a consistent ART for some species, it needs further development for others. The low results obtained for some domestic species, the high training needed and the equipment required have limited this technique to the production of elite specimens or for research purposes.

Embryo aggregation affects in vitro and in vivo equine-cloned embryo development.

Embryo aggregation has been demonstrated to improve cloning efficiency in mammals. However, since no more than three embryos have been used for aggregation, the effect of using a larger number of cloned zygotes is unknown. Therefore, the goal of the present study was to determine whether increased numbers of cloned aggregated zygotes results in improved in vitro and in vivo embryo development in the equine. Zona-free reconstructed embryos (ZFRE's) were cultured in the well of the well system in four different experimental groups: I. 1x, only one ZFRE per microwell; II. 3x, three per microwell; III. 4x, four per microwell; and IV. 5x, five ZFRE's per microwell. Embryo size was measured on day 7, after which blastocysts from each experimental group were either a) maintained in culture from day 8 until day 16 to follow their growth rates, b) fixed to measure DNA fragmentation using the TUNEL assay, or c) transferred to synchronized mares. A higher blastocyst rate was observed on day 7 in the 4x group than in the 5x group. Non-aggregated embryos were smaller on day 8 compared to those aggregated, but from then on the in vitro growth was not different among experimental groups. Apoptotic cells averaged 10% of total cells of day 8 blastocysts, independently of embryo aggregation. Only pregnancies resulting from the aggregation of up to four embryos per microwell went beyond the fifth month of gestation, and two of these pregnancies, derived from experimental groups 3x and 4x, resulted in live cloned foals. In summary, we showed that the in vitro and in vivo development of cloned zona-free embryos improved until the aggregation of four zygotes and declined when five reconstructed zygotes were aggregated.