The aim of this study was to carry out in vitro fertilization using spermatozoa selected with Androcoll-E™ and to evaluate the efficiency of the culture medium DMEM-F12 for in vitro embryo development in the llama. Twelve adult females from 18 superstimulated (67%) were used as oocyte donors. They were superstimulated with 1500 IU of eCG and after 5 days, received a single dose of buserelin. Twenty hours post-injection, follicular aspiration was conducted by flank laparotomy. Semen collections were performed under general anesthesia by electroejaculation of the male. The ejaculates were processed with a solution of collagenase (0.1%) and an Androcoll-E™ column was used to improve the sample. Sixty nine COCs were recovered from 79 aspirated follicles (87% recovery). Only expanded COCs were used (n = 67); they were randomly placed in groups of 1-5 in Fertil-TALP and the sperm suspension (20 × 10(6) live spermatozoa/ml) was added to each fertilization microdroplet. After 24 h, they were randomly placed in one of two culture media: SOF (n = 34) or DMEM-F12 (n = 33) and incubated for 6 days in humidified atmosphere of 5% CO(2) , 5% O(2) and 90% N(2) at 38°C. The blastocyst rate was 20% (7/34) in SOF medium (3 hatched, 2 expanded and 2 early blastocysts) and 15% (5/33) in DMEM medium (all expanded blastocysts). In conclusion, using Androcoll-E™ it is possible to select good quality spermatozoa from llama ejaculates for in vitro fertilization and to produce blastocysts in DMEM-F12 medium. This is also the first time that hatched llama blastocysts have been produced after culture in a defined medium such as SOFaa.

The aim of this study was to evaluate the developmental competence and pregnancy rate of llama hatched blastocysts produced in vitro using gametes from live animals and two different culture conditions. Fifteen adult females were superstimulated with 1500 IU of eCG, eleven (73%) responded to the treatment and were used as oocyte donors. Follicular aspiration was conducted by flank laparotomy. Semen collections were performed under general anesthesia by electroejaculation of the male. Sixty-six COCs were recovered from 77 aspirated follicles (86% recovery) and were randomly placed in Fertil-TALP microdroplets with the sperm suspension (20 × 10(6)live spermatozoa/ml). After 24 h, they were placed in SOFaa medium supplemented with FCS and randomly assigned to one of two culture conditions. Culture condition 1 (CC1) consisted of 6 days of culture (n=28) and culture condition 2 (CC2) consisted of renewing the culture medium every 48 h (n=35). In CC1, the blastocyst rate was 36% (10/28) and the hatched blastocyst rate was 28% (8/28) whereas in CC2, the blastocyst rate was 34% (12/35) and the hatched blastocyst rate was 20% (7/35) (p>0.05). No pregnancies were obtained after embryo transfer (ET) of CC1 blastocysts (0/8) while one pregnancy was obtained (1/7) after transferring a hatched blastocyst from CC2. Forty-two days after the ET, the pregnancy was lost. This study represents the first report of a pregnancy in the llama after intrauterine transfer of embryos produced by in vitro fertilization using gametes from live animals.

Normal mammalian early embryonic development involves apoptosis of blastomeres as a remodeling process during differentiation, starting at the blastocyst stage. Genomic DNA has been recently detected in the blastocele fluid of human embryos and has been amplified by real-time polymerase chain reaction (PCR) to diagnose the sex of in vitro-produced human embryos. This new approach varies from conventional preimplantation genetic diagnosis in that no cells are extracted from the embryo and only the blastocele fluid is aspirated and used as a DNA sample for diagnosis. In the present work, we investigated whether the blastocele fluid of equine preimplantation embryos contains nuclear DNA and whether this DNA could be used to diagnose the sex of the embryos by conventional PCR, using specific primers that target the TSPY and AMEL equine genes. The sex of 11 of 13 in vivo-produced embryos and of four of five in vitro-produced embryos was successfully diagnosed. The PCR amplification product was analyzed using genetic sequencing reporting that the DNA present in blastocele fluid was genomic. Additionally, after polyacrylamide gel electrophoresis and silver staining, the blastocele fluid from three different embryos produced a ladder pattern characteristic of DNA fragmented during apoptosis. Therefore, the results presented in this work report that blastocele fluid from in vivo-and in vitro-produced equine embryos contains nuclear DNA which is probably originated by apoptosis of embryonic cells, and this DNA could be used to diagnose the sex of preimlpantation embryos by conventional PCR This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

The aim of this work was to evaluate the use of air-dried spermatozoa for in vitro production of equine embryos and verify if sperm extract activation and in vivo culture improve in vitro embryo production. Cooled spermatozoa (control) and air-dried spermatozoa stored for 2, 14 or 28 days were used for ICSI sperm extract, or ionomycin was used for oocyte activation, and embryos were in vitro or in vivo (in mare's oviduct) cultured for 7 days. With in vitro culture, cleavage rate was higher when activating with sperm extract (P < 0.05). No differences in embryo development were seen between the two activation treatments nor between storage periods (P > 0.05). Blastocysts were obtained with cooled spermatozoa, and morulae were achieved using in vivo culture with 28-day storage spermatozoa and ionomycin-activated oocytes. When in vivo culture was performed, sperm DNA fragmentation was assessed using the sperm chromatin dispersion test and did not show statistical correlation with cleavage nor embryo recovery rates. In conclusion, equine embryos can be produced using air-dried spermatozoa stored for several weeks. Sperm extract activation increased cleavage rates but did not improve embryo development. In vivo culture allowed intrauterine stage embryos to be achieved.

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