Air-drying llama sperm affects DNA integrity

The objective of this study was to evaluate the effects of air-drying preservation on llama sperm DNA. Semen collections were carried out using electroejaculation under general anesthesia. A total of 16 ejaculates were processed from 4 males (n = 4, r = 4). Each sample was diluted 4:1 in a collagenase solution in TALP media, then incubated and centrifuged at 800 g for 8 min. The pellet was re-suspended to a concentration of 20 million sperm/ml in TALP. Then the samples were placed onto sterile slides forming lines and were left to dry under laminar flow for 15 min. After this, the slides were placed into Falcon centrifuge tubes and kept at 5°C. Sperm characteristics (motility, membrane function, viability and morphology) were evaluated in raw semen and in the air-dried samples kept at 5°C for 30 min. DNA evaluation (integrity and degree of chromatin condensation) was carried out in raw semen and in the air-dried samples after 30 min, 7, 14, 21, 30, and 60 days after preservation. To compare raw semen to the air-dried samples, a Wilcoxon test was used for all sperm characteristics except for DNA, where a paired Student t-test was applied. A split plot design was used to compare chromatin condensation between the different periods of preservation and a Kruskal Wallis test was used to compare DNA integrity. Motility, membrane function, viability and sperm with intact DNA decreased in the air-dried samples (p 0.05). No significant differences were observed in the percentage of sperm with condensed chromatin between the different periods of preservation (p > 0.05). On the other hand, a significant decrease in the percentage of sperm with intact DNA was observed as from day 7 of preservation (p < 0.05). In conclusion the air-drying process has a negative effect on llama sperm DNA, hence the media used will need to be improved to protect DNA and be able to implement this technique in this species.Fil: Carretero, Maria Ignacia. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Chaves, María Graciela. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; ArgentinaFil: Arraztoa, Claudia Cecilia. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fumuso, Fernanda Gabriela. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gambarotta, Mariana Carla. Universidad de Buenos Aires; ArgentinaFil: Neild, Deborah Margarita. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentin

Transfer of cooled llama embryos obtained from synchronized females

This study evaluated the efficiency of a synchronization protocol based on GnRH and PGF2α on embryo donor llamas for fixed timed mating and assessed the viability of embryos maintained at 5 ◦C and 15 ◦C for 24 h, using the Equitainer® and the Botu-BOX® as cooling devices respectively. Llamas were divided into four follicular wave groups: growth, dominance, static and regression; they received a GnRH analogue on day 0 followed by a second dose plus cloprostenol on day 8 and 15 and mating was indicated in females with a follicle ≥ 6 mm. Embryos were recovered 8 days post mating. Synchronization rate was 80% for the treated embryo donors, with no significant differences among groups. Uterine flushing was performed in 70% of the treated females (87.5% of mated llamas) and an embryo was recovered in 50%. Fourteen embryos were assigned randomly to 5 ◦C (Equitainer® group) and 15 ◦C (Botu-BOX® group) preservation for 24 h to be transferred later. In the Equitainer® group, we obtained 14% pregnancies and a female offspring was born. In the Botu-BOX® group, 28% resulted pregnant but subsequently pregnancies were lost. This protocol was effective for synchronizing follicles in growth phase in 80% of embryo donor llamas. In addition, cooling llama embryos using the Equitainer® and the Botu-BOX® as cooling devices to 5 ◦C and 15 ◦C respectively, preserves its morphology and viability for 24 h.Fil: Zampini, Enzo German. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; ArgentinaFil: Fernanda Veiga, María. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; ArgentinaFil: Arraztoa, Claudia Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; ArgentinaFil: Gallelli, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; ArgentinaFil: Moncalvo, Carla Evangelina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; ArgentinaFil: Gambarotta, Mariana. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Neild, Deborah Margarita. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; ArgentinaFil: Miragaya, Marcelo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; ArgentinaFil: Trasorras, Virginia Luz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; Argentin

In vitro inhibitory activity of IgY antibodies against Salmonella ser. newport isolated from horses

Equine salmonellosis is caused by several Salmonella serotypes, including Salmonella Newport, which cause enterocolitis and diarrhea. Treatment usually includes the administration of antibiotics. However, since multidrug-resistant Salmonella is commonly detected, alternative options to control the pathogen are needed. One of these options is the use of specific egg yolk antibodies (IgY) for passive immunotherapy. Thus, the aim of our work was to produce IgY antibodies against an equine S. Newport strain and assess their in vitro inhibitory activity. To this end, laying hens were immunized with an inactivated S. Newport strain by using either Freund's or Montanide adjuvant and egg yolk extracts were obtained. The levels of specific IgY antibodies against Salmonella in sera and egg extracts were determined by dot-blot and microagglutination. Besides, the IgY extracts were characterized by total protein analysis, SDS-PAGE, Western Blot, and inhibition of bacterial motility. IgY extracts showed high purity (87.7 to 91.8 %), high microagglutination titers, and the ability to inhibit the motility of the bacterium. The results using Montanide were similar to those using the traditional Freund's adjuvant. Thus, Montanide may also be a good adjuvant to produce IgY. IgY-technology represents a potential tool for the control of salmonellosis in horses.Instituto de PatobiologíaFil: Bustos, Carla Paola. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Bustos, Carla Paola. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bustos, Carla Paola. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Enfermedades Infecciosas; ArgentinaFil: Leiva, Carlos Leónidas. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Leiva, Carlos Leónidas. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gambarotta, Mariana. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Bioestadística; ArgentinaFil: Guida, Nora. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Enfermedades Infecciosas; ArgentinaFil: Chacana, Pablo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Chacana, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Cryoprotectant-free vitrification of llama spermatozoa: cryoloop vs sphere method, warmed rapidly or ultra-rapidly

Our objective was to determine the effect of vitrification using both cryoloop and sphere methods, combined with two warming protocols, on the percentage of motility, membrane integrity and DNA fragmentation/condensation of llama spermatozoa. Raw semen (n = 5; r = 3) was incubated with collagenase (0.1 % in Tyrode's Albumin Lactate Pyruvate, TALP, solution), centrifuged and diluted in TALP with or without 5% dimethylformamide (DMF). Two spermatozoa concentrations were used: 20 × 106 sperm/mL (cryoloops) and 5 × 106 sperm/mL (spheres). Two warming procedures were evaluated: a rapid method (30 s at 37 °C) and an ultra-rapid method (7 s at 75 °C, followed by 30 s at 37 °C). Percentages of sperm total motility, viability, membrane function, chromatin condensation and chromatin fragmentation were evaluated before and after vitrification and analyzed using Friedman's test. The only seminal parameters maintained following vitrification were: 1) chromatin condensation, in all evaluated protocols and 2) DNA integrity, with the spheres method using both warming protocols without cryoprotectants and warming rapidly with cryoprotectants. Spermatozoa vitrification is a simple and inexpensive cryopreservation method that was able to preserve llama sperm chromatin condensation and integrity, especially using the spheres method without cryoprotectants combined with warming at 37 °C for 30 s. However it failed to preserve spermatozoa motility, membranes and viability.Fil: Arraztoa, Claudia Cecilia. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Miragaya, Marcelo Horacio. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; ArgentinaFil: Chaves, María Graciela. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; ArgentinaFil: Carretero, Maria Ignacia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; ArgentinaFil: Santa Cruz, Romina Carla. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; ArgentinaFil: Giuliano, Susana María. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; ArgentinaFil: Gambarotta, Mariana Carla. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Neild, Deborah Margarita. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Area de Teriogenología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigación y Tecnología en Reproducción Animal; Argentin