Semen cryopreservation in South American camelids has a low efficiency. Post-thaw viability of sperm is low, and poor results are obtained when artificial insemination is performed with cryopreserved semen, impeding advances both in accelerated genetic progress and selection. This study aimed to describe the effect of a conventional method of camelid semen cryopreservation on the llama sperm ultrastructure during cooling and freezing, using transmission and scanning electron microscopy (TEM, SEM). Sperm motility, vigor, viability, and DNA integrity during those steps were also examined. Ejaculates from five fertile adult llama males were obtained by electroejaculation. For cooling, semen samples were washed with Hepes-balanced salt solution (HBSS), diluted in Tris-citric acid-fructose egg yolk extender (TCF-EY), and then cooled until 5 • C for 24 h. For freezing, sperm samples were washed with HBSS, diluted in TCF-EY and cooled until 5 • C for 2.5 h. Samples were equilibrated with TCF-EY, supplemented with 6% glycerol at 5 • C for 20 min, and then stored in liquid nitrogen for a month before thawing. TEM and SEM analyses were carried out on sperm samples prior to cryopreservation, after cooling down until 5 • C for 2.5 and 24 h, and after the freeze-thaw process. Ultrastructural injury was noticed during cooling, even though sperm motility, vigor, viability, and DNA integrity were not significantly affected. Analysis revealed plasma membrane and acrosome damage, loss of mitochondria, and axoneme and periaxonemal structure disorganization after 2.5 h of cooling. During freezing, a significant decrease in sperm motility and viability was observed after thawing. TEM and SEM revealed prominent signs of post-thawing damage. The plasma membrane was lost or exhibited various degrees of swelling, undulation, and perforations. Besides, the sperm presented vacuoles in the nucleus and broken acrosomes. Mitochondria in the midpiece showed vacuolization and structural disorganization. In conclusion, SEM and TEM revealed that cryopreservation induced ultrastructural damages in llama sperm that initiated during cooling and intensified during freezing. These details provide valuable data for further studies to minimize cryodamage in camelid sperm.

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Early gastrula stage embryos of the purple starfish, Pisaster ochraceus, were raised for 10 days in microgravity (µG) in an Aquatic Research Facility aboard the space shuttle Endeavour (STS 77). Controls consisted of embryos raised at 1 × g (1G) in flight and embryos raised at 1G on the ground. Experimental organisms and controls were fixed on mission days (MD) 3, 4, 5, 6, and 7 and one sample was returned alive. Comparison of the µG embryos with the 1G in-flight controls and ground controls suggests that there is little difference in size and overall development. Scanning electron microscopic examination of the development of the esophageal musculature showed that the pattern of development and differentiation was normal and was the same in both the in-flight and ground controls. The esophageal muscle cells of specimens returned alive after 10 days in µG contracted normally. Detailed transmission electron microscopic examination of MD 7 embryos revealed a decreased amount of sarcoplasmic reticulum in the µG embryos compared with both MD 7 1G in-flight and ground controls. These results suggest that while exposure to µG may slow muscle differentiation slightly, it has little overall effect on embryos/larvae of up to 7-8 days of development.Résumé : Des embryons de l'étoile de mer Pisaster ochraceus en début de gastrulation ont été gardés en élevage pendant 10 jours en état de microgravité (µG) dans le laboratoire de recherche aquatique à bord de la navette spatiale Endeavour (STS 77). Les témoins utilisés étaient des embryons élevés à 1 × g (1G) en vol et des embryons élevés à 1G au sol. Les animaux expérimentaux et les témoins ont été fixés aux jours 3, 4, 5, 6 et 7 de la mission et un échantillon a été retourné au sol vivant. La comparaison entre les embryons µG, les témoins 1G en vol et les témoins au sol a révélé que les conditions de µG entraînent peu de changement marqué de la taille ou du développement global des embryons. L'examen au microscope électronique à balayage a démontré que le développement de la musculature oesophagienne et la différenciation se déroulaient normalement, de la même façon chez les témoins en vol et les témoins au sol. Les cellules musculaires de l'oesophage des spécimens retournés vivants au sol après 10 jours dans des conditions de µG se contractaient normalement. L'examen détaillé au microscope électronique ordinaire des embryons fixés au jour 7 de la mission a révélé que le reticulum sarcoplasmique des embryons µG était moins abondant que celui des témoins 1G en vol ou au sol. Ces résultats indiquent que si l'exposition à la µG ralentit légèrement la différenciation musculaire, elle n'a pas d'effet global important sur les embryons/larves jusqu'aux jours 7-8 du développement.[Traduit par la Rédaction] Crawford and Martin 1650

Our aim was to update a standard technique for embedding bone samples, with or without implants, using methyl methacrylate (MMA) to obtain reliable optical microscopy images from mineralized bone tissues and bone-implant interfaces. In addition, comparative studies were carried out using different temperatures throughout the polymerization process. Twenty-two New Zealand rabbit calvaria and femur bone samples with or without implants were used. The samples were fixed in 10% buffered formalin, dehydrated in an ascending alcohol series, and infiltrated in methyl methacrylate solutions (I, II and III). The specimens were divided into three groups. Groups 1 and 2 were polymerized at 24°C ± 5°C, while Group 3 was polymerized at 60°C. Group 1 and 2 obtained homogeneous, transparent, crystalline polymerization. Group 3 presented multiple bubbles (ø 2 -3 mm), depressions, folds and even rupture of the vial container. The MMA bone samples polymerized at room temperature, showed good embedded, low hardness index, thin cuts, and effective staining.