Individual return to Leydig cell function after GnRH-immunization of boars.

Immunization of boars against GnRH inhibits synthesis of testicular anabolic steroids and the sex odour androstenone. Time between second vaccination and return of Leydig cell function is unknown. Six catheterized boars received the second dose (Improvac) at 26 weeks of age (day 0). Titre, LH, testicular steroids, and IGF-I were determined in blood until testosterone exceeded 0.5 ng/mL again in all boars. At week 10 the titre was low again. Return of testicular function was preceded by increased pulsatile secretion of LH but onset of steroid synthesis was delayed and highly variable (10-24 weeks) and was not related to the initial antibody titre. A major reason for delayed onset of Leydig cell function apparently is a variable refractoriness against LH.

Expression of the monocarboxylate transporter 1 (MCT1) in cells of the porcine intestine.

Uptake of energy into cells and its allocation to individual cellular compartments by transporters are essential for tissue homeostasis. The present study gives an analysis of MCT1 expression and its cellular occurrence in the porcine intestine. Tissue portions from duodenum, jejunum, ileum, colon ascendens, colon transversum and colon descendens were collected and prepared for immunohistochemistry, Western blot and real time RT-PCR. A 169bp porcine MCT1 cDNA fragment was amplified and published. MCT1 mRNA expression in the large intestine was 20 fold higher compared to the small intestine. Western blot detected a single protein band of 41kDa at a much higher amount of MCT1 protein in the large intestine vs. the small intestine. MCT1 protein was detected in mitochondrial fractions of the large but not the small intestine. Immunohistochemistry in the small intestine showed that immune cells in the lamina propria and in the lymphoid follicles primarily expressed MCT1 while in the colon epithelial cells were the main source of MCT1. In summary, cellular expression of MCT1 differs between epithelial cells in the colon and small intestine. A possible role of MCT1 for uptake of butyrate into immune cells and the overall role of MCT1 for intestinal immune cell function remains elusive.

An improved microtitre enzyme immunoassay to measure the boar taint steroid 5α-androst-16-en-3-one in blood plasma of pigs.

Androstenone (5α-androst-16-en-3-one) is a volatile steroid which is synthesized in boar testes and stored in high amounts in fat thus leading to an urine-like odor in pork. Whereas microtiter assays (MTA) exist for fat determination, measurement in blood with MTA was not yet possible. The system reported here is based on a specific antiserum and a horse radish peroxidase conjugate as tracer. For coating bovine serum albumin was avoided which otherwise would lead to unspecific binding. Blood plasma was extracted with n-hexane, solvent was evaporated in a vacuum centrifuge and the extract was taken up in buffer with 10% methanol. Assay sensitivity was 2.5pg/well (0.025ng/mL), specificity was confirmed by GC-MS (r=0.96; n=50) and inter- and intraassay variation were 4.9% and 5.9%, respectively. Thus an assay system is available for studies in pig production and in wildlife research due to pheromone activity also in other species.

Expression of 11beta-hydroxysteroid-dehydrogenase 2 in Sertoli cells of boar testes.

11beta-Hydroxysteroid-dehydrogenase 2 (11beta-HSD2) activity occurs in boar testes but it is not known which cell types express 11beta-HSD2 mRNA and protein. Therefore, testes samples were taken from mature boars. For immunocytochemistry and Western blot pig-specific antibodies were raised against a 10-amino acid peptide corresponding to amino acids 391-400 of the coding sequence. Quantitative PCR was performed in testis homogenates and additionally RT-PCR in samples collected by UV-single cell microdissection. Data show that in interstitial tissue 11beta-HSD2 is expressed in Leydig cells and additionally in blood capillaries. In tubuli, 11beta-HSD2 primarily is formed in Sertoli cells whereas occurrence in spermatogonia could not be definitely proven. Because glucocorticoid receptors were never found in boar Leydig cells, it is concluded that the expression of 11beta-HSD2 in several types of cells forms consecutive lines of defense to protect spermatogonia against glucocorticoid-induced apoptosis.

Short-term endocrine and metabolic reactions before and after second immunization against GnRH in boars.

Immunization of boars against GnRH inhibits synthesis of testicular steroids including androstenone (sex odour). Timing of the second vaccination (anamnestic reaction) should occur as late as possible to maintain anabolic effects of testicular hormones, but early enough to remove androstenone from body fat. Five catheterized boars received the second dose (Improvac) at age 22 weeks. Titre, hormones and parameters reflecting protein turnover were determined in blood. An increased antibody titre and drop of LH and steroids occurred within 5 days. Metabolism adapted after 7 days. Results from this study in conjunction with previous work suggest that after two doses of Improvac given 4 weeks apart, clearance of androstenone from body fat may be achieved as early as 3 weeks after the second vaccination. Thus, it might be possible to extend the duration of anabolic effect in male pig production.

Changes of testicular aromatase expression during fetal development in male pigs (Sus scrofa).

Male pig fetuses secrete considerable amounts of estrogens, but the location of aromatase activity within the fetal testis is not known. The location of aromatase expression was investigated by immunocytochemistry in fetal testes from week 6 (n = 5), weeks 10, 13, and 15 (each: n = 6) of gestation and additionally in neonates (n = 4). Blood was sampled from the umbilical artery of fetuses and jugular vein of neonates. Histological evaluation of testes involved morphological criteria and counting of Leydig cells, Sertoli cells, and gonocytes. Aromatase activity was localized immunocytochemically and quantified by the percentage of positive stained cells within the same cell type. Aromatase expression was further characterized by quantitative RT-PCR. Concentrations of estrogens, testosterone, FSH, and LH were measured in blood plasma. Total estrogens increased from week 10 to a maximum of 31.03 nmol/l in week 15. Increased testosterone concentrations were only measured at week 6 and were paralleled by slightly elevated estrogens. Thereafter, testosterone dropped and was low throughout. The increase of estrogens was not paralleled by a similar increase of FSH and LH but was related to the increase of the total number of Leydig cells. This increase was also found for mRNA expression. Both Leydig cells and gonocytes were identified as contributors to estrogen formation. Gonocytes were the main source of aromatase at week 10, when gene expression by Leydig cells is low due to the preparation of a wave of Leydig cell mitosis.

Compound subretinal prostheses with extra-ocular parts designed for human trials: successful long-term implantation in pigs.

BACKGROUND: Subretinal implants aim to replace photoreceptor function in patients suffering from degenerative retinal disease like retinitis pigmentosa by topically applying electrical stimuli in the subretinal space. This study-as a last step before upcoming human trials-explored a newly developed surgical technique for permanent implantation of complex subretinal implants with extra-ocular parts. METHODS: The implant consisted of a microphoto-diode array (MPDA) with 1550 electrodes and a 4x4 array of gold electrodes for direct electrical stimulation; both were mounted onto a polyimide foil for transscleral placement into the subretinal space. The foil carried connection lanes to a silicone cable that was implanted under the skin and led to a stimulator box in the animal's neck. Surgery was performed in 11 domestic pigs. Improved vitreo-retinal surgical technique consisted of a 180 degrees peripheral retinotomy and use of diathermy to penetrate the choroid in order to avoid choroidal haemorrhage. Subretinal forceps were used to place the implant safely onto the retinal pigment epithelium before the retina was flattened, peripheral laser photocoagulation was applied and the eye was filled with silicon oil. The implant was stabilized by a scleral fixation patch, use of a metal clamp with bone screws on the animal's skull and a tissue ring under the animal's skin in the neck. Behaviour was observed in the freely moving animals after direct subretinal electrical stimulation and funduscopy, optical coherence tomography, fluorescein angiography and histology were performed. RESULTS: All implants were successfully placed subretinally. In three animals a proliferative vitreo-retinopathy was observed after approximately 2 weeks. Otherwise, funduscopy and OCT demonstrated complete retinal attachment and FA showed no retinal vascular abnormalities over and around the implant. The animals showed clear behavioural reactions to electrical stimulation over the whole examination period. Histological examination failed to show any voltage-induced alteration in the cellular architecture of the retina overlying the stimulation electrodes. CONCLUSIONS: This study demonstrates the feasibility of a new surgical procedure for highly safe and controlled implantation of complex subretinal devices with extra-ocular parts. The new implant design proved to be safely implantable in free-moving pigs for an observation period of 4 weeks.

Rise of testosterone, nortestosterone, and 17beta-estradiol concentrations in peripheral blood plasma of pigs after sublingual application in vivo.

Application of endogenous anabolic steroids to meat producing animals is not allowed in the EU. In other countries application is practised due to a low oral activity based on an efficient first liver passage. This contrasts with pharmacological investigations where steroids were readily absorbed by the buccal and sublingual mucosa using absorption enhancers. An in vivo study was performed to clarify possible absorption after sublingual applications of one milligram portions of either testosterone (T), 17beta-estradiol (E), or nortestosterone (NT) in sesame oil to castrated male pigs (n=5) without specific delivery systems during anaesthesia. Blood samples were drawn using jugular vein catheters for 15 min before and 3h after application. Hormone concentrations were determined by Radioimmunoassay for T and E or Enzymeimmunoassay for NT. For all steroids a slight increase was measurable one minute after application. Maximal values for T, E, and NT were 2.5 ng/ml, 1.5 ng/ml and 4.2 ng/ml, respectively, and were observed after 10 min. The concentrations of the three steroids decreased slowly thereafter but were still significantly elevated 1-3h after application. Oral absorption of steroids without enhancers should be considered in risk analysis.

Flavor improvement in pork from barrows and gilts via inhibition of intestinal skatole formation with resistant potato starch.

Skatole originates from microbial processing of tryptophan in the large intestine of pigs and accumulates in adipose tissue. Formation may be inhibited by the anti-apoptotic function of butyrate formed out of raw potato starch. Two groups of pigs (each consisting of gilts and barrows) were fed from 30 to 110 kg life weight either a conventional diet (controls; n = 35) or an isocaloric diet containing 300 g of raw potato starch/kg of body weight (RS; n = 34). Skatole concentrations were measured in colon content, blood, and adipose tissue. Odor of cooked meat samples was evaluated by a test panel. RS reduced concentrations in colon content and blood plasma (P < 0.001). Back fat concentrations were decreased significantly from 25 to 1.40 ng/g (barrows; P < 0.001) and from 40 to 9 ng/g (gilts; P < 0.001). Odor rating (scale of 1-5 from very unpleasant to very pleasant) was 3.07 for low skatole concentrations and 2.66 for both medium and high skatole concentrations (P < 0.05).

Effects of estradiol infusion in GnRH immunized boars on spermatogenesis.

Active immunization of boars against gonadotropin-releasing hormone (GnRH) inhibits luteinizing hormone (LH) and testicular steroids, so that mitosis of spermatogonia is reduced and apoptosis increased. To clarify whether high amounts of estrogens which are synthesized in the boar testis support spermatogenesis, a group of 6 boars was immunized against GnRH and then infused for 7 weeks with estradiol (E2-17beta). For comparison, intact boars and immunized boars were infused with saline only. Testicular tissue was then analyzed by immunocytochemistry for apoptosis (TUNEL, EM), mitosis (Ki67), and estrogen receptor alpha (ER alpha). The specificity of ER alpha staining was confirmed by RT-PCR and Western blot. Immunization decreased LH and testosterone to minimal concentrations in immunized and E(2)17beta-infused immunized boars, whereas follicle-stimulating hormone (FSH) was not significantly altered. Estradiol decreased to base levels after immunization. Infusion increased E2-17beta in peripheral blood plasma of the immunized boars to physiological levels. Except for A-spermatogonia, all spermatogenic cells decreased after immunization by about 60%. After estradiol infusion, cell counts increased again and were intermediate between control and immunized boars. Mitosis of spermatogonia was reduced by nearly 50% due to immunization but was partly restored by E2-17beta infusion. Expression of ER alpha was localized in spermatogonia, suggesting stimulation of mitosis, which was further confirmed due to its predominant occurrence in stage I of the seminiferous epithelial cycle (main stage of cell division). Apoptosis was minimal in boars but elevated in the other 2 groups. Data showed that estrogens in physiological concentrations supported mitosis but were not sufficient to normalize sperm production because apoptosis was still high.

Interaction between lactation, photoperiodism and male effect in German Merino ewes.

A study with 93 German Merino ewes was performed from January until the end of March to clarify the relative importance of lactation, photoperiodism and ram effect on cyclic activity and lambing data. Ovarian activity was registered by progesterone concentrations in blood plasma three times weekly. Half of the ewes were kept under supplemental light (20 h/day) for the last 6 weeks of lactation and additionally 3 weeks post-weaning, the other half were kept under natural photoperiod but were weaned simultaneously. Thereafter, light was reduced to natural photoperiod and rams were introduced to half of the ewes, of both light reduced and photoperiod group. Ewes entered cyclicity during lactation gradually, but at weaning 56% of photoperiod ewes and 53% of supplemental light ewes were still acyclic. After weaning, resumption of cyclic activity before ram introduction was more pronounced (P<0.05) in the photoperiod group (75% cyclic) than in the supplemental light group (51% cyclic). Ram introduction led to cyclicity in all ewes. Light reduction without ram slightly increased cyclicity but 57% were still acyclic. In the photoperiod group without ram no ewe entered cyclicity and two ewes even ceased cycling again. Data show that German Merinos still have a remarkable lactational anoestrus but are extremely sensitive to ram. Light reduction has no direct effect on cyclicity but is likely to contribute to the elevated ovulation rate so that a combination with the ram effect led to a higher lambing rate (1.94) compared to photoperiod and ram (1.55).

Intraepithelial but not lamina propria lymphocytes in the porcine gut are affected by dexamethasone treatment.

It is well established that glucocorticoids are key regulators of the immune system and act as immunosuppressive agents in high concentrations. In the pig, effects on the gut immune system and trafficking of lymphocytes between tissues and blood plasma were not investigated so far. Twelve pigs of 70 kg were fed 0.4 mg portions of dexamethasone (Dexa) twice daily for 9 days or remained untreated (controls) and were sacrificed for tissue collection at the end of Dexa treatment. Another six pigs with jugular vein catheters were left untreated for 7 days (control period) and then received Dexa for 9 days. Blood was drawn twice during the control period and at days 3, 6 and 9 of the Dexa period for characterization of peripheral blood leukocytes. Cells were obtained from thymus, mesenteric lymph nodes, jejunal mucosa and Peyer's patches. Lymphoid cells from gut tissue were isolated from two fractions: the EDTA-fraction, containing the intraepithelial lymphocytes (IEL), and the Collagenase-fraction, containing the lamina propria lymphocytes (LPL). In all samples, cell counts and phenotypic characterization of cells by flow cytometry (FCM) were performed. In thymus, Dexa led to a more than 90% reduction of the absolute cell number, which was mainly found in the CD4+CD8+ subpopulation. Dexa effects on lymphocytes from mesenteric lymph nodes were less severe (50%) and led mainly to a decrease (71%) of B-lymphocytes. The number of lymphocytes in the EDTA-fraction (IEL) of the jejunal mucosa decreased significantly by 56% in the Dexa-treated animals compared to the controls, whereas the number of lymphocytes in the Collagenase-fraction (LPL) decreased only moderately. In the Peyer's patches, a decreasing tendency in the number of lymphocytes in the EDTA-fraction was observed which, however, was not significant. In blood, monocytes and granulocytes were significantly increased in an order of 60%. The data show that supraphysiological amounts of Dexa remarkably reduce cell numbers in thymus and also in the intraepithelial compartment of the jejunal mucosa and ileal Peyer's patches. In blood, a notable homeostasis was observed for several leukocyte populations whereas both monocytes and granulocytes increased.

Use of biotinylated 17beta-estradiol in enzyme-immunoassay development: spacer length and chemical structure of the bridge are the main determinants in simultaneous streptavidin-antibody binding.

17beta-estradiol (E2) concentrations are in the low pg/ml range in plasma. To develop a sensitive enzyme immunoassay (EIA) for E2-determination a highly specific antibody raised against a 6-carboxymethyl (CMO)-E2-bovine serum albumine conjugate was used. Based on 6-CMO-E2 and 6-amino-E2, four biotinylated tracers with two different spacer lengths between E2 and biotin were synthesized using biotinylation reagents in one step reactions. All amino-based tracers were unsuitable for assay development because the antibody binding was too weak compared to the analyte E2. For 6-CMO-based tracers the simultaneous binding of the tracer to the antibody and streptavidin seems to be the determining step in the procedure depending on incubation temperature and spacer lengths. While a short spacer of 9 carbon atoms was susceptible to room temperature, a longer spacer of 16 carbon atoms showed nearly the same results for incubation at 4 degrees C or at room temperature. The absolute detection limit of this system was 0.63 pg/well. For sample clean-up, porcine plasma was solvent-extracted and depending on the initial plasma volume further purified by solvent partition. Determination of reproducibility resulted in intraassay coefficients of variation of 13% and 5.3% for samples with E2-levels of 15 pg/ml and 236 pg/ml, respectively. Measurement of E2-spiked blood plasma revealed recoveries of 83% up to 100% for E2 concentrations between 50 pg/ml and 1000 pg/ml. Only for the lowest concentration (20 pg/ml) a recovery of 58% was observed. Correlation of the EIA with an established radio immunoassay resulted in r=0.991 using the same antibody.

Effects of resistant potato starch on odor emission from feces in Swine production units.

Odor emission from swine facilities is determined by microbial breakdown of amino acids or carbohydrates in the pig colon. It was the aim to influence apoptosis and thus amino acid availability for odor formation by feeding resistant starch (300 g kg(-1) feed) over the whole fattening period to 40 pigs. Concentrations of 12 key components (indoles, volatile fatty acids, methanethiol) were measured in feces and headspace over the slurry duct and compared to 40 normally fed controls in a separate compartment. Concentrations of substances resulting from amino acids were reduced in feces by 70% (indoles) and 8% (branched chain fatty acids) and in the headspace by 72% and 20%. Resistant starch only led to minor increases of straight chain fatty acid concentration. Maximal reduction occurred for 3-methyl-1H-indole (skatole) which is the main determinant of malodor so that the results point to promising strategies for reducing pig odor emission.

Development of a rapid and accurate method for the determination of key compounds of pig odor.

Sampling of odor substances in the emissions from swine production facilities is still the limiting step for routine odor quantification. Solid sorption techniques based on cartridges were developed for three categories of substances (indoles, volatile fatty acids and methylthiol) and were standardized to a sampling time of 15 min. These cartridges also trap dust particles which transport odor substances. Quantification was performed by RP-HPLC or GC. Reliability criteria revealed excellent values for sensitivity (lower ppb level) and repeatability (R.S.D. < 10%), thus they are comparable to fiber solid-phase micro extraction sorption techniques. Parallel determinations in feces and air revealed high correlations (r = 0.99, P < 0.01), so that microbial processes during digestion determine odor quality and hence provide a possibility to reduce odor via feeding.

Serum IgA-promoting effects induced by feed loads containing isolated deoxynivalenol (DON) in growing piglets.

Deoxynivalenol (DON), a Fusarium toxin belonging to the trichothecene group, has been reported to produce a variety of adverse health effects in farm animals, such as inhibition of protein synthesis, reduction of feed intake, and alteration of the immune system. In pigs, the effects of increasing levels of chemically pure DON in a semisynthetic diet on performance, health, and serum immunglobulin A (IgA) levels were examined. A diet, without grain components and trichothecene free (8 main trichothecenes), with doses of 0, 300, 600, and 1200 microg pure DON/kg was fed to 34 female pigs for a period of 8 wk after weaning under standardized conditions. Body weight gain and biochemical and hematological values in the blood and serum, including concentrations of IgA, blood glucose, cortisol, and insulinlike growth factor 1 (IGF-1), were determined. Increasing levels of DON in the feed induced a significant depression of glucose levels. Cortisol and IGF-1 levels were not significantly affected but differed between groups at the end of the experiment. A significant increase of IgA concentration in the serum even at a dosage level of 600 microg DON/kg feed was observed. This is the first report demonstrating in vivo that limited dosages of DON are able to stimulate IgA levels in the serum of growing piglets.