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7 Pág. ; Over the last century, several reproductive biotechnologies beyond the artificial incubation of eggs were developed to improve poultry breeding stocks and conserve their genetic diversity. These include artificial insemination (AI), semen storage, diploid primordial germ cell (PGC) methodologies, and gonad tissue storage and transplantation. Currently, AI is widely used for selection purposes in the poultry industry, in the breeding of turkeys and guinea fowl, and to solve fertility problems in duck interspecies crosses for the production of mule ducklings. The decline in some wild game species has also raised interest in reproductive technologies as a means of increasing the production of fertile eggs, and ultimately the number of birds that can be raised. AI requires viable sperm to be preserved in vitro for either short (fresh) or longer periods (chilling or freezing). Since spermatozoa are the most easily accessed sex cells, they are the cell type most commonly preserved by genetic resource banks. However, the cryopreservation of sperm only preserves half of the genome, and it cannot preserve the W chromosome. For avian species, the problem of preserving oocytes and zygotes may be solved via the cryopreservation and transplantation of PGCs and gonad tissue. The present review describes all these procedures and discusses how combining these different technologies allows poultry populations to be conserved and even rapidly reconstituted. ; This work received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 677353 IMAGE. Part of this work was supported by Zoitechlab S.L. (Arquimea group) -INIA contract CON18-141 ; Peer reviewed

14 Pág. ; The present work aimed to evaluate the chromatin compaction of rooster spermatozoa along the male reproductive tract, and to study the vas deferens lining cells, potentially involved in sperm maturation. Chromomycin A3 (CMA3) was used to determine the chromatin compaction of spermatozoa from testis (T), proximal (including epididymis, V1), intermediate (V2) and distal (V3) vas deferens, and ejaculate (E). Six Birchen Leonesa roosters were used. E was obtained in vivo by dorso-ventral massage. V1, V2 and V3 sperm were obtained post mortem (six pairs of vasa deferentia), by flushing. T was obtained by washing the testes, cut in halves. The fixed cells were stained with CMA3 and propidium iodide for flow cytometry assessment. Results showed higher (P P P. ; This research was funded by the INIA project RTA2017-00034-00-00. Part of this research received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 677353 IMAGE. ; Peer reviewed

9 Pàg. ; 1. Birchen and Blue Leonesa are two endangered chicken breeds mainly raised in Curueño Valley in North Spain. The establishment of a germplasm bank to guarantee the preservation of these breeds is needed. However, cockerels from different breeder flocks can show variance in semen cryoresistance.2. The following work focused on the sperm characterisation and cryopreservation of Birchen and Blue Leonesa cockerels from four different breeders. A total of 30 semen pools were analysed. Besides conventional sperm analysis, including motility by computer-aided sperm analysis (CASA) and DNA fragmentation by TUNEL, the present study tested a double staining method (MitoTrackerTM Green FM/propidium iodide). This gave simultaneous assessment of plasma and acrosomal and mitochondrial membranes, which were previously validated by SYBR-14/PI, CASA, aniline blue and TUNEL.3. No significant differences were found among fresh semen variables between breeds and breeders. For post-thawed variables, significant differences (P < 0.05) were found between breeders in sperm viability (58.0 ± 1.90 breeder D vs. 35.2 ± 7.41 breeder A, 37.2 ± 4.09 breeder B and 22.3 ± 5.92 breeder C) and DNA fragmentation (62.4 ± 9.91 breeder C vs. 31.8 ± 7.08 breeder B and 24.5 ± 5.49 breeder D). The lowest DNA fragmentation values for semen from breeder D birds were coincident with higher integrity of the mitochondrial membrane.4. The results revealed higher sperm cryoresistance in the cockerels from one of the breeders, possibly due to differences in management system (e.g. diet, housing, control of stress elements and pathogens, reproduction practices or maintenance of genetic diversity). These differences may determine the sperm freezability, and thus the effectiveness of developing a germplasm bank. ; r ; This research is part of a project that received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement Nº 677353 IMAGE. Part of this research was funded by the project ...