Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres.

Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the species to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. For this purpose, we compared in vitro development of synchronic (Sync) or asynchronic (Async) and asynchronic with a tetraploid (Async4n) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA fragmentation. Additionally, the developmental rates of Async4n aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. Async aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by Async4n aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by Async4n aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of two-cell-fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos.

Capacitation and acrosome reaction induction on thawed Dama dama deer spermatozoa: glycine effect as cryopreservation diluent supplement.

The aim of this research was to evaluate two different diluents for sperm cryopreservation and to study functional parameters in relation to the response to heparin, lysophosphatidylcholine and progesterone, in frozen-thawed semen of fallow deer (Dama dama) during the reproductive season (brama). In this way, fallow deer can be used as a biological model of endangered cervids. Semen was obtained by electroejaculation. Heparin, progesterone and lysophosphatidylcholine were used as capacitation and acrosome reaction inducers, respectively. Capacitation and acrosome reaction were evaluated by chlorotetracycline epifluorescence technique (CTC), membrane integrity by Hypo-osmotic swelling test (HOS) and viability and acrosome integrity by trypan blue stain/DIC. Data was analyzed by ANOVA and Tukey Test (P < 0.05). Semen was cryopreserved in different diluents and Fructose-Tris-Glycine extender was selected. Capacitation with heparin at different incubation times determined that the highest capacitation percentage was obtained at 45 minutes incubation. Progesterone (1 'M) and lysophosphatidylcholine in heparin capacitated sperm induced acrosome reaction (P < 0.05). This study contributes to improve cryopreservation methods and to increase the knowledge about capacitation and acrosome reaction in vitro in deer spermatozoa, allowing an advance in the development of reproductive biotechnologies.

Reproductive traits in captive and free-ranging males of the critically endangered Iberian lynx (Lynx pardinus).

The Iberian lynx (Lynx pardinus) is the most endangered felid in the world. Adequate genetic management of in situ and ex situ populations, and linkage between both, require knowledge on male reproductive biology and factors influencing it. We examined the influence of age, free-ranging versus captive conditions and seasonality on phenotypic, endocrine and semen traits, and links between reproductive traits and male fertility. Males had relatively small testes, produced low sperm numbers, a low proportion of normal sperm, and a high proportion of motile sperm. Young (2-year-old) males had lower testosterone levels, fewer sperm, and a lower proportion of motile and normal sperm than > or =4-year-old males. No major differences were found in semen traits before and after the mating season or between free-ranging and captive males, although the latter had better sperm motility. Males with larger relative testes weight and more sperm copulated more frequently, whereas males that produced more sperm with higher motility produced more cubs per female. In conclusion, small relative testes size and low sperm quality could indicate either low levels of sperm competition or high levels of inbreeding. Young males are probably subfertile; there is a slight trend for males in the captive breeding programme to have better semen quality than wild males, and males with higher sperm production are sexually more active and more fertile. These findings have major implications for decisions regarding which males should breed, provide samples for the genetic resource bank, or participate in programmes involving the use of assisted reproductive techniques.

Adult and yearling pampas deer stags (Ozotoceros bezoarticus) display mild reproductive seasonal patterns with maximum values in autumn.

The pampas deer is an endangered species, from which reproductive biology little is known. We aimed to describe and compare the reproductive seasonal patterns of adult and yearling pampas deer stags throughout the year, including morphological traits, testosterone concentration, sperm morphology and cryoresistance pattern changes. Six adult (AS) and five yearling (YS) stags were captured with anesthetic darts once in winter, spring, summer and autumn to study morphological variables, serum testosterone and semen. Adult males were heavier, their neck girth tended to be greater and their testosterone concentration was higher than in YS. Animals were heavier in summer and autumn. Neck girth and testosterone concentration were greater in autumn. Scrotal circumference, testicular volume and gonado-somatic index varied with seasons, decreasing from winter to spring, increasing in summer and remaining in greater values in autumn. Sperm quality had maximum values from summer to winter. However, the cryoresistance ratio of motility score was greater in spring. In conclusion, in the captivity conditions, pampas deer stags seems to present a light seasonal reproductive pattern, with maximum testis size, testosterone secretion and fresh semen quality in autumn. Nevertheless, sperm cryoresistance ratio seemed to remain stable along the year. Although YS were still growing, they achieved similar semen quality than AS.

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, Head Width, Acrosome Length, Midpiece Length, Midpiece Width 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.

Isolation of Primary Fibroblast Culture from Wildlife: the Panthera onca Case to Preserve a South American Endangered Species.

Cell line establishment of somatic cells is a valuable resource to preserve genetic material of rare, difficult-to-find, endangered and giant species like Jaguar (Panthera onca), the largest South American felid. This unit focuses on the isolation and culture of fibroblasts from Jaguar skin and muscle biopsies, and ear cartilage dissection immediately after death to preserve one of the several endangered species in this biome. These culture techniques enabled us to contribute 570 samples from 45 autochthonous and endangered species, including Jaguar. The fibroblasts obtained are a part of the Genetic Bank of Buenos Aires Zoo with the 6700 samples, including tissues such as muscle, ovarian, testicular, blood, fibroblast cultures, sperm, hair, and fluids and cells from 450 individuals of 87 different species. © 2016 by John Wiley & Sons, Inc.

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 degrees C, 5s; (II) 50 degrees C, 8s and (III) 37 degrees C, 10s. 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 1h in citrate-yolk at 42 degrees C, and FMP and FMR after 2h 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 2h 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.