Allurin and egg jelly coat impact on in-vitro fertilization success of endangered Albanian water frog, Pelophylax shqipericus.

Amphibian egg-jelly coat plays an important role in successful fertilization and development. Here, we ask whether proteins like allurin in the jelly coats of frog eggs might influence fertilization rate success. Using in vitro fertilization of Albanian water frog, Pelophylax shqipericus, we found that body cavity eggs or eggs deprived of jelly coat were not fertilized, compromising the success of in vitro fertilization procedure. When de-jellied eggs were inseminated with sperm suspension, the fertilization efficiency is dramatically decreased even inhibited, suggesting that the gel structure is one of the major factors in the achievement of fertilization in the frogs. Fertilization of de-jellied eggs with sperm pre-treated with egg jelly coat, restored the fertilization competency. Such a result suggests that egg jelly coat probably guides the sperm to the egg surface while maintaining the fertilization ability, contributing to a successful in vitro fertilization of Pelophylax shqipericus.

Characterization of Albanian water frog, Pelophylax shqipericus, sperm traits and morphology, by using phase contrast microscopy.

Mature spermatozoa traits and morphology of endangered Albanian water frog, Pelophylax shqipericus, have been characterized for the first time through phase contrast microscopy, as part of successful implementation of in vitro fertilization technique for this species. The basic morphology of P. shqipericus spermatozoa consists of an elongated, thick, smooth-edged, and solid-staining head, continuing with a thin and long tail which usually extends 2.48 times the head length. The acrosome was not clearly discernible so the measurements were done on the head as a whole, while the middle section was better visible. Average length of head, including the acrosome and midsection was estimated to be 11.78 µm ± 0.32, while the tail length resulted 29.24 ± 1.75 µm. The average thickness of the head was shown to be 3.45 µm. The total sperm length resulted to be 41.02 ± 1.83 µm. The average sperm concentration was estimated of 25.5 × 106 /ml. Sperm amount, survival rate and motility were also measured. The sperm survival rate was maximal immediately after preparation of the suspension and tended to decrease over time of storage, reaching 50% after 72 hr. Decreased sperm motility seemed to follow the same trend as sperm viability. Sperm traits resulted to be very similar both in size and in shape with those of "Lessonae" frog group, one of the lineages of Western Palearctic species complex, suggesting a strong phylogenetic relationship among these species.