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Apricot seeds due to the presence of cyanogenic glycoside amygdalin belong to the popular "alternative cancer cures", although anticancer effect of amygdalin remains controversial. This in vivo study points to the effect of long-term peroral administration of bitter apricot seeds on bone microstructure of rabbits since chronic amygdalin toxicity in relation to bone parameters has not been investigated yet. Rabbits (n = 16) were randomly divided into four experimental groups of 4 animals each. Three experimental groups S1, S2 and S3 received commercial feed for rabbits mixed with crushed bitter apricot seeds at doses 60, 300 and 420 mg/kg bw during five months, respectively. The control (C) group received no apricot seeds. The long-term consumption of apricot seeds had no impact on total body weight, femoral weight and femoral length of rabbits. Also, microcomputed tomography (3D analysis) of cortical and trabecular bone tissues did not reveal any significant impact of amygdalin toxicity on relative bone volume, BMD, cortical bone thickness, bone surface, trabecular number, thickness, and their separation. On the other hand, histological (2D) analysis demonstrated evident changes in cortical bone microstructure consistent with a decreased density of secondary osteons in the middle part of substantia compacta due to a replacement of Haversian bone tissue by plexiform bone tissue, vasoconstriction in the primary osteons' vascular canals, Haversian canals, and decreased sizes of secondary osteons in rabbits from S1, S2 and S3 groups. These negative changes are associated with different vascularization and biomechanical properties of cortical bones.

The purpose of this study was to investigate the impact of eggshell calcium (Biomin H

The purpose of this study was to investigate the effect of alcohol exposure on liver and kidney antioxidant systems in taurine exhibition during different time periods. Mice were divided into groups: I - control; II - alcohol (2.5 g/kg b.w.); III - taurine (42.84 mg/kg b.w.); and IV - alcohol + taurine. Treatments were provided for 24 h, 14 days, and 56 days. In the liver and kidney of the alcohol group, antioxidant enzyme (superoxide dismutase, catalase, and glutathione peroxidase) activities, reduced glutathione (GSH), and malondialdehyde (MDA) levels were decreased, as compared to the control group in all time periods. Taurine was found to be effectively inhibiting oxidative action of alcohol and increasing all the tested parameters in the liver (after 24 h) and kidney (after 24 h and 14 days). Moreover, the positive effect of taurine administration on GSH and MDA levels persisted in the kidneys of mice exposed to alcohol for 56 days. In conclusion, alcohol administration led to a significant influence on antioxidant system in the liver and kidney, but simultaneous intake of taurine, along with ethanol, partly attenuated the antioxidant changes in these organs.

Taurine, a sulphur - containing amino acid, has been termed a functional nutrient. Its synthetic form is a common ingredient in supplements and energy drinks. There is no information concerning taurine impact on bone microstructure after prolonged supplemental use. Also, differences in bone parameters of mice following taurine exposure are unknown. In this study, a detailed microstructure of compact and trabecular bone tissues of mice subchronically exposed to taurine was determined. Animals (n=12) were segregated into three groups: E1 group – mice received 20 mg/kg b.w. of taurine per day during 8 weeks; E2 group – mice were fed by taurine at a dose of 40 mg/kg b.w. for 8 weeks and a control (C) group. Decreased density of secondary osteons, increased sizes of primary osteon's vascular canals (P

This study examined for the first time whether bee bread (BB, consisting of monofloral rape bee pollen) could alleviate lipid derangements and reduced bone quality in Zucker diabetic fatty (ZDF) rats, which are considered an appropriate animal model for type 2 diabetes mellitus (T2DM) investigation. Adult ZDF rats were segregated into four groups: lean non-diabetic rats (L group), obese diabetic rats untreated (C group), and those treated with the BB at two doses (500 and 700 mg/kg body weight, respectively, B1 and B2 groups) for 10 weeks. Significantly reduced levels of total cholesterol and triglyceride were recorded in the B2 group versus the C group. In both BB-treated groups, significantly increased relative volume of trabecular bone and trabecular thickness, enhanced density of secondary osteons, accelerated periosteal bone apposition, and improved blood flow were observed. A positive effect of higher dose of BB on femoral weight and cortical bone thickness was also demonstrated. Our results suggest a promising potential of BB to ameliorate T2DM-related complications associated with lipid and bone damages.

Our study aimed to investigate subacute exposure to alcohol in relation to bone microstructure of mice. Animals from experimental (E) group drank a solution composed of 15 % ethanol and water for 14 days (one remodeling cycle), while those from control (C) group drank only water. In the compact bone of E group, decreased bone formation and increased porosity were observed which corresponds with lower levels of serum alkaline phosphatase and glutathione. Alcohol significantly increased sizes of primary osteon's vascular canals and decreased those of secondary osteons, Haversian canals. Relative bone volume, bone mineral density (BMD), relative bone volume without marrow cavity were also lower in E group. On the contrary, trabecular bone microstructure did not differ significantly between E and C groups. Liver function test showed higher levels of alanine aminotransferase, aspartate aminotransferase in alcohol-fed mice. Serum calcium, phosphate were significantly lower in E group. According to our study, only changes in compact bone microstructure of mice following one remodeling cycle were observed due to both direct and indirect effects of alcohol.

The present study aimed to elucidate the structural changes in testis and epididymis of adult rats following subchronic peroral administration of cadmium at 30 mg/L, diazinon at 40 mg/L, cadmium at 30 mg/L, and diazinon at 40 mg/L, respectively. At the end of 90-day experiment, the samples of the testes and epididymis were assayed by qualitative and quantitative histological methods. The testis and epididymis weights increased following exposure to cadmium and simultaneous exposure to cadmium and diazinon. Testicular damage following cadmium and diazinon coexposure was significantly less expressive than in groups with individual administration of these compounds. Cadmium caused a significant thickening of seminiferous epithelium, cellular degeneration, and necrosis. Desquamation of immature germ cells resulted in a significant increase of intraepithelial spaces and reduced tubule volume in all experimental groups. Vascular dilation and congestion were detected in the interstitial tissue. The changes in epididymal histology in the group exposed to cadmium and group exposed simultaneously included a reduction of epithelium, necrotic epithelial cells, vasoconstriction, and interstitial edema together with mononuclear cell infiltration. Results did not indicate a synergistic or any additional effect from the simultaneous administration of both toxicants. Further research is needed to determine the significance and the mechanism of the adverse effects.

Background Humans are ubiquitously exposed to multiple environmental contaminants. Consequences of combined action on the reproductive system remain unknown. This study aimed to assess single and joint effects of cadmium and diazinon exposure on sperm quality parameters. Methods Male adult Wistar rats were randomized into 4 groups of ten animals each. Group A was used as a control, animals from group B were exposed to cadmium (30 mg/L), rats from group C were administered with diazinon (40 mg/L), and rats from group D were exposed simultaneously to cadmium (30 mg/L) and diazinon (40 mg/L) via drinking water for 90 days. Sperm morphology and motility were evaluated using a bright field microscope and a computer-assisted semen analysis. Results The percentage of motile spermatozoa and morphologically normal sperm was markedly reduced in rats from the group B. Rats from the C group showed an increase in velocity parameters, amplitude of lateral head displacement, decrease in beat-cross frequency, and an increase in abnormal sperm morphology. Simultaneous coexposure to cadmium and diazinon increased distance and velocity parameters, and amplitude of lateral head displacement. Reductions were observed in straightness, linearity, wobble, and beat-cross frequency. The decreased normal sperm morphology rates were related to defects of the sperm tail. Conclusions Exposure to cadmium and diazinon at relatively low doses impairs sperm quality and can reduce male fertility. Cadmium and diazinon caused significant changes on sperm morphology with varying effects on motility patterns. These parameters were significantly higher in the group D as compared to the group C. The findings have important implications for reproductive risk assessment of combined exposures to multiple chemicals.

Summary The aim of this study was to investigate differences in compact bone tissue microstructure between New Zealand White transgenic rabbits with human blood clotting factor VIII gene and the non-transgenic ones. The transgene was under the transcriptional control of the whey acidic protein (WAP) promoter. Altogether, 42 femora were analysed. Specimens were prepared using standard histological equipment, producing thin sections approximately 80–100 μm. Areas, perimeters, minimum and maximum diameters of vascular canals of primary osteons, Haversian canals and secondary osteons were measured. Our results indicate the basic structural pattern of the bone tissue was primary vascular longitudinal in both groups of rabbits. However, a fibrolamellar bone tissue was identified only in the transgenic rabbits. The measured variables of the vascular canals of the primary osteons, the Haversian canals and the secondary osteons were higher in transgenic rabbits in comparison with non-transgenic ones in most cases and the differences were often statistically significant (P

Summary The purpose of this investigation was to study in detail compact bone tissue microscopic structure of adult cows and pigs with an emphasis to find an adequate key for the species identification. Altogether 18 femurs were analysed. Each of the bones was sectioned at the smallest breadth of their diaphysis. Specimens were prepared using standard histological equipment, producing thin sections approximately 80–100 μm thick. The qualitative differences between investigated species were examined in anterior, posterior, medial and lateral views of thin sections. The quantitative ones were counted using the specific computer software Scion Image. We measured the area, perimeter, minimal and maximal diameter of the Haversian canals, the Haversian systems and the vascular canals of primary osteons. After that a discriminant function analysis was used for the species identification. According to our results the basic structural pattern of femur diaphysis was primary vascular plexiform in both species. However, non-vascular bone tissue was identified in cows and resorption lacunae were found between the secondary osteons in pigs. The measured variables of the Haversian canals, the Haversian systems and the primary osteon's vascular canals were higher in most cases in cows. Classification functions for investigated species give a correct classification of 64.69% of cases. This percentage value can be increased by integrating conclusions from the qualitative analysis.

Background Patulin, a toxic mold metabolite, has been found as natural contaminant of processed fruits, most notably apples, apple juices and other apple-based products. A number of adverse health effects in humans and animals are associated with patulin intoxication. The current study was performed to analyse possible toxic effects of patulin on femoral bone microstructure in adult rabbits in detail. Fourteen clinically healthy four-month-old rabbits of both sexes (6 males and 8 females) were included in the study. Animals from the experimental groups (group E♂, n = 3; group E♀, n = 4) were injected intramuscularly with patulin at dose 10 μg/kg body weight two times a week for 28 days. The dose of patulin was estimated based on the maximum permitted level of patulin for apple products intended for infants and young children. Three males and four females without patulin administration served as controls (groups C♂ and C♀). Cortical bone thickness and qualitative and quantitative histological characteristics of compact bone tissue were investigated. Results Intramuscular applications of patulin significantly increased the thickness of cortical bone in both sexes of rabbits. In patulin-exposed males, an absence of primary vascular longitudinal bone tissue near the endosteal border was observed, which could be associated with intensive bone remodeling. Femoral diaphyses of females displayed a lower number of secondary osteons in the middle part of the substantia compacta, and occurrence of the osteons near the periosteum. This could indicate alterations in bone turnover. Histomorphometrical evaluations showed significantly increased sizes of the primary osteons’ vascular canals (P

Background Chronic exposure to cadmium (Cd), even at low concentrations, has an adverse impact on the skeletal system. Histologically, primary and secondary osteons as basic structural elements of compact bone can also be affected by several toxicants leading to changes in bone vascularization and mechanical properties of the bone. The current study was designed to investigate the effect of subchronic peroral exposure to Cd on femoral bone structure including histomorphometry of the osteons in adult male rats. In our study, 20 one-month-old male Wistar rats were randomly divided into two experimental groups. In the first group, young males received a drinking water containing 30 mg of CdCl2/L, for 90 days. Ten one-month-old males without Cd intoxication served as a control group. After 90 days of daily peroral exposure, body weight, femoral weight, femoral length, cortical bone thickness and histological structure of the femora were analysed. Results We found that subchronic peroral application of Cd had no significant effect on body weight, femoral length and cortical bone thickness in adult rats. On the other hand, femoral weight was significantly increased (P

Histological sections of femoral diaphysis from adult sheep were investigated in this study. The qualitative characteristics in microstructure of the compact bone were examined in anterior, posterior, medial and lateral views; the quantitative ones were assessed using the software Scion Image. Areas, perimeters, minimum and maximum diameters of the Haversian canals, the Haversian systems and the primary osteons' vascular canals were measured. Our results indicate that ovine diaphysis of the femur is occupied mainly with primary vascular plexiform (laminar, fibrolamellar) tissue. In addition, irregular and dense Haversian bone tissues were observed. All measured variables disposed lower values in comparison with other artiodactyls (cattle and pig).

Background The role of selenium (Se) on bone microarchitecture is still poorly understood. The present study aims to investigate the macroscopic and microscopic structures of femoral bone tissue in adult male rats after subchronic peroral administration of Se. Methods Twenty one-month-old male Wistar rats were randomly divided into two experimental groups. In the first group (Se group) young males were exposed to 5 mg Na2SeO3/L in drinking water, for 90 days. Ten one-month-old males without Se administration served as a control group. At the end of the experiment, macroscopic and microscopic structures of the femurs were analysed using analytical scales, sliding instrument, and polarized light microscopy. Results The body weight, femoral length and cortical bone thickness were significantly decreased in Se group rats. These rats also displayed different microstructure in the middle part of the femur, both in medial and lateral views, where vascular canals expanded into the central area of the bone while, in control rats, these canals occurred only near the endosteal surfaces. Additionally, a smaller number of primary and secondary osteons was identified in Se group rats. Histomorphometric analyses revealed significant increases for area, perimeter, maximum and minimum diameters of primary osteons’ vascular canals but significant reductions for all measured variables of Haversian canals and secondary osteons. Conclusions Se negatively affected the macroscopic and microscopic structures of femoral bone tissue in adult male rats. The results contribute to the knowledge on damaging impact of Se on bone.

Free-living wild rodents are usually used as indicators of pollution, with elements being determined in either whole body or in specific organs. In the present study, the accumulation of cadmium (Cd), copper (Cu), iron (Fe), and zinc (Zn) in kidney, liver, testis, uterus and bone of yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) trapped in a polluted area of Nováky, Slovakia was investigated. Yellow-necked mice and bank voles were collected using standard theriological methods for wood ecosystems. All animals were adults in good physical condition. Concentrations of Cd, Cu, Fe, and Zn in all analyzed organs were determined by atomic absorption spectrophotometry. The highest concentrations of Cd and Zn were found in the bone of both species while Cu and Fe accumulated most in the uterus. Significantly higher concentrations of Cd and Cu were detected in the liver of the bank vole in comparison with the yellow-necked mouse (P0.05). Similar significantly higher levels of Cd and Zn were found in the bone of the bank vole (P0.05) than in the yellow-necked mouse, while these rodents had significantly higher Cu and Fe concentrations (P0.05) in the kidney. Significantly higher levels of Fe and Zn were detected in the testis and uterus of bank voles, respectively. On the other hand, significantly higher concentration of Cu was found in the testis of yellow-necked mice. Results of this study suggest that bank voles are more sensitive heavy metal loaded bioindicators than yellow-necked mice.

Background Exposure to cadmium (Cd) is considered a risk factor for various bone diseases in humans and experimental animals. This study investigated the acute effects of Cd on femoral bone structure of adult male rats after a single intraperitoneal administration. Methods Ten 4-month-old male Wistar rats were injected intraperitoneally with a single dose of 2 mg CdCl2/kg body weight and killed 36 h after the Cd had been injected. Ten 4-month-old males served as a control group. Differences in body weight, femoral weight, femoral length and histological structure of the femur were evaluated between the two groups of rats. The unpaired Student's t-test was used for establishment of statistical significance. Results A single intraperitoneal administration of Cd had no significant effect on the body weight, femoral weight or femoral length. On the other hand, histological changes were significant. Rats exposed to Cd had significantly higher values of area, perimeter, maximum and minimum diameters of the primary osteons' vascular canals and Haversian canals. In contrast, a significant decrease in all variables of the secondary osteons was observed in these rats. Conclusions The results indicate that, as expected, a single intraperitoneal administration of 2 mg CdCl2/kg body weight had no impact on macroscopic structure of rat's femora; however, it affected the size of vascular canals of primary osteons, Haversian canals, and secondary osteons.

Background Free-living wild rodents are often used as zoomonitors of environmental contamination. In the present study, accumulation of cadmium (Cd), copper (Cu), iron (Fe), and zinc (Zn) in critical organs of yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) trapped in a polluted area in Nováky, Slovakia was investigated. Methods Yellow-necked mice (n = 8) and bank voles (n = 10) were collected using standard theriological methods for wood ecosystems. All animals were adult males in good physical condition. The concentrations of Cd, Cu, Fe, and Zn in the liver, kidney, and bone were determined by atomic absorption spectrophotometry. Results The highest concentrations of Cd and Zn were found in the bone of both species while Cu and Fe accumulated mainly in kidney or liver. Significant higher concentrations of Cd and Cu were detected in the liver of bank voles than in yellow-necked mice. Similar significant higher levels of Cd and Zn were found in the bone of bank voles. In contrast, significant higher concentrations of Cu and Fe were present in the kidney of yellow-necked mice. Conclusions In the yellow-necked mouse and bank vole, bone seems to accumulate Cd and Zn following prolonged exposure. On the contrary, kidney and liver store Cu and Fe after a long-term environmental exposure. In the present study, bank voles seemed to be more heavy metal loaded zoomonitors than yellow-necked mice.

Heavy metal content in the femora of yellow-necked mouse (Apodemus flavicollis) and wood mouse (Apodemus sylvaticus) caught in different polluted biotopes of a low hill level in Slovakia (Novaky and Kolinany) was investigated in the present study. Length, weight and histological structure of mouse bones have also been analysed. According to our results, higher concentrations of Cd, Ni, Fe, Cu and Zn were detected in the femora of A. flavicollis from Kolinany area. Similarly, we observed higher concentrations of Ni, Fe, Cu and Zn in the bones of A. sylvaticus trapped at the same biotope. Significant differences were observed for concentrations of Ni and Zn in both species (P < 0.05). The measured values for bone length and bone weight were higher in yellow-necked mice and wood mice from Novaky locality (P < 0.01). Histological observation of thin sections from femora of A. flavicollis and A. sylvaticus revealed an outer and inner non-vascular lamellar layer around a poorly developed reticular layer. We did not identify demonstrable changes in qualitative histological characteristics of the femora between the mice (A. flavicollis and A. sylvaticus separately) from different types of polluted environment. Also, no statistically significant differences for all the measured variables of primary osteons’ vascular canals were observed. Correlation analysis in yellow-necked mouse showed high positive relation between bone weight and bone length (r = 0.66), area and perimeter (r = 0.87) and perimeter and maximum diameter (r = 0.87). In wood mouse, high positive correlation between bone weight and bone length (r = 0.80), area and perimeter (r = 0.72), area and maximum diameter (r = 0.66) and perimeter and maximum diameter (r = 0.74) was found. Our results demonstrate slightly elevated accumulation of some heavy metals in the femora of yellow-necked mouse and wood mouse from Kolinany biotope and thus give an evidence of a contamination of the environment.

Background While gross morphological changes in the skeleton between males and females are well know, differences between sexes in the histomorphology are less known. It is important to have knowledge on the bone structure of rabbits, as this is a widely used species in biomedical research. A study was performed to evaluate the association between sex and the compact bone morphology of the femoral diaphysis in juvenile rabbits. Methods Seventeen clinically healthy 2–3 month-old rabbits (9 females, 8 males) were included in the study. The rabbits were euthanized and the right femur was sampled for analysis. 70–80 microns thick bone sections of the femoral diaphysis were prepared using standard histological equipment. The qualitative histological characteristics were determined according to internationally accepted classification systems while the quantitative parameters were assessed using the software Scion Image. Areas, perimeters, minimum and maximum diameters of primary osteons' vascular canals, Haversian canals and secondary osteons were measured. Additionally, blood plasma concentrations of progesterone, corticosterone, IGF-I, testosterone and estradiol were analyzed. Results Qualitative histological characteristics were similar for both sexes. However, variations of certain quantitative histological characteristics were identified. Measured parameters of the primary osteons' vascular canals were higher in males than for females. On the other hand, females had significant higher values of secondary osteons parameters. Differences in Haversian canals parameters were only significant for minimum diameter. Conclusion The study demonstrated that quantitative histological characteristics of compact bone tissue of the femoral diaphysis in juvenile rabbits were sex dependent. The variations may be associated with different growth and modeling of the femur through influence by sex-specific steroids, mechanical loads, genetic factors and a multitude of other sources. The results can be applied in experimental studies focusing on comparison of the skeletal biology of the sexes.

In order to develop an identification key for distinguishing between several mammalian species, bone structure of their compact bone tissue was analyzed using qualitative and quantitative characteristics. Ninety femora of adult male humans, pigs, cows, sheep, rabbits, and rats were studied. The average area, perimeter, minimum, and maximum diameter of 1863 Haversian canals and 1863 secondary osteons were measured using a digital image device. The observed data were first used to evaluate inter- and intraspecies diversity. After that, we applied a discriminant function analysis for differentiation of the species by these variables. Classification functions for investigated species give cross-validated correct classification rates for 76.17% of cases. This percentage value can be increased by integrating conclusions from the qualitative analysis.

The ryanodine receptor gene (RYR1) and the estrogen receptor gene (ESR) are the best commercially used markers for predisposition of stress susceptibility (malignant hyperthermia--MH) and increased litter size, respectively. A simplified method of simultaneous detection of MH and ESR genotypes has been developed. The method is based on simultaneous amplification of fragments of two genes by multiplex PCR and subsequent digestion of the products with two restriction enzymes. The PCR and the digestion could be performed in a single tube and all genotypes could be detected by electrophoretic separation on the same agarose gel. Thus, the development of the method can decrease the cost of the sample analysis and increase the speed and efficiency of the analysis. In our study, frequencies of mutated T allele of the RYR1 gene in Large White (LW), White Meaty (WM) and Landrace (L) were 0.11, 0.13, and 0.15, respectively. Frequencies of the preferred B allele of the ESR gene in the same breeds were 0.35, 0.26, and 0.06, respectively.

Concentrations of selected heavy metals in the femora and femoral bone structure of bank (Myodes glareolus) and common (Microtus arvalis) voles from different polluted biotopes in Slovakia (Koliňany and Novaky sites) were investigated. Length, weight, and histological structure of vole bones were also analyzed. We observed higher concentrations of lead (Pb), iron (Fe), copper (Cu), and zinc (Zn) in the bones of both species from the Koliňany site. Significant differences were observed in the concentration of Fe in bank and common voles (p

Acrylamide (AA) is one of the most common toxins in foods. Its effect on bone microstructure has not been investigated. The aim of our study was to analyze the impact of acute exposure to AA on femoral bone microstructure in mice. Adult animals were treated perorally with 2 doses of AA (E1 group, 1 mg/kg b.w.) in a 24-h period and with 3 doses of AA (E2 group, 1 mg/kg b.w.) in a 48-h period. Mice exposed to AA had smaller sizes of primary osteon's vascular canals. Secondary osteons were significantly smaller in mice from E2 group; however their increased number (from 38 % to 77 %) was identified in both E1 and E2 groups. In these groups, a higher number of resorption lacunae (from 100 % to 122 %) was also found. The values for bone volume, trabecular number were increased and that for trabecular separation was decreased in mice administered AA. Significantly higher value of bone surface was observed in mice from E1 group whereas trabecular thickness was increased in E2 group. The effect of AA on microstructure of compact and trabecular bone tissues is different. In our study, one dose of AA was used and acute effects of AA were investigated. Therefore, further studies are needed to study mechanisms by which AA acts on bone.

The aim of these studies was to compare some endocrine and non-endocrine characteristics of transgenic (carrying mammary gland-specific mWAP-hFVIII gene construct) and non-transgenic rabbits. The concentrations of corticosterone, progesterone, testosterone, estradiol, insulin-like growth factor I (IGF-I) and human factor VIII (hFVIII) in the blood plasma of adult females (9 months of age, 3rd generation transgenic animals), adult males, and young females (1-2 months of age, 4th generation of transgenic animals), as well as in the milk of lactating adult females, were analyzed by using RIA. In addition, litter size and body mass of pups born by transgenic and non-transgenic females from the 3rd generation were compared. Transgenic animals were compared with their non-transgenic siblings (the same genetic and epigenetic background). Transgenesis did not influence plasma hFVIII, but significantly increased corticosterone (in all animals), reduced IGF-I (in adult males and females), testosterone and estradiol, (in young females) and altered progesterone (increase in adult males and decrease in adult females) concentrations in blood plasma. In addition, transgenic females had higher milk concentrations of testosterone, but not progesterone or IGF-I than their nontransgenic sisters. These endocrine changes were not associated with changes in litter size. Transgenic male (but not female) pups have smaller body mass than control animals. These observations demonstrate the influence of transgenesis per se on the animal growth and endocrine system (secretion of reproductive and stress steroid hormones as well as growth factors) over four generations.