Semen biotechnology optimization for successful fertilization in Japanese quail (Coturnix japonica).

Among the reproductive biotechnologies needed to improve Japanese quail conservation and valorization, optimized conditions of semen methodologies including sampling, treatment, and artificial insemination are a prerequisite. However, they have been poorly developed due to specific physiological and behavioral features of the species. The aim of the present study was to optimize them, from semen collection/treatment up to artificial insemination procedures. We studied different parameters including semen preparation (individual/pooled, presence of foam, type and pH of extender) and zootechnical parameters (depth of insemination in the female tract, number of sperm inseminated, insemination frequency). We showed that the separation of semen from individual males was required to optimize fertility, as a prerequisite for future semen cryopreservation. The deleterious effect of mixed foam extract addition on the fertility level was demonstrated. These results highlight parameters involved in male copulatory competitions and in sperm post copulation selection. Furthermore, we took into account extender effects and standardized the zootechnical conditions of insemination. The depth of intravaginal insemination (1 cm) was a key factor, but not the number of sperm inseminated (15-60 million sperm/female). Finally, artificial inseminations with optimized conditions led to successful fertility rates (up to 80%) and a duration of the fertile period equivalent to that obtained by natural mating.

Chicken semen cryopreservation and use for the restoration of rare genetic resources.

For the past 50 yr, practices for ex situ preservation of endangered breeds have been extended. Semen and primordial germ cells, gonadic tissues have been frozen to create genetic stocks of chicken genetic diversity in cryobanks. Semen cryopreservation stays the preferred method since it is not invasive. Many protocols have been developed to cryopreserve chicken semen, but they give highly variable success rate. The aim of the present study was to standardize and prove the effectiveness of semen long-term storage for the restitution of lost families. We showed that semen straws stored for 18 yr in liquid nitrogen did not lose their fertilizing ability. We demonstrated the usefulness of cryopreserved semen stored in the French National Cryobank for the recovery of families of a subfertile experimental chicken line. In order to highlight the standardization of the cryopreserved method, different cryoprotectant protocols were also tested on a rare breed, freezing/thawing and insemination conditions were controlled. The best results were obtained using glycerol protocol, a sperm dilution of 1:4 (semen:extender). The insemination dose of 200 million sperm/female was as efficient as 400 million of sperm. Altogether, these results demonstrated the effectiveness of chicken semen long-term storage for the restoration of lost genetic resources and highlighted the importance of standardized chicken semen cryopreservation using procedures combining biophysical (cryoprotectants, freezing/thawing conditions) and zootechnical (artificial insemination) features.

The 5'-AMP-Activated Protein Kinase (AMPK) Is Involved in the Augmentation of Antioxidant Defenses in Cryopreserved Chicken Sperm.

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.