Sperm morphology in neotropical primates

The morphological and morphometric characterization of spermatozoa has been used as a taxonomic and phylogenetic tool for different species of mammals. We evaluated and compared the sperm morphometry of five neotropical primate species: Alouatta caraya, Ateles belzebuth and Ateles chamek of family Atelidae; and Cebus cay (=Sapajus cay) and Cebus nigritus (=Sapajus nigritus) of family Cebidae. After the collection of semen samples, the following parameters were measured on 100 spermatozoa from each specimen: Head Length, HeadWidth, Acrosome Length, Midpiece Length, MidpieceWidth and Tail Length. Considering the available literature on sperm morphometry, we gathered data of 75 individuals, from 20 species, 8 genera and 2 families. These data were superimposed on a phylogeny to infer the possible direction of evolutionary changes. Narrower and shorter spermatozoa seem to be the ancestral form for Cebidae, with a trend toward wider and larger heads in derived groups. The spermatozoa of Atelidae may show an increase in total length and midpiece length. Sperm heads would have become narrower in the more derived groups of Ateles. Sperm length may increase in the more derived species in both families. Our results are discussed in the context of sperm competition and sexual selection.Fil: Steinberg, Eliana Ruth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Ecología, Genética y Evolución. Grupo de Investigación de Biología Evolutiva; ArgentinaFil: Sestelo, Adrián J.. Ecoparque Interactivo. Laboratorio de Biotecnología Reproductiva; ArgentinaFil: Ceballos, Maria Beatriz. Ecoparque Interactivo. Laboratorio de Biotecnología Reproductiva; ArgentinaFil: Wagner, Virginia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Ecología, Genética y Evolución. Grupo de Investigación de Biología Evolutiva; ArgentinaFil: Palermo, Ana María. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Ecología, Genética y Evolución. Grupo de Investigación de Biología Evolutiva; ArgentinaFil: Mudry, Marta Dolores. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Ecología, Genética y Evolución. Grupo de Investigación de Biología Evolutiva; Argentin

Sperm characterization and identification of sperm sub-populations in ejaculates from pampas deer (Ozotoceros bezoarticus)

Pampas deer (Ozotoceros bezoarticus) is a native endangered species. Knowledge of the basic spermiogram characteristics and the morphometric descriptors is necessary to effectively develop sperm cryopreservation. In other species, sperm sub-population is related to sperm cryo-resistance. The objective was to provide a general description of the sperm, including sperm head morphometric descriptors, its repeatability, and the existence of sperm sub-populations. Sperm were obtained from adult males by electroejaculation during the breeding season. The motility score was 3.4±0.2 (mean±SEM) and progressive motility was 59.4±3.7%. Ejaculated volume was 413.9±51.0μl, the total number of sperm ejaculated was 321.2±55.4×106. Also, 63.3±3.1% of the sperm were morphologically abnormal and 23.7±2.3% had acrosome damage. The sperm head length was 7.6±0.01μm, width 4.4±0.01μm, area 28.1±0.07μm2 and the perimeter was 21.9±0.04μm. There was a positive relationship among morphometric descriptors and the motility score, overall motility and progressive motility. Also length (P=0.011), width (P=0.003), area (P=0.006) and perimeter (P=0.009) of sperm head obtained in two different collections were positively related. Overall, the low concentration, volume, overall quality and abnormal morphology, and wide variation of these variables may be a limitation for the development of sperm cryopreserved banks. There were three sperm sub-populations with different morphometric characteristics. The morphometric descriptors are maintained similarly among different collections. © 2014 Elsevier B.V

Effect of thawing procedure on cryosurvival of deer spermatozoa: Work in progress

The objective of this study was to evaluate the effect of the thawing procedure on deer semen freezability. Frozen semen from the Genetic Resource Bank (GRB) of the Zoological Park of Buenos Aires (Argentina) was used. Seven mature stags (two red deer, two Père David’s deer and three fallow deer) were used as semen donors. Semen was diluted with a TRIS-egg yolk medium, packed in 0.25 ml straws and frozen in nitrogen vapour. For thawing, the frozen straws were subjected to the following procedures: (I) 70 °C, 5 s; (II) 50 °C, 8 s and (III) 37 °C, 10 s. Freeze-thaw motility percentage (FMP) and spermatozoa rating (FMR) were determined subjectively. Viability and acrosome integrity (NAR) were also assessed and the hypo-osmotic swelling test (HOST) was used to assess membrane integrity. Freeze-thaw motility percentage, FMR and NAR were assessed after an incubation of 1 h in citrate-yolk at 42 °C, and FMP and FMR after 2 h of incubation under the same conditions. The thawing procedure did not have an effect on the seminal characteristics evaluated immediately after this process. However, differences in FMP after 2 h of incubation (P<0.05) were found between the procedures, with the best overall recovery rates after freezing and thawing found with the use of protocols II (intermediate thawing) and III (slow thawing). Therefore, thawing protocols II and III, those that provide intermediate and slow thawing rates, were the most beneficial for semen thawing of the different cervid species analysed in this study.This study was sponsored by research funds of the Ministerio Español de Ciencia y Tecnologı́a (AGL2000-0671) and by Grant 190/PA-35 from the Consejerı́a de Agricultura y Medio Ambiente of the Junta de Comunidades de Castilla-La Mancha (Spain). The authors thank ALGAR SA for lending us the Père David’s deer stags, and Ma Dolores Pérez-Guzmán, for her help during the analysis of the data. Ana J. Soler is the recipient of a scholarship from the Consejerı́a de Ciencia y Tecnologı́a de la Junta de Comunidades de Castilla-La Mancha.Peer reviewe

Tiger, Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona-Free Nuclear Transfer

The aim of this study was to evaluate three different cloning strategies in the domestic cat (Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning (iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger (Panthera tigris) donor cells. In experiment 1, zona-free (ZP-free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP-free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non-aggregated ones within Bengal and tiger groups (278.3 61.9 vs 516.8 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4-positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP-free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation.Fil: Moro, L. N.. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Jarazo, J. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Buemo, Carla Paola. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sestelo, A.. Jardín Botánico de Buenos Aires; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression

The aim of this study was to evaluate the capacity of domestic cat (Dc, Felis silvestris) oocytes to reprogram the nucleus of cheetah (Ch, Acinonyx jubatus) cells by interspecies SCNT (iSCNT), by using embryo aggregation. Dc oocytes were in vitro matured and subjected to zona pellucida free (ZP-free) SCNT or iSCNT, depending on whether the nucleus donor cell was of Dc or Ch respectively. ZP-free reconstructed embryos were then cultured in microwells individually (Dc1X and Ch1X groups) or in couples (Dc2X and Ch2X groups). Embryo aggregation improved in vitro development obtaining 27.4, 47.7, 16.7 and 28.3% of blastocyst rates in the Dc1X, Dc2X, Ch1X and Ch2X groups, respectively (P<0.05). Moreover, aggregation improved the morphological quality of blastocysts from the Dc2X over the Dc1X group. Gene expression analysis revealed that Ch1X and Ch2X blastocysts had significantly lower relative expression of OCT4, CDX2 and NANOG than the Dc1X, Dc2X and IVF control groups. The OCT4, NANOG, SOX2 and CDX2 genes were overexpressed in Dc1X blastocysts, but the relative expression of these four genes decreased in the Dc2X, reaching similar relative levels to those of Dc IVF blastocysts. In conclusion, Ch blastocysts were produced using Dc oocytes, but with lower relative expression of pluripotent and trophoblastic genes, indicating that nuclear reprogramming could be still incomplete. Despite this, embryo aggregation improved the development of Ch and Dc embryos, and normalized Dc gene expression, which suggests that this strategy could improve full-term developmental efficiency of cat and feline iSCNT embryos.Fil: Moro, Lucía Natalia. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Buemo, Carla Paola. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Jarazo, J.. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Sestelo, A.. Jardín Zoológico de Buenos Aires; ArgentinaFil: Veraguas, D.. Universidad de Concepción; ChileFil: Rodríguez Alvarez, L.. Universidad de Concepción; ChileFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentin