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      Finding an Effective Freezing Protocol for Turkey Semen: Benefits of Ficoll as Non-Permeant Cryoprotectant and 1:4 as Dilution Rate

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          Abstract

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          The most adopted biotechnology for the conservation of genetic resources in avian species is semen cryopreservation. Therefore, the identification of a reference cryopreservation procedure represents a key point for ensuring the long-term conservation of genetic diversity in birds, through the implementation of a semen cryobank. In this study, our goal was to discover an effective freezing protocol for Meleagris gallopavo in order to realize the first Italian semen cryobank of autochthonous chicken and turkey breeds within our project (TuBAvI). For this purpose, we investigated the effects of three non-permeant cryoprotectants (sucrose, trehalose, and Ficoll) and two dilution rates (1:2 and 1:4) on the in vitro cryosurvivability of turkey spermatozoa. After thawing, the best semen quality was found in semen frozen in the presence of Ficoll and diluted at a final rate of 1:4. This paper provides encouraging results, however further studies are programmed to standardize the semen cryopreservation protocol.

          Abstract

          The present study aimed to find an effective cryopreservation protocol for turkey semen through the combined use of dimethylsulfoxide (DMSO) and three non-permeant cryoprotectants (NP-CPAs), sucrose, trehalose, and Ficoll 70. In addition, the action of two dilution rates (1:2 and 1:4) were also investigated. Semen was processed according to two final dilution rates and the following treatments: Tselutin extender (TE)/DMSO (control), TE/DMSO + sucrose or trehalose 50, 100, 200, or 400 mM, and TE/DMSO + Ficoll 0.5, 0.75, 1, or 1.5 mM. In total 26 different combinations treatments were achieved. The diluted semen was filled up into straws and frozen on liquid nitrogen vapor. The post-thawing sperm quality was assessed by analyzing motility, membrane integrity, osmotic resistance, and DNA integrity. The results obtained revealed a significant effect of NP-CPA concentration on total and progressive motility, on most of the kinetic parameters, on membrane integrity and DNA integrity, while the post-thaw quality was less affected by dilution rate. The highest post-thaw quality for all sperm quality parameters assessed except curvilinear velocity (VCL) and DNA integrity were found in semen frozen with 1 mM Ficoll/1:4 ( p < 0.05). Our findings provide an important contribution for the identification of a reference procedure for turkey semen cryopreservation, in order to create the first national avian semen cryobank.

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          Avian semen cryopreservation: what are the biological challenges?

          J.A. Long (2006)
          The value of the ability to cryopreserve and store germplasm has long been recognized for indefinite preservation of genetic material, especially for at-risk populations. In contrast to domestic livestock species, cryogenic storage of poultry semen is not reliable enough for germplasm preservation. The relatively low fertilizing ability of frozen/thawed poultry sperm most likely results from physiological sensitivity to the cryogenic process coupled with the requirement for prolonged sperm functionality in the hen reproductive tract; however, the concept of defining these physiological challenges has been underemphasized. For example, alterations in membrane carbohydrate content and diminished energy production in frozen/thawed sperm have important implications for successful gamete interaction. Recent data suggests that both glycoconjugate content and adenosine triphosphate (ATP) generation are affected by cryopreservation. Moreover, susceptibility to the cryogenic process seems to vary among lines and strains of birds, as illustrated by line-specific differences in ATP concentrations of frozen/thawed sperm from pedigreed commercial layers. Research based on biochemical and molecular comparisons of sperm among lines may lead to identification of factors that influence the freezability of poultry semen.
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            Semen Cryopreservation for Ex Situ Management of Genetic Diversity in Chicken: Creation of the French Avian Cryobank 1

            The need for semen preservation in domestic birds is a result of the reduction in genetic variability of domestic bird livestock and of the increasing risk of line extinction for health and safety reasons. Cryopreservation of embryos and primordial germ cells (PGC) is not routinely feasible in birds. The project therefore involved semen frozen in optimal safety and traceable conditions. Whole blood samples were also frozen to provide samples of analyses of genomes and health status. The feasibility of using ex situ conservation, i.e., collecting biological material to be stored outside the usual production area of the species (ex situ genetic stock), to preserve and manage rare breeds was tested with 4 subfertile populations: 3 rare experimental lines used for research into energy metabolism (R+), growth (Y33), and immunity (B4/B4), reared under known health status and the oldest endangered patrimonial French breed, the Gauloise dorée with an unknown health status. A general infrastructure was set up for the health screening and remediation of diseases, collection and storage of frozen cells and 2 sites were created for the storage of frozen samples. The screening and remediation of diseases of the Gauloise dorée, which was contaminated with various Salmonella and Mycoplasma strains, was achieved by successive treatment of parents, incubated eggs and young chicks with Baytril followed by Tiamulin. For each line, 474 to 994 semen straws have been frozen, thawed, and the semen evaluated. Insemination of frozen-thawed semen into females of the same genetic origin or of an egg-type commercial breed produced chicks in every case. For the most subfertile lines, insemination with egg-type females significantly increased the reproductive success. In conclusion, we report on the benefits of a semen and blood cryobanking complex for the management of endangered lines and strains of domestic birds. Current stocks made possible the restoration of more than 96% of the initial genome. This project also provided technical solutions to resolve some of the health problems frequently encountered for gene preservation in poultry.
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              Predictors of success of semen cryopreservation in chickens.

              Semen cryopreservation is very important for the ex situ management of genetic diversity in birds but it is rarely used. This is partly because of the highly variable success rates, and this emphasizes the need for predictors of semen freezability. This study evaluated the ability of semen quality tests to predict the success rates of semen cryopreservation in chickens and the relationships between each test. Individual variations of in vitro quality tests of semen were compared to the fertility obtained with fresh and cryopreserved semen. The in vitro semen quality tests represented viability, integrity, motility (percentage of viable and morphologically normal cells (PVN); mass motility (MMOT) and different motion parameters including percentage of motile spermatozoa (PMOT)) and biophysical tests (OSM, resistance to osmotic stress; membrane fluidity (FLUID)). Different in vitro tests were significantly correlated between each other for fresh (MMOT, PVN and FLUID, many criteria of objective motility) and cryopreserved semen (MMOT, different objective motility parameters, PVN). Fertility was significantly correlated with PVN for fresh semen and PVN and different objective motility criteria for cryopreserved semen. Membrane fluidity, followed by PVN, PMOT and MMOT, measured on fresh semen samples was positively correlated with fertility obtained with cryopreserved semen. The combination of the first three tests explained 85% of the variability of fertility observed with cryopreserved semen. In conclusion, we showed that different in vitro tests of semen quality are of predictive value for the success rate of semen cryopreservation in the chicken, the most accurate being membrane fluidity.
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                Author and article information

                Journal
                Animals (Basel)
                Animals (Basel)
                animals
                Animals : an Open Access Journal from MDPI
                MDPI
                2076-2615
                03 March 2020
                March 2020
                : 10
                : 3
                : 421
                Affiliations
                [1 ]Department of Agricultural, Environmental and Food Sciences, University of Molise, 86100 Campobasso CB, Italy; michele.diiorio@ 123456unimol.it (M.D.I.); giusyrusco@ 123456gmail.com (G.R.); robertaiampietro@ 123456hotmail.it (R.I.)
                [2 ]Department of Agricultural and Environmental Science, University of Bari Aldo Moro, 70126 Bari BA, Italy; mariaantonietta.colonna@ 123456uniba.it
                [3 ]Department of Veterinary Medicine, University of Milan, 20122 Milano MI, Italy; luisa.zaniboni@ 123456unimi.it
                Author notes
                [* ]Correspondence: nicolaia@ 123456unimol.it ; Tel.: +39 0874 404697; Fax: +39 0874 404855
                Author information
                https://orcid.org/0000-0002-7454-4970
                https://orcid.org/0000-0001-9680-1264
                Article
                animals-10-00421
                10.3390/ani10030421
                7143073
                32138164
                bd080b95-5856-41c8-86a7-4f90f6d58988
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 18 January 2020
                : 01 March 2020
                Categories
                Article

                meleagris gallopavo,cryopreservation procedure,ficoll,dilution rate,sperm cryobank

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