Throughout three years of monitoring, female Hermann's tortoises (Testudo hermanni boettgeri) were observed during the active reproductive season. Males were present in the first year until the first mating. The percentage of females who laid eggs in each year and in each nest was observed. The eggs were individually tracked until hatching. After incubation, the eggshell mortality was analysed. We assessed the following parameters: fertilization and causes of death, such as infection and dehydration. We concluded that due to the absence of males, both the number of eggs and the number of nests per female decrease each year.
The chemical composition of the Hermann’s tortoise eggs was studied. Oleic acid presented more than half of the total fatty acid composition in the yolk. In the egg albumen, among the 18 different amino acids that were detected, glutamic acid and aspartic acid were the most common. The highest concentrations of the trace elements measured in the shell, albumen and yolk were iron.
In Hermann's tortoise, ELISA was used to analyse the hormone concentrations of progesterone, oestradiol and testosterone in the faeces (non-invasive sampling) of both genders and of different ages. This method can be used in sexually active tortoises in both genders to determine the activity of gonads based on hormone dynamics. In our research, we concluded that only in the egg laying period, based on the ratio of individual hormone levels in adult tortoise faeces, can gender be determined.
Despite the limited volume of biological material, genetic analysis showed that at the level of mitochondrial as well as nuclear DNA, the detected genetic diversity enabled identification of the genetic structure of the populations included in our study. In the analysis of 188 samples of Hermann's tortoise from five different geographical locations in Slovenia, Croatia, Serbia, Montenegro and Italy, we found three mitochondrial haplotypes that differ from each other at three nucleotide positions within the 12S rRNA gene.
We also found specimens demonstrating mtDNA heteroplasmy. Nuclear DNA genotyping was performed at 10 microsatellite loci: Test56, Test76, Test21, GmuA19, Leo21, GmuD51, Test71, Leo56, Leo71 and GmuB08. Based on the results of microsatellite analysis, the studied populations could be divided into clusters that matched the geographical origin of the samples. To answer the question of genetic determination of sex in Testudo hermanni, we sequenced
the entire genome of five sexually mature males and five sexually mature females. Analysis of the entire genomic nucleotide sequence did not support the existence of sex chromosomes in this species and did not indicate major structural variation in the autosomal genomic regions that have been associated with gender differentiation in other amphibian and reptile species.
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