Experiences with Hermann’s tortoise (Testudo hermanni) microchipping in Slovenia — Short communication

This study describes experiences obtained with microchipping of Hermann’s tortoises in Slovenia. Over a period of three years, a total of 5,128 Hermann’s tortoises from parental breeding stock were microchipped. Microchips were implanted subcutaneously in the left inguinal region. During the application of microchips, males were bleeding in 2.6% and females in 1.4% of the cases. Bleeding frequency was related to sex, animal size and environmental temperature at the time of microchipping. The presence of microchips was followed up over a period of several years. At the control check conducted a few years later, all previously microchipped tortoises were included. Out of the entire parental breeding stock, 235 (4.6%) had lost their microchips, thus 63 males (5.7%) and 172 females (4.3%) were unmarked. The possible reasons for microchip loss are migration or inactivity of the implanted microchips

Control of Hymenolepis nana infection as a measure to improve mouse colony welfare

After cannibalism had appeared in the reproductive units of a white mouse colony, treatment against confirmed Hymenolepis nana, a tapeworm with zoonotic potential, was performed on 67 mice in the reproductive and nursery units. Faecal droppings were evaluated by flotation and sedimentation methods. The sedimentation method revealed a higher number of positive results before, during and after the treatment, but the flotation method yielded some additional positive cases. In the reproductive unit, H. nana eggs were confirmed in 50% of the tested mice by the flotation and in 70% by the sedimentation method. In the nursery units, H. nana eggs were detected in 10.5% of the tested mice by the flotation and in 24.6% by the sedimentation method. A colony of mice was treated against the tapeworm H. nana with praziquantel and emodepside in doses of 2.574 mg praziquantel/100 g body mass and of 0.642 mg emodepside/100 g body mass. The content of the original pipettes (Profender®) was applied as a spot-on on the back of the neck in the area between the shoulders. The application was repeated three times at 14-day intervals. Seven days after the third therapy no H. nana was found in any of the tested mice in the reproductive or the nursery units. After the treatment, cannibalism was no longer observed. This treatment represented one of the steps aimed at improving animal welfare and preventing potential zoonotic disease. The public health significance of this cestode should receive more attention, especially among people who take care of mice, have them as pets, or feed them to reptiles

Some reproductive and population parameters in the Hermann\u27s tortoise (Testudo hermanni) in captivity

Skozi triletni monitoring smo v aktivni reprodukcijski sezoni preučevali samice grške kornjače (Testudo hermanni boettgeri). Samci so bili prisotni prvo leto, do prvega parjenja. Spremljali smo odstotek izleganja jajc pri samicah v posameznem letu in v posameznem gnezdu. Jajca smo individualno sledili do izvalitve. Po končani inkubaciji smo pregledali valilniški ostanek. Ocenili smo naslednje parametre: oplojenost in vzroke zamiranja kot so inficiranost in dehidriranost. Ugotovili smo, da se ob odsotnosti samcev vsako leto zmanjšuje tako število jajc kot število gnezd na samico. Proučevali smo kemijsko sestavo jajc grške kornjače. Oleinska kislina je predstavljala več kot polovico maščobno kislinske sestave v rumenjaku. V beljaku smo med 18 različnimi aminokislinami ugotavljali največ glutaminske in asparaginske kisline. Med elementi v sledeh smo v lupini, beljaku in rumenjaku izmerili najvišje koncentracije železa. Pri grških kornjačah obeh spolov in različne starosti smo v iztrebkih s postopkom ELISA ugotavljali koncentracije progesterona, estradiola in testosterona v iztrebkih (neinvaziven način vzorčenja). Pri spolno aktivnih kornjačah se ta metoda lahko uporablja za dokazovanje aktivnosti reproduktivnih organov pri obeh spolih na podlagi dinamike hormonov. Ugotovili smo, da se le v obdobju izleganja jajc na podlagi nivoja med posameznimi hormoni v iztrebkih odrasle kornjače lahko določi spol. Z genetsko analizo smo kljub omejenemu obsegu biološkega materiala pokazali, da tako na ravni mitohondrijske, kot tudi jedrne DNK, genetska pestrost omogoča identifikacijo genetske strukturiranosti populacij, ki so bile vključene v našo raziskavo. V analizi 188 vzorcev grške kornjače s petih različnih geografskih lokacij v Sloveniji, Hrvaški, Srbiji, Črni gori in Italiji smo našli tri mitohondrijske haplotipe, ki se med seboj razlikujejo na treh nukleotidnih pozicijah znotraj gena za 12S rRNK. Našli smo tudi osebke s heteroplazmijo mtDNK. Genotipizacijo jedrne DNK smo izvedli na 10 mikrosatelitnih lokusih: Test56, Test76, Test21, GmuA19, Leo21, GmuD51, Test71, Leo56, Leo71 in GmuB08. Na osnovi rezultatov analize mikrosatelitnih lokusov smo proučevane populacije lahko razdelili v gruče, ki se ujemajo z geografskim izvorom vzorcev. Za odgovor na vprašanje ali je spol pri Testudo hermanni determiniran z genetskimi dejavniki, smo sekvencirali celoten genom petih spolno zrelih samcev in petih spolno zrelih samic. Analiza celotnega genomskega nukleotidnega zaporedja ne potrjuje obstoja spolnih kromosomov in ne nakazuje večjih strukturnih sprememb v avtosomalnih genomskih regijah, ki so bile pri drugih vrstah dvoživk in plazilcev povezane z diferenciacijo spola.Throughout three years of monitoring, female Hermann\u27s 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\u27s 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\u27s 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

Monitoring of unhatched eggs in Hermannʼs tortoise (Testudo hermanni) after artificial incubation and possible improvements in hatching

The causes of embryonic mortality in Hermann’s tortoises (Testudo hermanni) during artificial incubation were determined. Total egg failure at the end of the hatching period was investigated. The hatching artefacts represented 19.2% (N = 3557) of all eggs (N = 18,520). The viability rate of incubated eggs was 80.8%. The eggs, i.e., embryos, were sorted according to the cause of unsuccessful hatching and subsequently analyzed. Some of the eggs were divided into two or more groups. Unfertilized eggs were confirmed in 61.0%, infected eggs in 52.5%, and eggs in various stages of desiccation in 19.1%. This group also included mummified embryos. Pseudomonas aeruginosa, Bacillus sp., Purpureocillium lilacinum, and Escherichia coli were frequently confirmed in infected eggs. Embryos were divided into three groups: embryos up to 1.0 cm—group 1 (2.2%), embryos from 1.0 cm to 1.5 cm—group 2 (5.4%) and embryos longer than 1.5 cm—group 3 (7.3%) of all unhatched eggs. Inability of embryos to peck the shell was found in 1.3%. These tortoises died shortly before hatching. Embryos still alive from the group 2 and group 3 were confirmed in 0.7% of cases. Dead and alive deformed embryos and twins were detected in the group 3 in 0.5% and 0.1% of cases, respectively. For successful artificial hatching, it is important to establish fumigation with disinfectants prior to incubation and elimination of eggs with different shapes, eggs with broken shells, and eggs weighted under 10 g. Eggs should be candled before and periodically during artificial incubation, and all unfertilized and dead embryos must be removed. Heartbeat monitor is recommended. Proper temperature and humidity, incubation of “clean” eggs on sterile substrate and control for the presence of mites is essential. Monitoring of the parent tortoises is also necessary