Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse

Abstract

Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P!0.05). In Experiment 3, blastocyst development rate was 6-11% after injection of sperm lyophilized from fresh or frozen-thawed semen, suspended in SE. In Experiment 4, sperm lyophilized 3.5 months or 1 week previously, suspended in SE, yielded similar blastocyst rates (6 and 3% respectively). Rates of normal pregnancy after transfer were 7/10 and 5/7 for embryos from control and lyophilized sperm treatments respectively. Three pregnancies from the lyophilized sperm treatments were not terminated, resulting in two healthy foals. Parentage testing determined that one foal originated from the lyophilized sperm; the other was the offspring of the stallion providing the sperm extract. Further testing indicated that two of five additional embryos in the lyophilized sperm treatment originated from the stallion providing the sperm extract. We conclude that both lyophilized stallion sperm and stallion sperm processed by multiple unprotected freeze-thaw cycles (as for sperm extract) can support production of viable foals. To the best of our knowledge, this is the first report on production of live offspring by fertilization with lyophilized sperm in a non-laboratory animal species.

Establishment of conditions for ovum pick up and IVM of jennies oocytes toward the setting up of efficient IVF and in vitro embryos culture procedures in donkey (Equus asinus)

“…However, it is difficult to conclude on the efficiency of DMEM-F12 medium for in vitro culture of jennies zygotes as the IVF technique likely interferes with the original quality of zygotes. DMEM-F12 medium has been used previously for in vitro culture of equine zygotes after ICSI with high cleavage rates [31,48,49]. One can speculate that this medium might be efficient for the culture of donkey zygotes as well, but this remains to be shown.…”

“…Several attempts to establish an efficient conventional IVF technique in the equine have been performed, but IVF rates remained quite low and IVF techniques were not repeatable [24][25][26][27][28][29][30]. Intracytoplasmic Sperm Injection (ICSI) has been widely adopted to generate horse embryos in vitro, both for scientific purposes and in the horse breeding industry [11,31]. However, ICSI requires expensive equipment and expertise in micromanipulation.…”

Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations

“…Culture throughout development in DMEM/F-12-based media at O16 mM glucose has been associated with the highest equine blastocyst development rates previously reported (up to 43% per injected oocyte; Hinrichs et al 2005, Ribeiro et al 2008, Jacobson et al 2010) and with normal embryo development after transfer to recipient mares (Choi et al 2011). In reconciling this with the indications of a detrimental effect of 5 mM glucose in a GB medium during early culture, it is possible that the very high glucose in DMEM/F-12 causes the inhibition of glucose transport, as has been described in mouse embryos in high-glucose environments (Moley et al 1998), whereas the lower concentration (5 mM) allows glucose transport and subsequently impairs metabolism.…”

“…ICSI ICSI was conducted as described in our previous reports (Choi et al , 2011 with minor modifications, using modified CZB media, as detailed previously (Choi et al 2003b). After maturation, the oocytes were denuded of cumulus by pipetting in 0.05% hyaluronidase (Sigma-Aldrich) in CZB-M, and those with a polar body were held in M199 with Earle's salts and 10% FBS in an atmosphere of 5% CO 2 in air at 38.2 8C before being used for ICSI with frozen-thawed sperm.…”

The Effect of Freeze Drying Using Different Media and Storage Temperatures on Some Parameters of Buffalo Bull Spermatozoa

“…Regardless of the protocol applied, cryopreservation has a damaging effect on sperm resulting in reduction of both motility and fertilizing capacity (Critser et al, 1987). Therefore, in spite of apparent reduction in motility, cells still viable and characterized by normal nucleus and centrosome integrity which are essential for the success of ICSI (Choi et al, 2011). Although freeze-drying was focused on proper preservation of structural and functional sperm characteristics, an intact sperm nucleus is a necessary part for success of embryo production Yanagimachi, 1998, Kusakabe et al, 2001).…”