Effects of vitamin B6 on growth, diarrhea rate, intestinal morphology, function, and inflammatory factors expression in a high-protein diet fed to weaned piglets1.

Vitamin B6 (VB6) is an important coenzyme factor which participates in many metabolic reactions, especially amino acid metabolism. There are few reports on how VB6 mediates weaned piglet intestinal health. This study purposed to investigate dietary VB6 effects on growth, diarrhea rates, and intestinal morphology and function in weaned piglets fed a high-crude protein (22% CP) diet. Eighteen 21-d-old weaned [(Yorkshire × Landrace) × Duroc] piglets with body weights of 7.03 ± 0.15 (means ± SEM) kg were randomly assigned into 3 VB6-containing dietary treatments. Vitamin B6 content was: 0, 4, and 7 mg/kg, respectively. The feeding period lasted 14 d. The results showed that no significant difference existed for the growth performance. The 7 mg/kg VB6 group had a tendency to decrease diarrhea rate (P = 0.065). Blood biochemical parameters analysis demonstrated that total protein, cholesterol, and high-density lipoprotein significantly increased in the 7 mg/kg VB6 group (P < 0.05). In the jejunum, no significant differences were detected for villus height, villus width, crypt depth, villus height and crypt depth ratios, and positive Ki67 counts and the mRNA expression of inflammatory cytokines. Vitamin B6 significantly increased the mRNA expression of SLC6A19 and SLC6A20 (P < 0.05) and decreased the mRNA expression of SLC36A1 (P < 0.05). In the ileum, VB6 significantly increased villus height and villus width (P < 0.05) while decreased positive Ki67 cell counts for 7 mg/kg VB6 group (P < 0.05). Vitamin B6 had significantly increased the mRNA expression of interleukin-1ß, tumor necrosis factor-α,cyclo-oxygen-ase-2, and transforming growth factor-ß (P < 0.05). Vitamin B6 also had significantly increased mRNA expression of SLC6A19, SLC7A6, SLC7A7, and SLC36A1 (P < 0.05). These findings suggest that dietary supplementation with VB6 may affect the intestinal morphology and absorption and metabolism of protein in weaned piglets fed a high-protein diet by altering the expression of intestinal inflammatory cytokines and amino acid transporters.

Hepatotoxicity and renal toxicity induced by gamma-radiation and the modulatory protective effect of Ficus carica in male albino rats.

It is well-known that gamma radiation initiates generation of free radicals which prompting serious cellular damages in biological systems. In the present study, we investigated the role of Ficus carica, a natural antioxidant substance, in modulating changes in liver and kidney functions, antioxidant enzyme's gene expression, and apoptosis, in male albino rats exposed to gamma radiation. A total of 40 rats were used in this experiment and divided equally into 4 groups: Group 1, rats administered distilled H2O (Control); Group 2, rats administered F. carica; Group 3, rats irradiated; and Group 4, rats treated with F. carica and irradiated. Groups 3 and 4 were exposed to whole-body gamma radiations at a dose level of 8 Gy and with a dose rate of 0.762 Gy/min. F. carica was administered to rats by gavage, for 3 consecutive weeks, before exposure to radiation. Five rats were sacrificed from each group at intervals of 24 and 72 h after cessation of treatment. The results revealed marked increases in alanine aminotransferase and aspartate aminotransferase levels in liver, a decrease in albumin level and increase in urea level in kidney. Irradiation resulted in cytotoxic effects as indicated by elevation in antioxidant enzyme's gene expression at 24 h, the opposite was observed at 72 h. Immunohistochemical analysis revealed that cytochrome c and p53 expressions significantly increased following exposure to radiation. Oral administration of F. carica pre-irradiation as a natural product plays a modulatory protective and anti-apoptotic role against cells damaged by free radicals induced by whole-body irradiation.

Maternal ß-hydroxy-ß-methylbutyrate (HMB) supplementation during pregnancy affects early folliculogenesis in the ovary of newborn piglets.

Beta-hydroxy-beta-methylbutyrate (HMB) is a leucine metabolite with protein anabolic effects. This study was designed to determine whether prenatal HMB treatment has an effect on oogenesis and folliculogenesis in the ovary of newborn piglets. HMB decreased the number of egg nests and primordial follicles and increased the pool of developing follicles compared to the control group. Although the percentage of TUNEL-positive oocytes within the egg nests was higher in HMB-treated group no increase in the Bax/Bcl-2 ratio and active caspase-3 expression was observed. Moreover, the granulosa cell proliferation index and StAR protein expression were higher in HMB-treated group. In contrast to the control group, the expression of E-cadherins was reduced after the HMB treatment. In addition, a significant increase in the serum level of gonadotropins and steroid hormones was detected in HMB-treated piglets. In conclusion, prenatal HMB treatment dysregulates hormonal homeostasis which impairs early folliculogenesis in piglets.

Resistance of colostrum-deprived domestic lambs to infection with deer adenovirus.

Seven colostrum-deprived, 3-4-wk-old Rambouillet-Hampshire lambs were inoculated via the mucous membranes with deer adenovirus (DAdV) and monitored for clinical signs for 21 d post-inoculation at which time animals were euthanized and postmortem examinations were performed. Pre-inoculation and post-inoculation serum samples were tested for antibodies to DAdV, ovine adenovirus 7, bovine adenovirus 7, and goat adenovirus 1. Evidence for DAdV infection was determined by virus isolation, PCR tests, and histopathology with immunohistochemistry tests for DAdV. No clinical signs or lesions consistent with adenoviral hemorrhagic disease (AHD) in deer were seen in the lambs, and the lambs did not seroconvert to DAdV. DAdV was not detected by PCR, virus isolation, or immunohistochemistry in any of the samples tested from the lambs. A positive control deer similarly inoculated with DAdV developed fatal AHD 1 wk post-inoculation. Our colostrum-deprived lambs did not become infected when inoculated with DAdV.

Molecular markers of putative spermatogonial stem cells in the domestic cat.

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43 days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.

Characterization of Porcine Epidemic Diarrhea Virus Isolate US/Iowa/18984/2013 Infection in 1-Day-Old Cesarean-Derived Colostrum-Deprived Piglets.

Porcine epidemic diarrhea virus (PEDV) was first recognized in North America in April 2013 and has since caused devastating disease. The objective of this study was to characterize disease and viral detection associated with an original North American PEDV isolate inoculated in neonatal piglets. Thirty-six 1-day-old cesarean-derived and colostrum-deprived piglets were randomly assigned to the control (n = 16) or challenged group (n = 20); the latter were orogastrically inoculated with 1 ml of US/Iowa/18984/2013 PEDV isolate titered at 1 × 10(3) plaque-forming units per milliliter. Rectal swabs were collected from all piglets prior to inoculation and every 12 hours postinoculation (hpi) thereafter, with 4 control and 5 challenged piglets euthanized at 12, 24, 48, and 72 hpi. One piglet had a positive real-time quantitative polymerase chain reaction test on rectal swab at 12 hpi, and all remaining piglets were positive thereafter, with highest viral quantities detected at 24 and 36 hpi. Diarrhea was evident in 30% and 100% of challenged piglets at 18 and 24 hpi, respectively. Viral antigen was detected in enterocytes by immunohistochemistry in the duodenum and ileum of piglets euthanized at 12 hpi and was apparent throughout the small intestine of all piglets thereafter, with villus height:crypt depth ratios consistently below 4:1. Viremia was confirmed in 18 of 20 pigs at euthanasia. Clinical disease was severe and developed rapidly following infection with an original North American PEDV isolate, with lesions, viremia, and antigen detection possible by 12 hpi.

Expression and Localization of Aquaporin 4 and Aquaporin 5 along the Large Intestine of Colostrum-Suckling Buffalo Calves.

Aquaporins (AQPs) are membrane channel proteins that play a role in regulating water permeability in many tissues. To date, seven isoforms of AQPs have been reported in the gastrointestinal tract in different mammalian species. In contrast, both tissue distribution and expression of AQPs are unknown in the buffalo. The purpose of this study was to investigate the expression of both AQP4 and AQP5 mRNAs and their relative proteins in the large intestinal tracts of buffalo calves after colostrum suckling using reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. Our results revealed a diversified tissue AQP4 and AQP5 immunolocalization accompanied by their highest expression in the tissues of colostrum-suckling buffalo calves confirmed by Western blotting. In particular, AQP4 was distributed along the endothelium and enterocytes while AQP5 in the endocrine cells. These findings provide direct evidence for AQP4 and AQP5 expression in the large intestine, suggesting that different AQPs collaborate functionally and distinctively in water handling during intestinal development, especially during the first period after delivery.

Effect of colostrum and milk on small intestine expression of AQP4 and AQP5 in newborn buffalo calves.

Functional studies indicate differences in newborn gastrointestinal morphology and physiology after a meal. Both water and solutes transfer across the intestinal epithelial membrane appear to occur via aquaporins (AQPs). Given that the physiological roles of AQP4 and AQP5 in the developing intestine have not been fully established, the objective of this investigation was to determine their distribution, expression and respective mRNA in the small intestine of colostrums-suckling buffalo calves by using immunohistochemistry, Western blot, and reverse transcriptase-PCR analysis. Results showed different tissue distribution between AQP4 and AQP5 with the presence of the former along the enteric neurons and the latter in the endocrine cells. Moreover, their expression levels were high in the ileum of colostrum-suckling buffalo calves. The data present a link between feeding, intestinal development and water homeostasis, suggesting the involvement of these channel proteins in intestinal permeability and fluid secretion/absorption during this stage of development after birth.

Cloning and distribution of neuropeptide W and its receptors in pigs.

Neuropeptide W (NPW), a novel hypothalamic peptide, is an endogenous ligand for the orphan G protein-coupled receptors GPR7 (NPBWR1) and GPR8 (NPBWR2). Although several studies have implicated NPW in the regulation of feeding and energy metabolism in many species, the precise physiological function of NPW in pigs remains unclear. In this study, we cloned and sequenced NPW, GPR7, and GPR8 cDNA from pigs. NPW, GPR7, and GPR8 mRNA expression was quantified in the pig brain and peripheral tissues by semiquantitative reverse transcriptase polymerase chain reaction. Immunohistochemistry showed that NPW protein expression was limited in the brain and abundant in peripheral tissues. These results suggest that NPW is involved in the regulation of various physiological functions in pigs. The molecular and morphological data from this study provide a basis for further research on the functions of NPW in pigs.

The effects of dietary verbascoside on blood and liver oxidative stress status induced by a high n-6 polyunsaturated fatty acids diet in piglets.

Twenty-four weaned female Hypor piglets (10.9 ± 0.1 kg mean BW) were used to evaluate the antioxidant effect of a natural extract, titrated in verbascoside, on blood and liver oxidative status in relation to a high intake of n-6 PUFA, inducing oxidative stress. Piglets were assigned to 1 of 3 experimental groups; the first group was fed a diet with 9% sunflower oil (T1) and the second received the sunflower oil diet supplemented with 5 mg of verbascoside/kg feed from Verbenaceae extract (Lippia spp.; T2). The third group was fed a control diet (CTR), in which an isoenergetic replacement of oil by starch was done. Blood samples were collected at the beginning and the end of the trial (30 d). At the end of the trial, the animals were slaughtered and the liver specimens were collected. Oxidative stress markers, including total antiradical activity, superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activities, were determined in blood samples. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transferase (GGT) plasma levels were also evaluated. Immunohistochemistry and western blot analyses were performed in liver to evaluate heat shock protein (Hsp) 70, Hsp90, and Kupffer and Ito cell activation. Liver activities of SOD, GPX, and CAT were also determined. Total antiradical activity in blood and red blood cells were affected (P < 0.01) by dietary treatments. The n-6 PUFA supplementation at a high dosage for 30 d induced oxidative stress, decreasing total antiradical activity in blood and red blood cells (CTR vs. T1 + T2; P < 0.01) and plasma CAT activity (CTR vs. T1 + T2; P = 0.088) and increasing ALT value (CTR vs. T1 + T2; P < 0.01). Also, in liver, the CAT and GPX activities tended to be lower in pigs fed n-6 PUFA diets than pigs fed a control diet (CTR vs. T1 + T2; = 0.090 and = 0.085, respectively). The liver samples presented a normal architecture and no Ito and Kupffer cell activations were observed. In liver, the SOD activity tended to be lower in the T1 group (P = 0.064) than in the CTR and T2 groups. Moreover, the level of Hsp70 was higher (P < 0.01) in the T1 group than the CTR and T2 groups. These data suggest that the dose of dietary verbascoside partially restores the antioxidant status of the liver without affecting the systemic responses to oxidative stress induced by a high-fat diet.

A novel pathogenic mechanism for cerebellar lesions produced by Solanum bonariense in cattle.

Intoxication with Solanum bonariense in cattle causes cerebellar cortical degeneration with perikaryal vacuolation, axonal swelling, and death primarily of Purkinje cells, with accumulation of electron-dense residual storage bodies in membrane-bound vesicles. The pathogenesis of this disease is not fully understood. Previously, we proposed that inhibition of protein synthesis in Purkinje cells among other altered metabolic pathways could lead to cytoskeletal alterations, subsequently altering cell-specific axonal transport. In the present study, immunohistochemical and histochemical methods were used to identify neuronal cytoskeletal alterations and axonal loss, demyelination, and astrogliosis in the cerebellum of intoxicated bovines. Samples of cerebellum from 3 natural and 4 experimental cases and 2 control bovines were studied. Immunoreactivity against neurofilament (NF)-200KDa confirmed marked loss of Purkinje neurons, and phospho-NF protein, ß-tubulin, and affinity reaction against phalloidin revealed an altered perikaryal distribution of neuronal cytoskeletal proteins in the remaining Purkinje cells in intoxicated cattle. Reactive astrogliosis in every layer of the cerebellar cortex was also observed with anti-glial fibrillary acidic protein immunohistochemistry. In affected cattle, demyelination and axonal loss in the cerebellar white matter, as well as basket cell loss were demonstrated with Klüver-Barrera and Bielschowsky stains, respectively. Based on these results, we propose that neuronal cytoskeletal alterations with subsequent interference of the axonal transport in Purkinje cells may play a relevant role in the pathogenesis of this neurodegenerative disorder, and also that demyelination and axonal loss in the cerebellar white matter, as well as astrogliosis in the gray matter, likely occur secondarily to Purkinje cell degeneration and death.

Immunization with recombinant 3-1E protein in AbISCO®-300 adjuvant induced protective immunity against Eimeria acervulina infection in chickens.

Immunostimulating complexes (ISCOMs), a kind of novel antigen presenting system, could enhance immune protection by antigen presentation. AbISCO®-300 comprising purified saponin, cholesterol and phosphatidyl choline is an effective ISCOM adjuvant. To evaluate the immune protection of recombinant 3-1E protein against Eimeria acervulina infection, chickens were immunized with recombinant 3-1E protein in combination with AbISCO®-300 or recombinant 3-1E protein alone in this study. The protective immunity was assessed with body weight gain, fecal oocyst output, detection of intestinal IgA positive cells and percentages of CD3(+), CD4(+) or CD8(+) intestinal intraepithelial lymphocytes (IELs). Chickens vaccinated with different doses of recombinant 3-1E protein plus AbISCO®-300 showed higher percentages of CD3(+), CD4(+), and CD8(+) intestinal IELs, increased positive expression rate of intestinal IgA, increased body weight gains and decreased oocyst shedding compared with recombinant 3-1E protein-only vaccinated groups. The results showed that immunization with various doses of the recombinant 3-1E protein in AbISCO®-300 adjuvant enhanced immune protection against avian coccidiosis.

Cerebellar ataxia suspected to be caused by Oxytropis glabra poisoning in western Mongolian goats.

In the last five years in western Mongolia, a neurological disorder and resultant economic loss have developed in goats, sheep, cattle and horses: association of the disease with ingestion of Oxytropis glabra, a toxic plant, was suggested. Affected goats showed neurological signs, including ataxia, incoordination, hind limb paresis, fine head tremor and nystagmus. Three goats, one with moderate clinical signs and the other two with severe clinical signs, were necropsied and examined to describe and characterize the histologic, immunohistochemical and ultrastructural lesions. Although no gross pathological changes were observed in a variety of organs including the central nervous system of these goats, microscopic examination of the cerebellum demonstrated degenerative changes in all these goats, such as vacuolar changes and loss of Purkinje cells, torpedo formation in the granular layer, increased number of spheroids in the cerebellar medulla, and loss of axons and myelin sheaths of Purkinje cells. The chemical analysis of the dried plant detected 0.02-0.05% (dry weight basis) of swainsonine. This is the first report describing the clinical and pathological findings in Mongolian goats suspected to be affected by O. glabra poisoning.

Effect of vitamin C on growth of caprine spermatogonial stem cells in vitro.

The genetic manipulation of spermatogonial stem cells (SSCs) can be used for the production of transgenic animals in a wide range of species. However, this technology is limited by the absence of an ideal culture system in which SSCs can be maintained and proliferated, especially in domestic animals like the goat. The aim of this study therefore was to investigate whether the addition of vitamin C (Vc) in cell culture influences the growth of caprine SSCs. Various concentrations of Vc (0, 5, 10, 25, 40, and 50 µg/mL(-1)) were added to SSC culture media, and their effect on morphology and alkaline phosphatase activity was studied. The number of caprine SSC colonies and area covered by them were measured at 10 days of culture. The expression of various germ cell and somatic cell markers such as VASA, integrins, Oct-4, GATA-4, α-SMA, vimentin, and Thy-1 was studied to identify the proliferated cells using immunostaining analyses. Further, the intracellular reactive oxygen species (ROS) level was measured at the 3rd, 6th, and 9th day after culture, and expression of Bax, Bcl-2, and P53, factors involved in the regulation of apoptosis, were analyzed on the 7th day after culture using reverse transcription polymerase chain reaction and quantitative real-time polymerase chain reaction. The results showed that the SSCs formed compact colonies and had unclear borders in the different Vc-supplemented groups at 10 days, and there were no major morphologic differences between the groups. The number and area of colonies were both the highest in the 40 µg/mL(-1) Vc group. Differential expression of markers for germ cells, undifferentiated spermatogonia, and testis somatic cells was observed. Cultured germ cell clumps were found to have alkaline phosphatase activity regardless of the Vc dose. The number of Thy-1- and Oct-4-positive cells was the most in the 40 µg/mL(-1) Vc group. Moreover, the level of ROS was dependent on the Vc dose and culture time. The Vc dose 40 µg/mL(-1) was found to be optimum with regard to decreasing ROS generation, and increasing the expression of the antiapoptotic gene Bcl-2 and decreasing the expression of the proapoptotic genes Bax and P53. In conclusion, the addition of 40 µg/mL(-1) Vc can maintain a certain physiological level of ROS, trigger the expression of the antiapoptosis gene Bcl-2, suppress the proapoptotic gene P53 and Bax pathway, and further promote the proliferation of caprine SSCs in vitro.

Enteral leptin administration affects intestinal autophagy in suckling piglets.

Leptin has been shown to play an integral role in the endocrine regulation of metabolism. Moreover, a substantial amount of this peptide has been found in colostrum and milk. The aim of the study was to investigate the effects of exogenous leptin, administered intragastrically, on the process of autophagy and the changes in cell hyperplasia and hypertrophy in the small intestine mucosa. Three groups (n = 6) of neonatal piglets were used in the study. The pigs were fed either by their sows (sow-reared piglets) or with only milk formula, or with milk formula together with leptin administered via a stomach tube (10 µg/kg BW) every 8 h for 6 d. We have shown that pure milk formula feeding significantly elevates (P < 0.05) autophagy compared with that observed in sow-reared piglets. Compared with the control group, feeding milk formula supplemented with leptin resulted in a significant decrease (P < 0.05) in immunodetection of microtubule-associated protein 1 light chain 3, as well as significantly accelerated epithelial cell renewal (P < 0.05). We demonstrated that autophagy is involved in the remodeling of the small intestine mucosa and that leptin, when administered enterally, may be an important factor for its regulation.

Induction of cytochrome P450 enzymes in primary equine hepatocyte culture.

In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments. Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration-response curves for induction with rifampicin were created. Williams' medium E showed the best results on morphology and viability of the hepatocytes and was therefore used for the following induction experiments. Cells cultured in Dulbecco's Modified Eagle Medium were not inducible. Incubation with rifampicin increased the CYP activity in two different hepatocyte preparations in a dose dependent manner (EC50=1.20 µM and 6.06 µM; Emax=4.1- and 3.4-fold induction). No increase in CYP activity was detected after incubation with dexamethasone or phenobarbital. The hepatocyte culture conditions established in this study proved to be valuable for investigation of the induction of equine CYPs. In further studies, other equine drugs can be evaluated for CYP induction with this in vitro system.

Decreased IgA+ B cells population and IgA, IgG, IgM contents of the cecal tonsil induced by dietary high fluorine in broilers.

Fluoride is an environmental and industrial pollutant that affects various organs in humans and animals. The cecal tonsil is an important component of the mucosal immune system and performs important and unique immune functions. In the present study, we investigated the effects of dietary high fluorine on the quantities of IgA+ B cells in the cecal tonsil by immunohistochemistry, and the immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) contents in the cecal tonsil by ELISA. A total of 280 one-day-old avian broilers were divided into four groups and fed on a corn-soybean basal diet as control diet (fluorine 22.6 mg/kg) or the same diet supplemented with 400, 800 and 1,200 mg/kg fluorine (high fluorine groups I, II and III) in the form of sodium fluoride, respectively, throughout a 42-day experimental period. The results showed that the quantities of IgA+ B cells were lower (p < 0.05 or p < 0.01) and the IgA, IgG, and IgM contents were decreased (p < 0.05 or p < 0.01) in high fluorine groups II and III in comparison with those of control group. It was concluded that dietary fluorine, in the 800-1,200 mg/kg range, could reduce the numbers of the IgA+ B cells and immunoglobulin contents in the cecal tonsil, implying the local mucosal immune function was ultimately impacted in broilers.

Effects of genistein on oestrogen and progesterone receptor, proliferative marker Ki-67 and carbonic anhydrase localisation in the uterus and cervix of gilts after insemination.

Soya products are routinely fed to domestic animals as an important source of protein. The aim of this work was to study how the phytooestrogen genistein, supplemented at a feed relevant level, affects the morphology and distribution of reproductive hormone receptors, proliferative activities and carbonic anhydrase (CA) in the uterus and cervix of gilts. Eleven gilts were fed a soya-free diet. Six were given genistein (1 mg/kg bw) twice daily for eight days starting three days before expected oestrus. Five gilts were used as controls. All gilts were inseminated (AI) one day after signs of standing oestrus and euthanized three days after AI. Samples from the uterus and cervix were processed for morphometric evaluation, immunohistochemical localisation of oestrogen receptors α and ß (ERα and ERß), progesterone receptor (PR), proliferative marker Ki-67 and histochemical localisation of CA. Nuclear staining for ERß was detected in surface epithelial, glandular and some stromal cells in the uterus and in the cervix surface epithelial cells. ERα and PR were observed in surface epithelium, subepithelial stromal cells and smooth muscle cells of uterus and cervix, and glandular cells of the uterus. Ki-67 positive cells were recorded in uterine and cervical surface epithelium and subepithelial stromal layer. CA was mainly confined to glandular cells of the uterus. Immunohistochemical results were evaluated using semi-quantitative image analysis. Statistic comparison between groups revealed no differences. However, intra-treatment evaluation and correlations indicate that the supplementation of genistein modulates the expression pattern of all receptors and Ki-67, which may induce cellular activities in both the uterus and cervix of early pregnant gilts.

Effects of the phytoestrogen, genistein, and protein tyrosine kinase inhibitor-dependent mechanisms on steroidogenesis and estrogen receptor expression in porcine granulosa cells of medium follicles.

The use of soy-based products in pig diets had raised concerns regarding the reproductive toxicity of genistein, the predominant isoflavone in soybeans. Genistein was reported to exhibit weak estrogenic activity but its mechanism of action is not fully recognized. The aim of the study was to examine the in vitro effects of genistein on (1) progesterone (P(4)) and estradiol (E(2)) secretion by porcine granulosa cells harvested from medium follicles, (2) the viability of cultured granulosa cells, and (3) the mRNA and protein expression of estrogen receptors α and ß (ERα and ERß) in these cells. In addition, to verify the role of protein tyrosine kinase (PTK)-dependent mechanisms possibly involved in genistein biological action, we tested the effects of lavendustin C, the nonsteroidal PTK inhibitor, on granulosa cell steroidogenesis. We found that genistein inhibited (P < 0.05) basal P(4) secretion by granulosa cells harvested from medium follicles of pigs. In contrast, lavendustin C did not affect basal P(4) secretion by the cells. Moreover, genistein increased (P < 0.05) basal granulosal secretion of E(2). In contrast, lavendustin C did not alter basal E(2) secretion by porcine granulosa cells. In addition, we demonstrated that genistein increased mRNA and protein expression of ERß (P < 0.05) in the examined cells. The expression of ERα mRNA was not affected by genistein and ERα protein was not detected in the cultured granulosa cells of pigs. In summary, the genistein action on follicular steroidogenesis in pigs involved changes in the granulosal expression of ERß. However, the genistein action on P(4) and E(2) production by granulosa cells harvested from medium follicles did not seem to be associated with PTK.

Effects of maternal and dietary selenium (Se-enriched yeast) on the expression of Sel P and apoER2 of germ cells of their offspring in goats.

In this experiment the effect of maternal dietary selenium on the expression of Sel P and apoER2 of goat offspring was studied. The experiment was conducted on 119 Taihang Black Goats randomly divided into 4 groups which were fed with a basal diet, supplemented with 0 (control), 0.5, 2 and 4 mg kg(-1) DM Se. Testis samples were collected from young male of each treatment group at the end of the study (30 d after weaning) for mRNA expression using real-time PCR and for protein expression by immunohistochemistry assay. A significant decrease was observed in mRNA expression of Sel P and apoER2 in the testis of the Se-deficient (Group 1) and the Se-excess (Group 4) compared with that in Groups 2 and 3. A similar trend of the protein expression of Sel P and apoER2 was also found. These data indicate that maternal and dietary selenium has an effect on the expression of Sel P and apoER2 in testis of their offspring. In addition, both groups were similar suggesting that the relationship between Sel P and apoER2, and apoER2 is a receptor of Sel P in the seminiferous epithelium to uptake the selenium.