Gamma-aminobutyric acid (GABA) can affect physiological processes in preimplantation embryos via GABAA and GABAB receptors.

Purpose: Several widely used substances (e.g., some therapeutics or food supplements) can act on gamma-aminobutyric acid (GABA) receptors, and we investigated whether the activation of these receptors could affect the preimplantation embryo. Methods: Transcripts of all GABA receptor subunits and selected proteins were examined using quantitative RT-PCR and immunohistochemistry. To analyze the effects of receptor activation, in vitro culture of mouse preimplantation embryos with natural and synthetic GABA receptor ligands was used. Results: We detected nine GABA receptor transcripts in mouse blastocysts and 14 GABA receptor transcripts in ovulated oocytes. The results of this study indicate that ionotropic GABAA receptors can be formed from α5, ß3, and γ3 (or δ, π) subunits, GABAA-ρ receptors can be formed from ρ2 subunits and metabotropic GABA receptors can be formed from GABAB1b and GABAB2 subunits in mouse blastocysts. Supplementing the culture medium with GABA at concentrations of 2-10 mM or with specific GABAA and GABAB receptor agonists (at concentrations of 10-100 µM) significantly increased the proportion of dead cells in blastocysts. The GABA-induced effects were prevented by pretreatment of embryos with GABAA and GABAB receptor antagonists. Conclusion: The results of this study indicate that GABA and synthetic GABA receptor ligands can negatively affect preimplantation embryos via GABAA and GABAB receptors.

Dietary Supplementation of Flaxseed (Linum usitatissimum L.) Alters Ovarian Functions of Xylene-Exposed Mice.

The aim of the performed study was to examine the ability of xylene, flaxseed, and their combinations to affect morphological and endocrine indexes of murine ovaries. The 72 indexes of secondary and tertiary follicular cells, oocytes, corpora lutea, and ovarian stroma have been quantified: diameter, markers of proliferation PCNA and apoptosis caspase 3, receptors to FSH, oxytocin, estrogen (alpha and beta), and progesterone. In addition, concentrations of the ovarian hormones progesterone, estradiol, and IGF-I in the blood, as well as their production by isolated ovaries cultured with and without gonadotropins (FSH + LH mixture), were determined using histological, immunohistochemical, and immunoassay analyses. The character of xylene and flaxseed effects on ovarian functions in mice depended on the stage of ovarian folliculogenesis. It was shown that flaxseed could mitigate and prevent the major (63%) effects of xylene on the ovary. In addition, the ability of gonadotropins to affect ovarian hormone release and prevent its response to xylene has been shown. The effects of these additives could be mediated by changes in the release and reception of hormones. These observations suggest that flaxseed and possibly gonadotropins could be natural protectors of a female reproductive system against the adverse effects of xylene.

Glutamate can act as a signaling molecule in mouse preimplantation embryos†.

Free amino acids are present in the natural environment of the preimplantation embryo, and their availability can influence early embryo development. Glutamic acid is one of the amino acids with the highest concentrations in female reproductive fluids, and we investigated whether glutamic acid/glutamate can affect preimplantation embryo development by acting through cell membrane receptors. Using reverse transcription-polymerase chain reaction, we detected 15 ionotropic glutamate receptor transcripts and 8 metabotropic glutamate receptor transcripts in mouse ovulated oocytes and/or in vivo developed blastocysts. Using immunohistochemistry, we detected the expression of two α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits, three kainate receptor subunits, and member 5 metabotropic glutamate receptor protein in blastocysts. Extracellular concentrations of glutamic acid starting at 5 mM impaired mouse blastocyst development, and this fact may be of great practical importance since glutamic acid and its salts (mainly monosodium glutamate) are widely used as food additives. Experiments with glutamate receptor agonists (in combination with gene expression analysis) revealed that specific AMPA receptors (formed from glutamate receptor, ionotropic, AMPA3 [GRIA3] and/or glutamate receptor, ionotropic, AMPA4 [GRIA4] subunits), kainate receptors (formed from glutamate receptor, ionotropic, kainate 3 [GRIK3] and glutamate receptor, ionotropic, kainate 4 [GRIK4] or glutamate receptor, ionotropic, kainate 5 [GRIK5] subunits), and member 5 metabotropic glutamate receptor (GRM5) were involved in this effect. The glutamic acid-induced effects were prevented or reduced by pretreatment of blastocysts with AMPA, kainate, and GRM5 receptor antagonists, further confirming the involvement of these receptor types. Our results show that glutamic acid can act as a signaling molecule in preimplantation embryos, exerting its effects through the activation of cell membrane receptors.

Maternal overweight increased sensitivity of mouse preimplantation embryos to oxidative stress in vitro.

The aim of this study was to compare the sensitivity of mouse preimplantation embryos obtained from mothers with different body conditions to an environment with increased oxidative stress. An intergenerational dietary model based on mouse overfeeding during the intrauterine and early postnatal period was used to produce females with increased body fat content (≥ 11 %). Three different sources of oxidative stress were applied: 0.01 mM 2,2'-Azobis (2-methylpropionamidine) dihydrochloride (AAPH), free radical-generating compound; 5 mM l-Buthionine-sulfoximine (BSO), glutathione synthesis inhibitor; and 0.01 mM Sodium nitroprusside dihydrate (SNP), nitric oxide donor. Two-cell embryos isolated from controls (with 7 %-8 % body fat content) and overweight mice were cultured in vitro with selected compounds until blastocyst formation. Development of two-cell embryos isolated from overweight dams was negatively affected by the presence of BSO and SNP (P < 0.01). Similar impact was recorded in two-cell embryos obtained from control mothers only after exposure to BSO (P < 0.05). Fluorescence analysis of blastocysts recovered from overweight dams revealed reduced total cell numbers after AAPH and BSO treatment, and increased incidence of cell death after BSO and SNP. In the controls, negative impact on blastocyst quality, represented by reduced cell number, was observed only after BSO. Immunofluorescence evaluation of freshly-recovered zygotes and two-cell embryos showed that those obtained from overweight dams displayed significantly lower fluorescence signal intensity of Glutathione peroxidase 8 than those from control dams. In conclusion, the results suggest that preimplantation embryos originating from overweight mice might be more vulnerable to oxidative stress than those originating from control females.

Dietary flaxseed's protective effects on body tissues of mice after oral exposure to xylene.

Xylene is a common pollutant in the environment that enters the body of animals and humans in various ways, but most often through the respiratory tract and adversely affects their overall health. However, xylene effects after oral exposure have not been sufficiently studied. This study aimed to investigate the effects of xylene exposure on the mouse organism and to identify possible beneficial effects of flaxseed on such exposure. Eighty mice were divided into four groups: control group C (basal diet + no xylene exposure), group X (oral exposure by 400 mg/kg/day xylene), group F (10% flaxseed supplementation of basal diet), and group XF (10% dietary flaxseed + oral exposure by xylene). Experimental trial took 14 days. Clinical examination, spectroscopic analysis of tissue aminotransferases, total lactate dehydrogenase (TLDH), and acetylcholinesterase (AchE) activities, electrophoretic analysis of LDH isoenzymes, western blot and immunohistochemical analysis of apoptosis as well as routine histology of the kidneys and jejunum, and transmission electron microscopy of the liver were performed. Marked restlessness in group X and high weight losses in mice of all groups were recorded during the experiment. Xylene promoted apoptosis (caspase-3 expression) without causing marked structural changes in the liver and jejunum, although renal cortex structure was affected adversely. In the brain, liver, and kidney of mice, xylene increased levels of liver transaminases, LDH, and decreased AchE activities, reflecting cell membrane damage. Flaxseed feeding improved animal behaviour, leakage of enzymes and prevented selected tissue toxic damage induced by xylene by protecting cell membrane integrity and fluidity and by suppressing apoptosis. These results point at the protective effect of flaxseed consumption on mice.

Apoptotic cells in mouse blastocysts are eliminated by neighbouring blastomeres.

Apoptosis is a physiological process that occurs commonly during the development of the preimplantation embryo. The present work examines the ability of apoptotic embryonic cells to express a signal promoting their phagocytosis, and quantifies the ability of neighbouring, normal embryonic cells to perform that task. Microscopic analysis of mouse blastocysts revealed phosphatidylserine externalization to be 10 times less common than incidence of apoptotic cells (as detected by TUNEL). In spite of the low frequency of phosphatidylserine-flipping (in inner cell mass, no annexin V staining was recorded), fluorescence staining of the plasma membrane showed more than 20% of apoptotic cells to have been engulfed by neighbouring blastomeres. The mean frequency of apoptotic cells escaping phagocytosis by their extrusion into blastocyst cavities did not exceed 10%. Immunochemically visualised RAC1 (an enzyme important in actin cytoskeleton rearrangement) was seen in phagosome-like structures containing a nucleus with a condensed morphology. Gene transcript analysis showed that the embryonic cells expressed 12 receptors likely involved in phagocytic process (Scarf1, Msr1, Cd36, Itgav, Itgb3, Cd14, Scarb1, Cd44, Stab1, Adgrb1, Cd300lf, Cd93). In conclusion, embryonic cells possess all the necessary mechanisms for recognising, engulfing and digesting apoptotic cells, ensuring the clearance of most dying blastomeres.

Different response of embryos originating from control and obese mice to insulin in vitro.

The aim of the present work was to investigate the impact of maternal obesity on DNA methylation in ovulated oocytes, and to compare the response of in vitro-developing preimplantation embryos originating from control and obese mice to insulin. An intergenerational, diet-induced obesity model was used to produce outbred mice with an increased body weight and body fat. Two-cell and eight-cell embryos recovered from obese and control mice were cultured in a medium supplemented with 1 or 10 ng/ml insulin until blastocyst formation. In the derived blastocysts, cell proliferation, differentiation, and death rates were determined. The results of immunochemical visualization of 5-methylcytosine indicated a slightly higher DNA methylation in ovulated metaphase II oocytes recovered from obese females; however, the difference between groups did not reach statistical significance. Expanded blastocysts developed from embryos provided by control dams showed increased mean cell numbers (two and eight-cell embryos exposed to 10 ng/ml), an increased inner-cell-mass/trophectoderm ratio (two-cell embryos exposed to 1 ng/ml and eight-cell embryos exposed to 10 ng/ml), and a reduced level of apoptosis (two and eight-cell embryos exposed to 10 ng/ml). In contrast, embryos originating from obese mice were significantly less sensitive to insulin; indeed, no difference was recorded in any tested variable between the embryos exposed to insulin and those cultured in insulin-free medium. Real-time RT-PCR analysis showed a significant increase in the amount of insulin receptor transcripts in blastocysts recovered from obese dams. These results suggest that maternal obesity might modulate the mitogenic and antiapoptotic responses of preimplantation embryos to insulin.

Dietary Zinc and Fibre Source can Influence the Mineral and Antioxidant Status of Piglets.

The study investigated the effect of dietary zinc glycine chelate and potato fibre on the absorption and utilisation of Zn, Cu, Fe, and Mn; the activity of Zn-containing enzymes (superoxide dismutase, SOD; alkaline phosphatase, ALP); and zinc transporter concentrations (metalothionein1, MT1; zinc transporter1, ZnT1) in tissues, with a special emphasis on the small intestine. Twenty-four barrows (Danbred × Duroc) were randomly allotted to four diets (supplemented with 10 g/kg of crude fibre and 120 mg Zn/kg) that consisted of cellulose and either zinc sulphate (C) or zinc glycinate (ZnGly), or contained potato fibre supplemented with ZnSO4 (PF) or ZnGly (PF + ZnGly). Feeding PF can influence the Zn absorption in the small intestine due to reduced zinc transporters MT1 and ZnT1 in the jejunum. The activity of antioxidant enzyme SOD and liver ZnT1, and duodenal iron concentrations were increased in the PF treatments. Dietary ZnGly did not significantly influence the Zn distribution, but it may alter the absorption of Fe and Mn. Given the elevated content of thiol groups and the Zn/Cu ratio in plasma, as well as the altered SOD activity and MT content in the tissues, we can conclude that feeding PF and ZnGly can influence the mineral and antioxidant status of growing piglets. However, further research is needed in order to elucidate the effect of both dietary sources on the transport systems of other minerals in enterocytes.

In vitro exposure to pyrethroid-based products disrupts development of mouse preimplantation embryos.

The objective of this study was to evaluate the potential toxicity of pyrethroids (deltamethrin, permethrin, fenvalerate, λ-cyhalothrin), commercial pyrethroid-based products DECIS EW 50 (deltamethrin mixture), TOP SPOT ON STRONGER (permethrin mixture), as well as related secondary ingredients on mouse preimplantation embryo development. Two-cell stage embryos were in vitro cultured with addition of the listed chemicals until blastocyst formation. All active pyrethroids negatively affected embryonic development at 1000 µM concentration. Decreased quality of obtained blastocysts in permethrin, fenvalerate and λ-cyhalothrin-treated embryos was revealed as well. Deltamethrin showed harmful impact on embryo development at 100 µM concentration. Lower concentrations of pyrethroids (1, 10 µM) had no effect on embryo development. The presence of DECIS EW 50 containing deltamethrin at 100 µM caused degeneration of all embryos. Similarly, TOP SPOT ON STRONGER containing 100 µM of permethrin impaired embryonic development and quality of obtained blastocysts. Evaluated secondary ingredients (butylhydroxyanisol, butylhydroxytoluen, butylparaben and cyclohexanone) at corresponding concentrations showed damaging impact on preimplantation embryo development as well. Our results indicate that the embryotoxic potential of active pyrethroids is relatively low, whereas pyrethroid-based products have relatively high potential to impair mouse preimplantation development. Embryotoxicity of commercial products is probably attributable to the presence of secondary ingredients.

Glucocorticoid receptor isoforms and effects of glucocorticoids in ovulated mouse oocytes and preimplantation embryos†.

To investigate possible involvement of glucocorticoid receptor (GR) in mediating effects of maternal stress or therapeutically administered glucocorticoids on early embryo, we analyzed the expression of GR subtypes in ovulated mouse oocytes and preimplantation embryos. RT-PCR analysis results showed that GRα and GRγ transcripts are relatively highly expressed in mouse oocytes, and both transcripts are present at lower amounts in preimplantation embryos. We also detected low expression of two other splice variants, GRß and a transcript orthologous to the human GR-P subtype, mainly at the blastocyst stage. Using western blot analysis, we detected several GR protein bands that differed in size between oocytes and preimplantation embryos. To compare the effects of corticosterone (a major endogenous glucocorticoid in rodents) and dexamethasone (a synthetic glucocorticoid) on early embryos, we cultured mouse preimplantation embryos in the presence of these glucocorticoids. Corticosterone showed a strong inhibitory effect on embryo development (starting from a 50 µM concentration), without a significant influence on apoptosis incidence. On the other hand, dexamethasone induced apoptosis in early embryo cells (starting from a 1.5 µM concentration), and its effect on embryo development was less detrimental than that found with the same dose of corticosterone. In summary, our results showed that different GR subtypes are expressed in ovulated mouse oocytes and preimplantation embryos and that the composition of GR subtypes changes during early embryo development. Moreover, we found significant differences in the effects of the two glucocorticoids on early embryo development, which might be associated with activation of different GR subtypes.

Fipronil causes toxicity in mouse preimplantation embryos.

In this study the possible toxicity of phenylpyrazole fipronil, the related commercial product FIPRON spot-on as well as FIPRON spot-on secondary ingredients on the developmental capacities and quality of mouse preimplantation embryos was evaluated. During in vitro tests, isolated two-cell stage embryos were cultured in media with addition of the listed chemicals until blastocyst formation. Stereomicroscopic evaluation of in vitro produced embryos showed that fipronil at 1 µM and higher concentration negatively affected embryonic development. Fluorescence staining revealed that the obtained blastocysts displayed lower numbers of blastomeres at 10 µM concentrations and elevated incidence of cell death from 1 µM concentration. The presence of FIPRON spot-on at a concentration equivalent to 10 µM of fipronil caused massive degeneration of all embryos. Secondary ingredients (butylhydroxyanisolum, butylhydroxytoluenum) at corresponding concentrations negatively impacted the development and quality of preimplantation embryos as well. During in vivo tests (daily oral administration of fipronil during the preimplantation period) in embryos collected from treated mouse females, significantly elevated incidence of cell death was observed even at the acute reference dose. Fipronil impaired the development and quality of mouse preimplantation embryos in both in vitro and in vivo tests. Embryotoxicity of the commercial product FIPRON spot-on was potentiated by the presence of secondary ingredients.

Overweight negatively affects outcome of superovulation treatment in female mice.

Superovulatory response is characterized by a high degree of variability and unpredictability. The aim of the present experimental study was to examine whether the amount of maternal body fat can influence the efficiency of ovarian stimulation with gonadotropins. Female mice of two body condition types, normal and obese, produced in a standardized two-generation model, were subjected to ovarian stimulation using eCG and hCG followed by natural mating. Produced ova and embryos were recovered on day 1 and day 4 of pregnancy respectively, and several quantitative, qualitative and developmental parameters were evaluated in them. The overall response of mouse females with normal and elevated amounts of body fat to superovulation was similar: They produced almost the same numbers of ova and embryos on average. Conversely, a higher number of immature oocytes, non-fertilized mature oocytes and lower-stage zygotes were collected from fat females. In both groups, the majority of fertilized oocytes was able to cleave and reach the higher stages of development. However, in the group of fat mice, a lower number of blastocysts was collected, and these blastocysts showed increased incidence of apoptotic cell death. In conclusion, although the response of normal and fat mice to superovulatory treatment was similar, the quality and developmental capacities of produced ova were lower in the group of fat donors.

Exposure to neonicotinoid insecticides induces embryotoxicity in mice and rabbits.

The potential toxicity of neonicotinoids (thiacloprid, acetamiprid, thiamethoxam and clothianidin) as well as related commercial products Calypso 480SC (thiacloprid mixture), Mospilan 20SP (acetamiprid mixture) and Agita 10WG (thiamethoxam mixture) on developmental capacities and quality of preimplantation embryos was evaluated. During in vitro tests, isolated 2-cell stage mice embryos were cultured in media with various concentrations of active compounds or commercial products until blastocyst formation. As found using stereomicroscopic examination, all neonicotinoids at highest (100µM) concentration negatively affected embryonic development (P<0.001). Fluorescence staining revealed that the blastocysts obtained displayed lower numbers of blastomeres and elevated incidence of cell death. Thiacloprid and acetamiprid decreased quality of blastocysts also at 10µM concentration. From the tested products only Calypso 480SC containing 10µM of thiacloprid showed harmful impact on embryo quality. In an experiment using rabbit embryos, similar negative effect of thiacloprid in vitro was recorded. In vivo testing confirmed that blastocysts collected from thiacloprid-treated mice displayed lower total cell counts than blastocysts from controls. The sensitivity of embryonic cells to neonicotinoids is in the order of thiacloprid>acetamiprid, thiomethoxam>clothianidin. Thiacloprid impairs development and quality of both mouse and rabbit preimplantation embryos, and shows embryotoxicity even at acute reference dose.

The Responses of Mouse Preimplantation Embryos to Leptin In Vitro in a Transgenerational Model for Obesity.

The aim of the present study was to test the hypothesis that leptin can directly mediate the negative effect of maternal obesity on preimplantation embryos. As previously shown, maternal obesity retards early embryonic development in vivo and increases the incidence of apoptosis in blastocysts. When two-cell embryos isolated from control and obese mice were transferred to identical (leptin free) conditions in vitro, no differences in any growth or quality parameters were recorded, including apoptosis incidence in blastocysts. Embryos isolated from control mice responded to transfer to environments with a high concentration of leptin (10 ng/mL) with a significant increase in arrest at the first or subsequent cell cycle. However, the majority of non-arrested embryos developed into blastocysts, showing morphology comparable to those cultured in the leptin-free group. On the other hand, the exposure of embryos isolated from obese mice to high leptin concentration in vitro did not retard their development. Furthermore, these embryos developed into blastocysts, showing a lower incidence of apoptosis. In vivo-developed blastocysts recovered from obese mice showed elevated expression levels of the proapoptotic gene BAX and the insulin-responsive glucose transporter gene SLC2A4. In conclusion, elevated leptin levels have both positive and negative effects on preimplantation embryo development in vitro, a response that likely depends on the body condition of the embryo donor. Moreover, these results suggest that leptin acts as a survival factor rather than an apoptotic inductor in embryonic cells. Since no elevations in the expression of the leptin receptor gene (LEPR) or fat metabolism-associated genes (PLIN2, SLC27A4) were recorded in blastocysts recovered from obese mice, the role of leptin in mediating the effects of obesity on embryos at the peripheral level is likely lower than expected.

Effect of neonatal ß(3)-adrenoceptor agonist CL 316,243 treatment on body fat accumulation and intestinal alkaline phosphatase activity in rats from reduced nests.

INTRODUCTION: The aim of this study was to evaluate the effects of early life administration of ß3-adrenoceptor agonist (CL 316,243) on somatic and feeding parameters, obesity development as well as small intestinal enzyme activity in 21- and 40-day-old overfed male Sprague-Dawley rats. MATERIAL AND METHODS: To induce postnatal overnutrition, litter size was reduced to 4 pups/litter (small litters, SL); while in normally-nourished groups (NL) the litter size was adjusted to 10 pups/litter. From days 5 to 15, half of the suckling pups from NL and SL groups received CL 316,243 subcutaneously. From 21st to 40th day, in the post-weaning period, both control (NL, SL) and CL 316,243-treated (NL-CL, SL-CL) rats had free access to standard diet and water. Body composition was determined by magnetic resonance imaging, blood pressure was measured using non-invasive tail-cuff, and intestinal alkaline phosphatase (AP) activity was assessed by histochemistry. RESULTS: At 21 and 40 days of age the SL rats showed higher body mass, displayed higher adiposity and had significantly increased duodenal and jejunal AP activity compared with NL animals. On day 21, NL and SL rats treated with CL 316,243 showed significantly less fat deposition and jejunal AP activity than the non-treated controls. In contrast, treatment-related changes in adiposity and AP activity were not observed in 40-day-old NL-CL, SL-CL rats. CONCLUSIONS: These results indicate that early pharmacological intervention with CL did not permanently influence physiological processes involved in body weight/fat regulation, which resulted in the development of early-programmed overweight status in SL rats after weaning.

Effect of repeated fasting/refeeding on obesity development and health complications in rats arising from reduced nest.

BACKGROUND: Overnutrition during postnatal life represents a risk factor for later obesity and associated metabolic disorders. AIM: We investigated the interaction between postnatal and later-life nutrition on body composition, blood pressure and the jejunal enzyme activities in male Sprague-Dawley rats. METHODS: From birth, we adjusted the number of pups in the nest to 4 (small litters-SL; overfeeding) or to 10 pups (normal litters-NL; controls), and from day 50 until 70, the SL (SL-R) and NL (NL-R) rats were subjected to 1 day fasting and 1 day refeeding cycles (RFR). Their body composition was determined by magnetic resonance imaging, and enzyme activity was assayed histochemically. RESULTS: At 50 and 70 days, SL rats were found to be overweight (p < 0.001), with higher adiposity (p < 0.001) and blood pressure (p < 0.01). Moreover, despite significantly decreased daily food intake during RFR (SL-R 39 %, NL-R 23 %), higher fat deposition (p < 0.001) and blood pressure (p < 0.05) was detected in SL-R rats. Activity of alkaline phosphatase (AP) functionally involved in lipid absorption was significantly higher in SL than NL rats (p < 0.001) but substantially decreased in RFR groups (SL-R p < 0.001, NL-R p < 0.01). However, despite these enzymatic adaptations to reduced food intake, the SL-R rats displayed significantly higher AP activity in comparison with NL-R rats (p < 0.01) on day 70. CONCLUSIONS: Our results demonstrate that postnatal overfeeding predisposes the ontogeny of intestinal function, which may promote the probability of obesity risk. Accordingly, in these animals, efficient fat deposition and elevated blood pressure were not diminished in response to dietary restrictions in later life.

Long-term effect of altered nutrition induced by litter size manipulation and cross-fostering in suckling male rats on development of obesity risk and health complications.

OBJECTIVE: We investigated the long-term effect of pre-weaning nutrition on positive and/or adverse regulation of obesity risk and health complications in male Sprague-Dawley rats. DESIGN AND SUBJECT: Two experimental models were used in the present work: (1) To induce postnatal over- or normal nutrition, the litter size was adjusted to 4 (small litters-SL) and to 10 pups (normal litters-NL) in the nest, (2) in suckling pups at day 10, we used cross-fostering to identify the effect of altered dietary environment on their future body fat regulation, food intake, blood pressure, and the duodenal and jejunal alkaline phosphatase activity. After weaning, these control (NL, SL) and cross-fostered (NL-SL, SL-NL) groups were exposed to standard laboratory diet. RESULTS: On day 50, the SL in comparison with NL rats became heavier and displayed enhanced adiposity accompanied by significantly increased systolic blood pressure (19%) and duodenal (16%) and jejunal (21%) alkaline phosphatase (AP) activity. The impact of pre-weaning over-nutrition of NL-SL pups was associated with long-lasting positive effect on obesity. In contrast, SL-NL rats submitted until weaning to the opposite normalized feeding condition on day 50 showed significantly decreased fat deposition (21%), systolic blood pressure (20%), and AP activity in duodenum and jejunum (14%). CONCLUSIONS: These results contribute to a better understanding of how early-acquired dietary habits determine the attenuation or prevention of obesity development in later life and can provide some benefit for optimizing the future dietary strategies in young and adult obese individuals.

Effect of antibiotic treatment on intestinal microbial and enzymatic development in postnatally overfed obese rats.

OBJECTIVE: To investigate the effect of the microbiota-induced changes and early overfeeding after amoxicillin administration (a) in suckling pups via their dams up to 15 days of lactation and (b) in weaned pups on intestinal microbial/functional adaptability and obesity development in male Sprague-Dawley rats. DESIGN AND METHODS: Postnatal nutrition was elicited by adjusting the number of pups in the nest to 4 (small litters [SLs]) and 10 (normal litters [NLs]), while from days 21 to 40, both groups were fed with a standard diet. The numbers of Bacteroides/Prevotella (BAC) and Lactobacillus/Enterococcus (LAB) in the jejunum and colon were determined by fluorescence in situ hybridization technique, and jejunal alkaline phosphatase (AP), α-glucosidase and aminopeptidase activity was assayed histochemically. RESULTS: On day 40, the SL in comparison with NL animals displayed excess weight/fat gain accompanied by higher LAB and lower numbers of BAC, and with permanently higher AP activity. Moreover, these acquired changes continued in SL vs. NL rats and were not influenced by antibiotic treatment, which induced significant decrease in the quantity of LAB and BAC. CONCLUSIONS: These findings highlight the role of early life overfeeding upon the gut microbial/functional ontogeny and allow to distinguish their potential involvement in later risk of obesity.

Developmental changes of gut microflora and enzyme activity in rat pups exposed to fat-rich diet.

The aim of this study was to investigate the effect of a high-fat (HF)/energy diet on the intestinal microbiota, the alkaline phosphatase (AP) activity, and related parameters of growth and obesity during the suckling and weaning periods in male Sprague-Dawley rats. From birth, nutrition in suckling pups was manipulated by feeding rat dams either HF or a standard diet, and then after weaning, by exposure of experimental pups to the HF, and control rats to normal diet. On days 15, 20, 40 the numbers of 2 microbial groups, i.e., Bacteroides/Prevotella (BAC) and the Lactobacillus/Enterococcus (LAB) in the jejunum, were determined by fluorescent in situ hybridization technique, and the AP activity was assayed histochemically. During all investigated periods HF pups gained body fat more rapidly than control animals, but from weaning they displayed significantly stunted growth resulting in final body weight loss. Obesity in HF rats was also accompanied by higher LAB and lower numbers of BAC and with permanently higher AP activity. Correlation of these data showed significant negative correlation between LAB, AP, and weight gain and energy efficiency, and significant positive correlation of BAC and AP activity with body fat. These data support the concept that postnatal nutritional experience represents an important factor affecting the ontogeny of intestinal microbial communities and intestinal function. These acquired changes could be a component of regulatory mechanisms involved in adverse and/or positive consequences of HF diet for adiposity, body weight, and energy-balance control in later life.