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.

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Weight and growth data on 5,136 lambs, from 104 sires and 1,552 dams were collected during 5 yr (13 lambing seasons) of selection for high weaning weight in a single flock under range conditions. Ewes were culled based on fertility and replaced by ewe lambs selected for high weaning weight. Heritabilities of birth weight, weaning weight (30 d), preweaning daily gain, postweaning daily gain, and weight at 90 d, respectively, were .13, .09, .03, .15, and .11. Genetic correlations were generally high. Genetic and phenotypic improvements of weaning weight were 22 +/- 3.0 and 198 +/- 11.3 g per lambing season, respectively, with three lambing seasons per year. At the end of the experiment, phenotypic mean weaning weight was 2.4 kg higher than the initial values. Estimated and theoretical responses were similar in the sire population. At the 13th lambing season, average breeding value of 30-d weight of the ram population was 600 g higher than the mean initial breeding value in the base population. Projected average breeding value of the lamb population under random mating at the 23rd lambing season was equal to 404 g.

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.

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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The generally accepted method of long-term sperm preservation is freezing in liquid nitrogen. However, it is not always available. Other techniques have shown to preserve sperm for a short period of time that can be used for intracytoplasmic sperm injection (ICSI). Mouse offspring have been produced after ICSI with sperm stored in a high osmolarity medium. Also, human embryos were obtained by ICSI with air-dried sperm. Recently, equine blastocysts have been obtained by ICSI with lyophilized sperm. In our study, cleavage rate was evaluated after ICSI of equine oocytes using air-dried sperm or sperm stored in a high osmolarity medium. Oocytes were obtained from slaughterhouse ovaries by scraping individual follicles and transported in a portable incubator at 38�C for 15 h in TCM-199 buffered with HEPES and supplemented with glutamine, sodium pyruvate, LH (Bioniche Animal Health, Inc., Beltville, Ontario, Canada), FSH (Bioniche Animal Health, Inc.), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), and 10% FBS (GIBCO, Grand Island, NY, USA). On arrival, they were cultured for 6 more hours in 5% CO2 in air at 38�C in microdroplets of the same medium, without HEPES. Sperm was collected from 2 stallions of proven fertility. The ejaculate was diluted in Kenney extender, centrifuged, and the pellet was resuspended in HEPES-TALP (H-TALP). Three sperm treatments were used: (C) Control: ejaculated motile sperm processed as described above; (1) Air-dried sperm: obtained from spreading sperm on a sterile slide and drying it for 10 min in a laminar flow chamber; (2) Sperm in a high osmolarity medium: ejaculated motile sperm resuspended in H-TALP with high osmolarity (800 mOsmol). Samples from groups 1 and 2 were stored at 5�C for 2 to 3 days before being used for sperm injection. Only oocytes with an intact cytoplasm and a visible polar body were selected for injection and randomly assigned to each experimental group. Each MII oocyte was injected with 1 sperm cell and activated in Ionomicin for 10 min and DMAP for 3 h. Injected oocytes were cultured in DMEM : F10 1 : 1 with 10% FBS at 38�C in 7% O2 and 5% CO2 for 48 h. The number of cleaved embryos was recorded. Data was analyzed by chi-square test. A total of 135 MII oocytes were injected. The cleavage rate in group 1 was significantly lower than in the control group (31/71, 43.66% vs. 28/38, 73.68%) (P &lt; 0.05). No differences were observed between group (2) and control (19/29, 73.07% vs. 28/38, 73.68%) (P &gt; 0.05). This is the first report of equine oocytes fertilized by ICSI with air-dried sperm or with sperm kept in high osmolarity medium. These simple sperm preservation techniques might be an alternative option when liquid nitrogen is not available. Further studies will determine if it is possible to obtain pregnancies or even healthy offspring.