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.
The ability to survive cryopreservation varies in spermatozoa from different bird species. Among the biological factors potentially responsible for such differences, species variations in membrane fluidity have a role in the restoration of the physiological state after freezing. Membrane fluidity may be assessed by measuring fluorescence polarization anisotropy with a fluorescent dye. Anistropy values are proportional to membrane rigidity and consequently inversely proportional to membrane fluidity. In the present study, polarization anisotropy of spermatozoa originating from species differing in the freezability of their semen (chicken, turkey and guinea fowl) was measured in addition to lipid composition (cholesterol/phospholipid ratio), sperm viability (membrane permeability to eosine) and morphological integrity before and after cryopreservation. The percentages of viable and normal spermatozoa in fresh sperm were highest in the chicken (87%), lowest in guinea fowl (64%), and intermediate in turkeys (69%). Anisotropy values were highest in guinea fowl (0.205), lowest in chickens (0.155), and intermediate in turkeys (0.180). As a consequence, membrane fluidity was highest in chickens and lowest in guinea fowl. Cryopreservation significantly decreased sperm viability and morphological integrity and increased anisotropy in all species but did not change the inter species hierarchy. Initial cholesterol/phospholipid ratios were lower in chickens than in guinea fowl, and intermediate in turkeys (0.25, 0.26 and 0.29, respectively). Cryopreservation induced a severe decrease in cholesterol/phospholipid ratios in turkeys and guinea fowl. Sperm membrane fluidity in chickens, turkeys and guinea fowl behaves as an indicator of sperm freezability in these species. Inter species differences for this parameter may be partly explained by differences in initial cholesterol/phospholipids content of spermatozoa. On the other hand, the rigidifying process induced by cryopreservation is not related to lipid damage by the same mechanisms.
Semen cryopreservation is a unique tool for the management of animal genetic diversity. However, the freeze-thaw process causes biochemical and physical alterations which make difficult the restoration of sperm energy-dependent functions needed for fertilization. 5’-AMP activated protein kinase (AMPK) is a key sensor and regulator of intracellular energy metabolism. Mitochondria functions are known to be severely affected during sperm cryopreservation with deleterious oxidative and peroxidative effects leading to cell integrity and functions damages. The aim of this study was thus to examine the role of AMPK on the peroxidation/antioxidant enzymes defense system in frozen-thawed sperm and its consequences on sperm functions. Chicken semen was diluted in media supplemented with or without AMPK activators (AICAR or Metformin [MET]) or inhibitor (Compound C [CC]) and then cryopreserved. AMPKα phosphorylation, antioxidant enzymes activities, mitochondrial potential, ATP, citrate, viability, acrosome reaction ability (AR) and various motility parameters were negatively affected by the freeze-thaw process while reactive oxygen species (ROS) production, lipid peroxidation (LPO) and lactate concentration were dramatically increased. AICAR partially restored superoxide dismutase (SOD), Glutathione Peroxidase (GPx) and Glutathione Reductase (GR), increased ATP, citrate, and lactate concentration and subsequently decreased the ROS and LPO (malondialdehyde) in frozen-thawed semen. Motility parameters were increased (i.e., + 23% for motility, + 34% for rapid sperm) as well as AR (+ 100%). MET had similar effects as AICAR except that catalase activity was restored and that ATP and mitochondrial potential were further decreased. CC showed effects opposite to AICAR on SOD, ROS, LPO and AR and motility parameters. Taken together, our results strongly suggest that, upon freeze-thaw process, AMPK stimulated intracellular anti-oxidative defense enzymes through ATP regulation, thus reducing ROS and lipid peroxidation, and consequently partially restoring several essential sperm functions and leading to a better quality of cryopreserved sperm.
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