Empirical Selection of Informative Microsatellite Markers within Co-ancestry Pig Populations Is Required for Improving the Individual Assignment Efficiency.

The Lanyu is a miniature pig breed indigenous to Lanyu Island, Taiwan. It is distantly related to Asian and European pig breeds. It has been inbred to generate two breeds and crossed with Landrace and Duroc to produce two hybrids for laboratory use. Selecting sets of informative genetic markers to track the genetic qualities of laboratory animals and stud stock is an important function of genetic databases. For more than two decades, Lanyu derived breeds of common ancestry and crossbreeds have been used to examine the effectiveness of genetic marker selection and optimal approaches for individual assignment. In this paper, these pigs and the following breeds: Berkshire, Duroc, Landrace and Yorkshire, Meishan and Taoyuan, TLRI Black Pig No. 1, and Kaohsiung Animal Propagation Station Black pig are studied to build a genetic reference database. Nineteen microsatellite markers (loci) provide information on genetic variation and differentiation among studied breeds. High differentiation index (FST) and Cavalli-Sforza chord distances give genetic differentiation among breeds, including Lanyu's inbred populations. Inbreeding values (FIS) show that Lanyu and its derived inbred breeds have significant loss of heterozygosity. Individual assignment testing of 352 animals was done with different numbers of microsatellite markers in this study. The testing assigned 99% of the animals successfully into their correct reference populations based on 9 to 14 markers ranking D-scores, allelic number, expected heterozygosity (HE) or FST, respectively. All miss-assigned individuals came from close lineage Lanyu breeds. To improve individual assignment among close lineage breeds, microsatellite markers selected from Lanyu populations with high polymorphic, heterozygosity, FST and D-scores were used. Only 6 to 8 markers ranking HE, FST or allelic number were required to obtain 99% assignment accuracy. This result suggests empirical examination of assignment-error rates is required if discernible levels of co-ancestry exist. In the reference group, optimum assignment accuracy was achievable achieved through a combination of different markers by ranking the heterozygosity, FST and allelic number of close lineage populations.

Ovarian hormone dependence of alpha(1)-adrenoceptor activation of the nitric oxide-cGMP pathway: relevance for hormonal facilitation of lordosis behavior.

The ovarian hormones estradiol (E(2)) and progesterone (P) facilitate rat lordosis behavior in part by regulating the expression of and signal transduction by adrenoceptors in the hypothalamus (HYP) and preoptic area (POA). The major adrenoceptor subtype mediating E(2) and P facilitation of lordosis is the alpha(1)-adrenoceptor. In the present studies, we tested the hypotheses that (1) alpha(1)-adrenoceptors in the HYP enhance lordosis responses by activating the nitric oxide (NO)-cGMP signaling pathway, and (2) coupling of alpha(1)-adrenoceptors to this signal transduction pathway is hormone-dependent. Basal levels of cGMP were significantly higher in HYP and POA slices from animals treated with E(2) and P when compared with slices from ovariectomized controls or females treated with only E(2) or P. When slices of HYP and POA from ovariectomized female rats were incubated with norepinephrine or the selective alpha(1)-adrenoceptor agonist phenylephrine, cGMP accumulation was observed only if slices had been derived from females treated with both E(2) and P before experimentation. Moreover, alpha(1)-adrenoceptor stimulation of cGMP synthesis was blocked by an inhibitor of NO synthase, confirming that these receptors act by NO-mediated stimulation of soluble guanylyl cyclase. Behavioral studies demonstrated further that the cell-permeable cGMP analog 8-bromoadenosine-cGMP reverses the inhibitory effects of the alpha(1)-adrenoceptor antagonist prazosin on lordosis behavior in E(2)- and P-treated female rats. Thus, the NO-cGMP pathway mediates the facilitatory effects of alpha(1)-adrenoceptors on lordosis behavior in female rats, and previous exposure of the HYP and POA to both E(2) and P are required to link alpha(1)-adrenoceptors to this pathway.

The women's health initiative estrogen replacement therapy is neurotrophic and neuroprotective.

The current study investigated the neurotrophic and neuroprotective action of the complex formulation of conjugated equine estrogens (CEEs), the most frequently prescribed estrogen replacement therapy in the United States and the estrogen replacement therapy of the Women's Health Initiative. Morphologic analyses demonstrated that CEEs significantly increased neuronal outgrowth in hippocampal, basal forebrain, occipital, parietal and frontal cortex neurons. Dose-response analyses indicated that the lowest effective concentration of CEEs exerted the maximal neurotrophic effect with greatest potency occurring in hippocampal and occipital cortex neurons. CEES induced highly significant neuroprotection against beta amyloid(25-35), hydrogen peroxide and glutamate-induced toxicity. Rank order of potency and magnitude of CEE-induced neuroprotection in the brain regions investigated was hippocampal neurons > basal forebrain neurons > cortical neurons. In hippocampal neurons pre-exposed to beta amyloid(25-35), CEEs halted Abeta(25-35)-induced cell death and protected surviving neurons from further cell death induced by Abeta(25-35). Because CEEs are the estrogen replacement therapy of the Women's Health Initiative, results of the current study could provide cellular mechanisms for understanding effects of CEEs on cognitive function and risk of Alzheimer's disease derived from this prospective clinical trial.

Cyclic GMP may potentiate lordosis behaviour by progesterone receptor activation.

The purpose of this study was to test the hypothesis that cGMP acts as a progesterone substitute to facilitate lordosis in oestrogen-primed rats. Female Sprague-Dawley rats underwent stereotaxic surgery to place a 26-gauge guide cannula into the third ventricle. Bilateral ovariectomy was done at the same time as stereotaxic surgery. Five days later ovariectomized rats were primed with 2 microg estradiol benzoate 24 and 48 h prior to behaviour testing. Some animals were further injected with 200 microg progesterone 4 h before behaviour testing. A nitric oxide synthase inhibitor infused into the third ventricle before progesterone administration significantly reduced lordosis performance. 8-Bromo-cGMP, a cell permeable cGMP analogue, or saline vehicle was infused into the third ventricle of hormone-primed animals approximately 4 h prior to the first of 3-h behaviour tests. This cGMP analogue facilitated lordosis behaviour. We next used KT5823, a highly specific inhibitor of protein kinase G (PKG), to test the hypothesis that cGMP action is mediated by this kinase. In this experiment, KT5823 was infused 15 min before progesterone. KT5823 significantly decreased lordosis behaviour. RU486, a progesterone receptor antagonist, was used to assess whether the stimulatory effects of cGMP are mediated through the progesterone receptor. Oestrogen-primed animals were injected with 5 mg of RU486 or vehicle 60 min before infusion with 8-bromo-cGMP. RU486 significantly attenuated cGMP-facilitated lordosis behaviour. These data show that cGMP facilitates lordosis through activation of PKG and the progesterone receptor.

Hormonal integration of neurochemical and sensory signals governing female reproductive behavior.

This review focuses on findings from our laboratory regarding mechanisms by which the ovarian steroid hormones, estradiol (E2) and progesterone (P), act in the hypothalamus (HYP) to regulate the expression of lordosis, an important component of female reproductive behavior in rats and many other species. The first section summarizes recent work suggesting that cGMP, perhaps via P-receptor activation, may be an intracellular mediator of the facilitatory actions of a variety of hormones and neurotransmitters on lordosis behavior in E2-primed rats. In the second section, we focus on E2 and P regulation of norepinephrine (NE) neurotransmission in the HYP. We review evidence that ovarian hormones act both peripherally and centrally to determine whether NE is released in the HYP in response to copulatory stimuli. We also suggest that the steroid milieu determines the cellular responses of hypothalamic neurons to released NE, favoring the activation of pathways implicated in the facilitation of both lordosis behavior and the preovulatory gonadotropin surge. It is likely that E2 and P have similar actions on other neurotransmitter and neuromodulator systems, thereby maximizing the probability that females are sexually receptive during the periovulatory period.

Effects of nitric oxide on stimulated release of norepinephrine from female rat hypothalamic slices.

Norepinephrine (NE) is an important neurotransmitter involved in ovarian steroid hormone regulation of female reproductive function in rats. Nitric oxide (NO) has also been suggested to be an essential mediator of gonadotropin-releasing hormone release and of lordosis behavior of female rats. These studies used a superfusion system to investigate the hypothesis that NO regulates [3H]NE release in the preoptic area (POA) and hypothalamus (HYP), brain regions that mediate ovarian steroid effects on reproductive function. The NO synthase inhibitors N-nitro-L-arginine and NG-nitro-L-arginine methyl ester did not modify either basal or N-methyl-D-aspartate (NMDA)-stimulated NE release in either brain region of ovariectomized, hormone-treated or control animals. The NO precursor L-arginine (L-Arg) reduced NMDA-stimulated NE release in POA but had no effect on KCl- or electrically-stimulated release. L-Arg did not influence basal or evoked release of [3H]NE from HYP slices. Sodium nitroprusside (SNP), a NO-generating compound, blocked the release of NE in response to NMDA stimulation but not in response to KCl or electrical stimulation. Thus, SNP is probably reducing NE release by acting as an NMDA antagonist rather than via NO production. There was a tendency for administration of both estrogen and progesterone to ovariectomized females to facilitate NMDA-stimulated NE release, particularly in the POA. Our data suggest that NO does not mediate basal or NMDA-stimulated NE release in rat POA and HYP. Therefore, NO regulation of lordosis behavior and gonadotropin release in female rats is probably not exerted at the level of NE release.

Attachment of Mycoplasma gallisepticum to the tracheal epithelium of fowls.

An electron microscopical study of fowl trachea infected with Mycoplasma gallisepticum showed slight to moderate changes at the cellular level. Mycoplasmas were demonstrated extra-cellularly and showed triple membranes, intracytoplasmic spaces, fibrillar nuclear areas and blebs. Blebs were seen attached to the plasma membrane of the epithelial cells.

Trihalomethane formation potentials of organic pollutants in wastewater discharge.

Drinking water resources contaminated by wastewater discharge could have great risk of trihalomethane (THM) formation upon chlorine disinfection during treatment. In the present study, THM formation potentials (THMFP) of the organic pollutants in the raw sewage and treated secondary effluent collected from f ull-scale wastewater treatment plants were characterised. The organic matter remaining in the secondary effluent had higher unit carbon THMFP in comparison with that in the raw sewage. However, owing to removal of organic pollutants in the treatment works, the overall THMFP of the wastewater was reduced significantly throughout the biological treatment process. Using XAD-8 resin, organic materials in the wastewater samples were separated into hydrophobic and hydrophilic fractions. Hydrophobic organic, which possesses higher THMFP than hydrophilic organic, was the predominant THM precursor in wastewater discharge. Ozonation could be used to alter the properties of organic matter, particularly the hydrophobic fraction, and reduce its THMFP.

Genetic variation and phylogenetics of Lanyu and exotic pig breeds in Taiwan analyzed by nineteen microsatellite markers.

The Lanyu pig is an indigenous miniature pig breed on Lanyu Islet near Taiwan, with a mitochondrial DNA genetic lineage remote from Asian and European pig breeds. The unknown population genetic structure and increased inbreeding among the small population of conserved Lanyu pigs is now of great conservation concern. Additionally, the presence for more than a century of exotic pig breeds in Taiwan has made gene introgression from exotic pig breeds into Lanyu pigs very possible. The present study thus aimed to investigate nuclear genetic variation within the conserved Lanyu pigs and the phylogenetic relationship and possible genetic introgression between Lanyu and exotic pig breeds by determining the polymorphism of 19 microsatellite loci. In the neighbor-joining tree constructed from 7 pig breeds based on Cavalli-Sforza and Edward chord genetic distances, 3 major clades were recognized, in which the Asian and European breeds were separately clustered into 2 clades with a 93.0 and 99.9% bootstrap confidence value, respectively. All individuals of the Lanyu breed formed a unique subclade within the Asian clade based on the distance of the proportion of shared alleles, -ln(ps), suggesting that the Lanyu breed possesses a unique nuclear genetic structure and that no nuclear gene introgression from exotic breeds into the conserved Lanyu pigs has occurred in recent history. Fifteen of 19 microsatellite loci deviated from Hardy-Weinberg equilibrium (by Wright's statistic), suggesting a significant loss of heterozygosity in the conserved population. The valuable nuclear genetic structure and phylogenetic information should assist future conservation and population management of Lanyu pigs.

Interpopulation and intrapopulation maternal lineage genetics of the Lanyu pig (Sus scrofa) by analysis of mitochondrial cytochrome b and control region sequences.

The Lanyu pig is an indigenous breed from the Lanyu Islet, which is southeast of Taiwan. Two herds of Lanyu pigs were introduced from the Lanyu Islet into Taiwan in 1975 and 1980. The current population of conserved Lanyu pigs consists of only 44 animals with unknown genetic lineage. The Lanyu pig possesses a distinct maternal genetic lineage remote from Asian and European pigs. The present study aimed to understand the phylogenetic relationship among conserved Lanyu, Asian, and European type pigs based on the cytochrome b coding gene, to ascertain the maternal lineage and genetic diversity within the conserved Lanyu pigs, and to address whether genetic introgression from exotic or Formosan wild pigs had occurred in the conserved Lanyu pigs. Entire mitochondrial genomes of both types of Lanyu pig comprised 2 ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes. Only 2 haplotypes of the mitochondrial DNA (mtDNA) control region and cytochrome b were identified in the conserved Lanyu pig herds. When maximum likelihood trees were constructed, the Type I Lanyu mitochondrial genes formed a unique clade with a large pairwise distance of both cytochrome b and the control region from Asian and European type breeds, Formosan wild pigs, and exotic breeds. Significant loss of genetic diversity of mtDNA within the conserved Lanyu pigs was demonstrated by low haplotype and nucleotide diversities, supported by Fu and Li's D* neutrality test (1.44055; P < 0.05). The mtDNA control region sequences of extant pigs in the Lanyu Islet, however, showed high haplotype and nucleotide diversity, and clustered with exotic pigs. These results indicate no maternal lineage mtD-NA gene introgression from Formosan wild pigs and introduced exotic pigs to conserved Type I Lanyu pigs, and a severe loss of heterozygosity of mtDNA in conserved Lanyu pigs. The remaining extant pigs on the Lanyu Islet have been introgressed with exotic breeds. Strategies for future conservation of native Lanyu pigs are now even more urgent and important.

The type I Lanyu pig has a maternal genetic lineage distinct from Asian and European pigs.

The Lanyu pig is an indigenous breed from Lanyu Islet, located south-east of Taiwan, with phenotypic characteristics distinctive from other pig breeds in Asia and Europe. Based on geographic considerations, the Lanyu pig may have originated from mainland China, Austronesia or the Ryukyu Islands. In the present study, polymorphism of the mitochondrial DNA control region sequence was used to clarify phylogenetic relationships among two herds of Lanyu pigs imported before 1980 from Lanyu Islet into Taiwan and reared in isolation on two different farms. Two distinct mitochondrial control region haplotypes were found. The type I Lanyu sequence appeared independently as a unique clade different from Asian and European pig sequences, while the type II Lanyu sequence was clustered within the major Asian clade. The pairwise distances between the major Asian clade vs. the type I Lanyu and European clades were 0.01726 +/- 0.00275 and 0.01975 +/- 0.00212 changes per site respectively. Estimates of divergence time suggest that the type I Lanyu sequence split from the major Asian pig clade in prehistoric times. The type II Lanyu mtDNA shares a close genetic lineage with Japanese Satsuma and New Zealand Kune Kune mtDNA with pairwise distances of 0.00095 +/- 0.00000 and 0.00192 +/- 0.00000 respectively, indicating gene flow between Lanyu Islet, Japan and Oceania in recent times. Together these results indicate that the type I Lanyu pig has a genetic lineage separate from Asian-type pigs, while the type II Lanyu sequence may represent a more recent introgression of modern Asian pigs.

A potential role of cyclic GMP in the regulation of lordosis behavior of female rats.

Nitric oxide (NO) has been suggested to play a crucial role in the regulation of lordosis behavior via stimulation of guanylyl cyclase to synthesize cyclic GMP. Whalen and Lauber (1986, Neurosci. Biobehav. Rev. 10, 47-53) hypothesized that hormones and pharmacological agents known to facilitate lordosis in estrogen-primed rodents act through cyclic GMP. The compound 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) has been shown to selectively inhibit NO-stimulated cyclic GMP production. In the present study, we investigated the effects of ODQ on lordosis behavior. Female rats were implanted with a guide cannula aimed at the lateral or third ventricles by stereotaxic surgery, and their ovaries were bilaterally removed. Five days later, animals were injected subcutaneously with 2 microg estradiol benzoate at 48 and 24 hr, and 200 microg progesterone 4 hr before behavioral testing. ODQ or vehicle (1 microl) was administered at the time of progesterone treatment or 20 min before lordosis testing. ODQ significantly decreased lordosis quotients and the quality of lordosis at both intervals of drug infusion. Locomotor activities, measured by line crossing and rearing, were not affected by ODQ. ODQ also inhibited cyclic GMP accumulation in response to NMDA stimulation in hypothalamic and cerebellar slices in vitro. We conclude that cyclic GMP produced by NO generation is an important modulator of female rat sexual behavior.

Single and mixed infections of avian infectious bronchitis virus and Mycoplasma gallisepticum.

Comparative studies of the clinical signs, pathological changes, multiplication of the pathogens and serological responses were made of groups of chickens infected with either or both avian infectious bronchitis virus and Mycoplasma gallisepticum. Electron microscopic examination of thin sections of infected tracheas showed that the multiplication of M. gallisepticum was greatly enhanced in the tracheas of chickens which had been previously or simultaneously infected with avian infectious bronchitis virus. The enhanced multiplication of mycoplasmas resulted in more severe clinical signs, pathological effects and serological responses. These findings confirm the general belief that avian infectious bronchitis infection may precipitate latent mycoplasma infection. Similar experiments were made in the allantoic sacs of 10-day embryonated hens' eggs. The results again show that there was at least a one hundred-fold increase in the multiplication of M. gallisepticum in eggs simultaneously or previously infected with avian infectious bronchitis virus. These findings emphasize the importance of using mycoplasma-free eggs for the production of virus vaccines.

The effect of maternal immunity, age at vaccination and doses or live vaccines on immune response to Newcastle disease.

The short life span of domestic fowls necessitates the use of early vaccination, but the response is affected by the immaturity of the immune mechanism and by the presence ofmaternal immunity: this results in an uneven immunity of the large and continually changing poultry population. This paper compares the response of groups of chicks from vaccinated and non-vaccinated hens after vaccination at various ages with Newcastle Disease vaccines of different pathogenicity (lentogenic and mesogenic strains). The results show that: 1. maternal immunity has a great effect on the pathogenicity of the vaccine viruses and the subsequent immune response; 2. the older the chicks are at the time of vaccination, the better is their immune response; 3. for vaccines of low pathogenicity the immune response can be enhanced by increasing the dose of the virus irrespective of the presence of maternal immunity.