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Whitesboro, TX, United States

Choi Y.H.,Texas A&M University | Varner D.D.,Texas A&M University | Love C.C.,Texas A&M University | Hartman D.L.,Hartman Equine Reproduction Center | Hinrichs K.,Texas A&M University
Reproduction | Year: 2011

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. © 2011 Society for Reproduction and Fertility. Source


Choi Y.-H.,Texas A&M University | Norris J.D.,Texas A&M University | Velez I.C.,Texas A&M University | Jacobson C.C.,Texas A&M University | And 3 more authors.
Theriogenology | Year: 2013

The presence of heterogenous mitochondria from the host ooplast affects the acceptance of offspring obtained by somatic cell nuclear transfer. This might be avoided by obtaining oocytes from selected females, but is then complicated by low numbers of available oocytes. We examined the efficiency of equine somatic cell nuclear transfer using oocytes recovered by transvaginal aspiration of immature follicles from 11 mares. Use of metaphase I oocytes as cytoplasts and of scriptaid (a histone deacetylase inhibitor) treatment during oocyte activation were evaluated to determine if these approaches would increase blastocyst production. In experiment 1, blastocyst development was 0/14 for metaphase I oocytes and 4/103 (4%) for metaphase II oocytes. Three blastocysts were transferred to recipient mares, resulting in two pregnancies and one live foal, which died shortly after birth. In experiment 2, blastocyst development was 2/47 (4%) for control oocytes and 1/83 (1%) for scriptaid-treated oocytes. No foals were born from two blastocysts transferred in the control group. The blastocyst from the scriptaid treatment resulted in birth of a live foal. In conclusion, this is apparently the first report of production of a viable cloned foal from oocytes collected from immature follicles of live mares, supporting the possibility of cloning using oocytes from selected mares. © 2013 Elsevier Inc. Source


Choi Y.H.,Texas A&M University | Velez I.C.,Texas A&M University | Hartman D.L.,Hartman Equine Reproduction Center | Bliss S.B.,Hartman Equine Reproduction Center | And 3 more authors.
Theriogenology | Year: 2011

Effective cryopreservation of expanded equine blastocysts (> 300 μm in diameter) has been difficult, perhaps due to the volume of blastocoele fluid or the presence of the equine embryonic capsule. Recently, we reported normal viability of equine embryos after trophoblast biopsy, which resulted in blastocyst collapse. The present study addressed the effect of biopsy and resultant breach of the capsule and blastocyst collapse on survival of expanded equine blastocysts after vitrification. First, non-biopsied, small embryos (< 300 μm) were vitrified in fine-diameter microloader pipette tips using dimethylsulfoxide-containing medium (DM) or ethylene glycol-containing medium (EG). A third group was vitrified with EG, but was warmed using sucrose (EG/s). Embryos in the DM and EG/s treatments grew in culture after vitrification, and established pregnancies after transfer (3 of 12 and 3 of 6, respectively). Expanded blastocysts 300-730 μm in diameter were then biopsied and vitrified; rates of normal pregnancy (detection of embryonic heartbeat) after warming and transfer were 2 of 16 (13%) and 6 of 13 (46%) for DM and EG/s treatments, respectively (P = 0.05). Within the EG/s treatment, it appeared that greater loss of blastocoele fluid after biopsy was associated with higher survival. Therefore, an altered ("Central") biopsy technique was used to aspirate blastocoele fluid, followed by vitrification in EG/s. Pregnancy rates were 1 of 8 (13%) for embryos cultured after warming and 4 of 7 (57%) for embryos transferred immediately after warming (P = 0.1). Finally, expanded blastocysts 407 to 565 μm in diameter were biopsied from the periphery, and blastocoele fluid was removed with gentle suction. After vitrification with EG/s, this resulted in a rate of normal pregnancy of 5 of 7 (71%). These findings demonstrated that blastocoele collapse and vitrification in fine-diameter pipettes allowed successful cryopreservation of expanded equine blastocysts. © 2011 Elsevier Inc. Source


Choi Y.H.,Texas A&M University | Gustafson-Seabury A.,Texas A&M University | Velez I.C.,Texas A&M University | Hartman D.L.,Hartman Equine Reproduction Center | And 3 more authors.
Reproduction | Year: 2010

The equine embryo possesses a capsule that is considered essential for its survival. We assessed viability after breaching the capsule of early (Day 6) and expanded (Day 7 and 8) equine blastocysts by micromanipulation. The capsule was penetrated using a Piezo drill, and trophoblast biopsy samples were obtained for genetic analysis. Pregnancy rates for Day-6 embryos, which had intact zonae pellucidae at the time of recovery, were 3/3 for those biopsied immediately after recovery and 2/3 for those biopsied after being shipped overnight under warm (∼28 °C) conditions. The pregnancy rates for encapsulated Day-7 expanded blastocysts were 5/6 for those biopsied immediately and 5/6 for those biopsied after being shipped overnight warm. Two of four encapsulated Day-8 blastocysts, 790 and 1350 μm in diameter, established normal pregnancies after biopsy. Nine mares were allowed to maintain pregnancy, and they gave birth to nine normal foals. Biopsied cells from eight embryos that produced foals were subjected to whole-genome amplification. Sex was successfully determined from amplified DNA in 8/8 embryos. Identification of disease-causing mutations matched in the analyses of 6/6 samples for the sodium channel, voltage-gated, type IV, alpha subunit (SCN4A) gene and in 6/7 samples for the peptidylprolyl isomerase B (PPIB) gene, in embryo-foal pairs. Thus, the capsule of the equine embryo can be breached without impairing viability. Further work is needed to determine whether this breach is transient or permanent. These findings are relevant to the understanding of equine embryo development and to the establishment of methods for micromanipulation and embryo cryopreservation in this species. © 2010 Society for Reproduction and Fertility. Source

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