Housing European Ground Squirrels (Spermophilus citellus) for an Ex Situ Conservation Program.

European ground squirrel (Spermophilus citellus) populations have declined precipitously over the last 70 years. Its protection cannot be ensured solely by protecting its habitat; it is also necessary to protect the animals ex situ. In our study, within a European ground squirrel species protection program, we examined two elements of indoor housing technology. Knowledge of the animals' needs is essential for captive housing and breeding success, so in our tests, the animals could freely choose both nest-building materials and feed. In the nest material preference test, the animals could choose from three materials with different structures: paper, Lignocel and hay. In the feed preference test, the animals could also choose from three types of feed: commercial rabbit feed, complete rabbit feed and a natural feed mixture. The first two feeds were in granulated format, and the third was a grain feed mix. Among the nesting materials, they preferred hay, which allowed them to build better-quality nests. Among the feeds, they preferred the grain feed mix, the composition closest to their natural feed, and it was the only one that contained animal protein. Our results contribute to the successful maintenance and breeding the European ground squirrel in captivity.

The Role of PRLR Gene Polymorphisms in Milk Production in European Wild Rabbit (Oryctolagus cuniculus).

One of the problematic points of rabbit breeding is that the nutritional requirements of the kits are not fully satisfied by the does' milk production from the third week of lactation onwards. The prolactin receptor gene has a significant effect on reproductive processes, and its polymorphisms have been associated with milk production in several species (cattle, goats, sheep, and buffalo). The European wild rabbit (Oryctolagus cuniculus), has a more diverse genetic background compared to domesticated lines. In the course of our study, sequencing of the 1210 bp long segment of the PRLR gene promoter region was accomplished. We detected four point mutations (SNP1-407G > A, SNP2-496G > C, SNP3-926T> and SNP4-973A > C) and one microsatellite at position 574. In our population, the four SNPs were segregated into four genotypes: AACCCCCC, GGGGTTAA, AAGGTTAC, and GGGGTCAC. Our results show that the genotype in the homozygous form is associated with higher milk production (1564.7 ± 444.7 g) compared to the other three genotypes (AACCCCCC 1399.1 ± 326.8 g; GTGACCTT 1403.8 ± 517.1 g; GGGGTCAC 1220.0 ± 666.2 g), and the short microsatellite repeat (167 bp) also coincides with significantly higher milk production (1623.8 ± 525.1 g). These results make the marker-assisted selection possible also for domesticated lines.

Parentage testing and looking for single nucleotide markers associated with antler quality in deer (Cervus elaphus).

To provide a cost-efficient parentage testing kit for red deer (Cervus elaphus), a 63 SNP set has been developed from a high-density Illumina BovineHD BeadChip containing 777 962 SNPs after filtering of genotypes of 50 stags. The successful genotyping rate was 38.6 % on the chip. The ratio of polymorphic loci among effectively genotyped loci was 6.5 %. The selected 63 SNPs have been applied to 960 animals to perform parentage control. Thirty SNPs out of the 63 had worked on the OpenArray platform. Their combined value of the probability of identity and exclusion probability was 4.9 × 10 - 11 and 0.99803, respectively. A search for loci linked with antler quality was also performed on the genotypes of the above-mentioned stags. Association studies revealed 14 SNPs associated with antler quality, where low-quality antlers with short and thin main beam antlers had values from 1 to 2, while high-quality antlers with long and strong main beams had values between 4 and 5. The chance for a stag to be correctly identified as having high-value antlers is expected to be over 88 %.

Sex-specific differences in Juniperus communis: essential oil yield, growth-defence conflict and population sex ratio.

In plants, biomass and nutrient allocation often generate trade-offs between the different biochemical pathways conflicting the utilization of the common source among growth, reproduction and chemical defence. However, in dioecious plant species, these trade-off patterns could appear as a more contrasted problem between males and females due to the dissimilar reproduction investment. Generally, the growth ratio is higher in males than females, while females have a stronger defence than males. To understand the possible role of the sex-specific dissimilarities within the growth-defence conflict framework, we investigated the possible causes of the high variance of the essential oil yield in a dioecious evergreen species, Juniperus communis. Specifically, we tested the correlations between the essential oil yield with other individual-specific traits (e.g. sex, age), the presence of the growth-defence trade-off, and the differential growth and survival patterns between males and females through an extensive field survey with sample collection in three natural populations (Kiskunság National Park, Hungary). The individual-specific essential oil yield was also measured and served as a proxy to describe the degree of chemical defence. We found that the essential oil yield showed strong and consistent sex-specific patterns decreasing with age in adults. Contrary to the predictions, the males showed a consistently higher yield than the females. We also observed a growth-defence trade-off in males but not in females. Consistently with the growth-defence conflict hypothesis, the populations' sex ratio was male-biased, and this pattern was more evident with ageing modifying the demographic structure due to the sexually dissimilar lifespan. Our juniper study revealed a contrasting and unique essential oil accumulation driven by the complex allocation trade-off mechanisms within individuals, which could be a flexible and adaptive defence response against the increasing biotic and abiotic environmental stresses exacerbated under global climate change.

The influence of familiarity and temperature on the huddling behavior of two mouse species with contrasting social systems.

Huddling with other individuals is an effective way to reduce heat loss. This cooperative behavior requires that the individuals tolerate each other's presence at least for a certain time or under certain circumstances. In our study, we investigated the effects of ambient temperature and familiarity on the huddling behavior of two closely related mouse species, the mound-building mouse (Mus spicilegus) and the house mouse (Mus musculus musculus). While their geographic distribution overlaps, their social systems differ in many aspects. Whereas house mice are territorial, mound-building mice tolerate each other and live in groups during winter. In laboratory experiments we found that familiarity and ambient temperature influenced the huddling behavior of both species. Familiar individuals were more likely to huddle, but while mound-building mice did so at all temperatures, huddling in house mice increased with lower temperatures. Our results are consistent with the previous knowledge about these species' social systems and might provide us with more details about their sociality. Investigating huddling behavior might be a good way to measure social tolerance between individuals within a species and compare social systems of different species.

Milkweed control by food imprinted rabbits.

Many species of invasive plants are spreading out rapidly in Europe. The common milkweed occupies increasingly more area. Being poisonous, most animals will not graze on it however rabbits would be an effective organism for the biological control of milkweed. Rabbit kittens can learn the maternal diet in various ways. They prefer aromatic foods which their mother had eaten during pregnancy or lactation period, -even if it is poisonous- but they can also learn the maternal diet from the fecal pellets deposited by the mother into the nest during the nursing events. The present study was aimed to investigate if rabbit kittens can learn that the common milkweed is a potential food also. In the first 10days of their lives kits got fecal pellets originating from individuals having fed on common milkweed previously. When weaned on day 28 postpartum, these pups preferred the milkweed in the 3-way food choice test, opposite to the control group. Most surprisingly in a second experiment it was also shown that the common milkweed was also preferred by the kittens if their mother ate it not during, but one month before pregnancy.

Long-term under-masculinization in male rabbits due to maternal stress is reversed by prenatal administration of testosterone.

It is well established that in mammals prenatal exposure to exogenous testosterone has a masculinizing effect on female morphology and behavior. Fewer studies, however, have been conducted in males on this subject, and the results are controversial. In the present study, we investigated the long-term effect of administering extra prenatal testosterone (testosterone proprionate; TP) on adult male domestic rabbits' morphology and behavior using two different control groups, non-treated and vehicle injected mothers. Unexpectedly, administering the vehicle alone had a clear under-masculinizing effect on all morphological and behavioral measures; lower body mass, smaller anogenital distance and smaller chin glands, lower chin-marking activity and greater timidity. Administration of TP counteracted this effect in a dose-dependent manner such that animals exposed to the highest dose prenatally showed values on the morphological and behavioral measures equivalent to but not greater than the non-treated control group. We conclude (1) that additional testosterone beyond what male fetuses produce in utero does not result in increased masculinization, and thus, that male fetuses are less susceptible prenatally to hormonal effects than females, and (2) that presumably stress-related effects of administering the vehicle alone resulted in under-masculinization, which could be recovered by the prenatal administration of TP. These results may partly account for the contradictory findings of previous studies, and indicate the importance of including both non-treated and sham- (vehicle) treated controls in future experiments.

Anogenital distance and condition as predictors of litter sex ratio in two mouse species: a study of the house mouse (Mus musculus) and mound-building mouse (Mus spicilegus).

The Trivers - Willard hypothesis (1973) suggests that the maternal condition may affect the female's litter size and sex ratio. Since then other factors had been found. Previous findings revealed in the case of some mammalian species, that females with larger anogenital distance have smaller litters, while the sex ratio is male-biased. That has only been demonstrated in laboratory animals, while the genetic diversity of a wild population could mask the phenomenon seen in laboratory colonies. We examined the connection between morphological traits (weight and anogenital distance) and the reproductive capacity of two wild mice species, the house mouse and the mound-building mice. We showed in both species that anogenital distance and body weight correlated positively in pre-pubertal females, but not in adults. Neither the house mouse nor the mound-building mouse mothers' weight had effect on their litter's size and sex ratio. Otherwise connection was found between the mothers' anogenital distance and their litters' sex ratio in both species. The results revealed that females with larger anogenital distance delivered male biased litter in both species. The bias occurred as while the number of female pups remained the same; mothers with large anogenital distance delivered more male pups compared to the mothers with small anogenital distance. We concluded that a female's prenatal life affects her reproductive success more than previously anticipated.

Anogenital distance as a predictor of attractiveness, litter size and sex ratio of rabbit does.

In the case of some mammalian species, females with larger anogenital distance (AGD) have smaller litters, where the sex ratio is male-biased. The first aim of the present study was to test whether this phenomenon exists in the Lagomorph group, especially in rabbits. The results revealed that does with large AGD have significantly smaller and lighter litters with a male biased sex ratio; with fewer females but not more males. The second aim of the present study was to test whether males differentiate between females differing in their AGD and are thus capable of choosing the more fertile and fecund does. Males showed a stronger response to the chin marks of females with small AGD than to the marks of females with large AGD. Our results suggest that variation in the prenatal hormonal environment, reflected through variation in AGD, could have long-term consequences on mate choice and population dynamics.

Development of the social behavior of two mice species with contrasting social systems.

The house mouse (Mus musculus) and the mound-building mouse (M. spicilegus) differ in their mating and social systems. The M. musculus is polygynous and females tend to breed cooperatively while M. spicilegus is known to be monogamous and famous for its unique cooperative behavior; the communal overwintering. Mus spicilegus is considered as a highly aggressive species in the genus Mus. In the present study, we attempted to analyze the development of aggressive and sociable behavior in these species and to discover how familiarity with the target moderates the development of sociable and aggressive behaviors and moderates the differences between the species. Dyadic social interaction tests in neutral cages were performed on 21-, 60-, and 120-day-old mice of both genders and both species. Each individual was tested against a sibling and an unfamiliar individual at all three ages. Our results showed that the development of aggressive and sociable behavior with age differed between the species and sexes; however, familiarity with the other mouse moderated the effect of species on aggression. At 21 days, both genders of M. spicilegus were more aggressive against strangers than siblings. This became true of both species at 60 days. When facing a stranger, both sexes of M. spicilegus were more aggressive than M. musculus at 120 days. However, when facing a sibling, neither gender of M. spicilegus was more agonistic than M. musculus, indicating that either kinship or early social experiences elicit tolerance.

Both gas chromatography and an electronic nose reflect chemical polymorphism of juniper shrubs browsed or avoided by sheep.

Chemical polymorphism may contribute to variation in browsing damage by mammalian herbivores. Earlier, we demonstrated that essential oil concentration in juniper, Juniperus communis, was negatively associated with herbivore browsing. The aim of the present study was to characterize the volatile chemical composition of browsed and non-browsed J. communis. By using either gas chromatography with flame ionization detection (GC-FID) or an electronic nose device, we could separate sheep-browsed or non-browsed juniper shrubs by their essential oil pattern and complex odor matrix. The main components of the essential oil from J. communis were monoterpenes. We distinguished three chemotypes, dominated either by α-pinene, sabinene, or δ-3-carene. Shrubs belonging to the α-pinene- or sabinene-dominated groups were browsed, whereas all individuals with the δ-3-carene chemotype were unused by the local herbivores. The electronic nose also separated the browsed and non-browsed shrubs indicating that their odor matrix could guide sheep browsing. Responses of sheep could integrate the post-ingestive effects of plant secondary metabolites with sensory experience that stems from odor-phytotoxin interactions. Chemotype diversity could increase the survival rate in the present population of J. communis as certain shrubs could benefit from relatively better chemical protection against the herbivores.

Testosterone treatment of pregnant rabbits affects sexual development of their daughters.

The intrauterine position (IUP) may affect the sexual development of a female fetus in litter-bearing mammals. Females gestating between two males (2M) develop into masculinized adults compared to females without contiguous males (0M). Fetuses are known to secrete different types and amounts of steroid hormones during the prenatal period, and diffusing steroids may affect sexual differentiation of their neighbors, too. Exposure of elevated testosterone (T) levels generally results in masculinized anatomy and behavior in females of several rodent species. Our recent study showed that IUP-dependent masculinization is also present in the domestic rabbit, which shows large variation in sex related traits as adults. The aim of the present study was to test if the IUP effect in rabbits can be induced by exogenous testosterone treatment. By administering different doses of testosterone propionate (TP) to pregnant rabbits, and following anatomical and behavioral development of their female offspring, we obtained dose-dependent increase both in the anogenital distance (AGD) and chin-marking behavior of prenatal T exposed females. The effects of treatment corresponded to the variation due to intrauterine position in our previous study, namely, exposure to T of known external origin resulted in similar tendencies of masculinization in rabbit females as the in utero proximity to male siblings. Our results suggest that the IUP effects may have similar physiological bases across different mammalian taxa including Lagomorphs.

Diuretic treatment affects the length of torpor bouts in hibernating European ground squirrels (Spermophilus citellus).

During the hibernation season, hibernating mammals show a sequence of torpor bouts that are interrupted by periodic arousals and brief normothermic periods. The functional significance of periodic arousals is still uncertain. We hypothesized that the imbalances in water economy may play a role in the timing of periodic arousals in hibernating species. We applied furosemide, a diuretic drug, to assess whether hibernating European ground squirrels respond to elevated urine production by shortening their torpor bouts. Urine production in the treated squirrels increased and led to more frequent arousals, presumably to restore water balance by recovery of lost water from blood and tissues. The length of the subsequent normothermic phase was not affected by the diuretic treatment. Body mass change correlated primarily with the amount of voided urine. Although our study did not identify the underlying mechanism, our results support the view that water economy, and water loss may play a role in the timing of periodic arousals.

Intrauterine position influences anatomy and behavior in domestic rabbits.

In several rodent species, the sexual differentiation of a female offspring is known to be affected in utero by the testosterone produced in adjacent male littermates. The aim of this study was to investigate the effect of male neighbors on the sexual differentiation in domestic rabbits. For this, the intrauterine position (IUP) of a female offspring from unilaterally ovariectomized, multiparous mothers was determined by their birth order. Depending on the sex of the adjacent fetuses, pups were divided into 4 groups: 1. Males. 2. 2 M females (females with 2 adjacent males), 1 M females (females with 1 male neighbor), and 0 M females (females with zero adjacent male). Pups' anogenital distance (AGD) was measured at birth and on Day 180 postpartum, when spontaneous chin marking activity was also measured. Our results revealed that AGD was a reliable indicator of sex as male pups had larger AGD than females, both at birth and later on. Adjacent male fetuses had significant effect: the more adjacent male fetuses females have had the longer AGD they possessed. AGD at birth was a good predictor of AGD and behavior of adults, as 2 M does showed the longest AGD and the highest chin marking activity among females. We concluded that, similarly to rodents, proximity to males in utero affects both anatomy and behavior in rabbits.

Essential oil yield and composition reflect browsing damage of junipers.

The impact of browsing on vegetation depends on the relative density and species composition of browsers. Herbivore density and plant damage can be either site-specific or change seasonally and spatially. For juniper (Juniperus communis) forests of a sand dune region in Hungary, it has been assumed that plant damage investigated at different temporal and spatial scales would reflect selective herbivory. The level of juniper damage was tested for a possible correlation with the concentration of plant secondary metabolites (PSMs) in plants and seasonal changes in browsing pressure. Heavily browsed and nonbrowsed junipers were also assumed to differ in their chemical composition, and the spatial distribution of browsing damage within each forest was analyzed to reveal the main browser. Long-term differences in local browsing pressure were also expected and would be reflected in site-specific age distributions of distant juniper populations. The concentrations of PSMs (essential oils) varied significantly among junipers and seasons. Heavily browsed shrubs contained the lowest oil yield; essential oils were highest in shrubs bearing no damage, indicating that PSMs might contribute to reduce browsing in undamaged shrubs. There was a seasonal fluctuation in the yield of essential oil that was lower in the summer period than in other seasons. Gas chromatography (GC) revealed differences in some essential oil components, suggesting that certain chemicals could have contributed to reduced consumption. The consequential long-term changes were reflected in differences in age distribution between distant juniper forests. These results confirm that both the concentration of PSMs and specific compounds of the essential oil may play a role in selective browsing damage by local herbivores.

Arousal, but not nursing, is necessary to elicit a decreased fear reaction toward humans in rabbit (Oryctolagus cuniculus) pups.

Rabbits that are handled at the time of feeding during the first week postpartum show reduced fear of humans later in their lives as compared to unhandled controls. Effective handling has been shown to be confined to a sensitive period. Our study aimed to investigate if feeding itself (provided by a second doe, 6 hr after the standard nursing time) affects the levels of fear of humans later in life. Our results showed that (a) the prenursing state of excitement is only characteristic of the standard nursing and is not elicited by a second feeding 6 hr past the usual nursing time, repeated daily across the first week postpartum; and (b) handling linked to a second feeding 6 hr after the standard nursing does not reduce fear responses toward humans at weaning. We conclude that the aroused state, per se, is essential for the reduction of a rabbit's fear response toward humans provoked by early handling.