Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model." However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasite's replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

Growth of long bones in European and Japanese quail from the 13th day of incubation to day 35 post-hatch.

This study described the growth, morphometric, biomechanical, and chemical properties of the femur, tibiotarsus, and tarsometatarsus of European and Japanese quail. Analyses were performed at 13 and 15 days of incubation, at hatch, and at 4, 7, 10, 14, 21, 28, and 35 days post-hatch (n=6/subspecies/period). Bone specimens were analyzed by cone-beam computed tomography, biomechanical assays, chemical analyses, and histomorphometry. Variables were fitted by the Gompertz function and its derivative or assessed using the analysis of variance. Analysis of the derivative of Gompertz curves showed that the growth behavior of the tarsometatarsal bone was similar between quail subspecies, and the femur and tibiotarsus of European quail increased first in width and then in length, whereas the opposite occurred in Japanese quail. There was an interaction between quail subspecies and days of growth on femoral, tarsometatarsal, and tibiotarsal bone densities. Femoral and tibiotarsal cross-sectional areas were influenced by the interaction of quail subspecies and day of growth. Interaction effects were significant for breaking strength and phosphorus percentage. European and Japanese quail have different femoral and tibiotarsal growth patterns, especially in the first few days after hatching, whereas tarsometatarsal growth is similar between subspecies.

Productive performance, breast growth and digestive system development in European quail subjected to post-hatch fasting for different periods.

This study assessed the effect of different periods of post-hatch fasting on animal performance and breast and digestive system growth in European quail. Quail chicks were distributed in a completely randomized design, with four fasting periods (0, 24, 36, and 48 hs) and four replications of 40 birds per treatment. In 1 to 14-day-old chicks, weight gain decreased with increasing fasting time. Compensatory gain was observed from 15 days of age onward. Fasted quail had a lower length and relative weight of the digestive system than fed animals for up to 14 days. Histologically, the duodenal villus height was significantly lower in 3-day-old quail fasted for 36 hs than in those fasted for 48 hs, but this effect was not observed at 7 days. Scanning electron microscopy showed no differences in the small intestinal mucosa between fasted and fed birds at 3 days of age. Post-hatch fasting reduced the relative weight of the breast in quail aged 1 to 14 days but did not affect type IIa and IIb fiber diameter at 35 days. On the basis of these results, it is recommended that European quail raised for meat should not be fasted for more than 48 hs post-hatch.

Growth of breast muscles in European and Japanese quail raised in meat production system: a morphological analysis.

Growth curves have been described in the quail but with no attention to the muscle composing of the breast. The description of the characteristics of growth curves to body weight and to breast muscle was the aim of this study. Morphological development of Musculus supracoracoideus and Musculus pectoralis in European and Japanese quail was assessed from the final incubation of to 35 days. Gompertz models were adjusted with maximum growth rates to body weight, breast weight, and Musculus pectoralis and supracoracoideus weight at 17.6; 22.2; 23.5, and 21.4 days. The European quail had a higher body and breast weight at maturity. Musculus supracoracoideus developed faster in both subspecies but with larger Musculus pectoralis. Both musculus had a greater number of fibers type IIA and largest fibers IIB, with quadratically increasing in fiber diameter with age in both subspecies and muscles. At 35 days, results of meat quality indicated similarity between genders and subspecies, with darker and redness breast meat in Japanese quail. In conclusion, breast weight gain was a result of type IIA and IIB fiber hypertrophy in both muscles and, despite the difference in size and aptitude, Japanese and European quail showed similar body and muscle growth patterns.

Genome-wide CRISPR screen identifies genes synthetically lethal with GRA17, a nutrient channel encoding gene in Toxoplasma.

Toxoplasma gondii is a parasite that replicates within a specialized compartment called the parasitophorous vacuole (PV), which is surrounded by the PV membrane (PVM). To obtain essential nutrients, Toxoplasma must transport molecules across the PVM, a process mediated by the secreted parasite proteins GRA17 and GRA23. These proteins form pores in the PVM through which small molecules can diffuse in and out of the PV. GRA17 and GRA23 are synthetically lethal, suggesting that at least one pore type is essential for parasite survival. In the 'nutrient sensitized' Δgra17 strain it is likely that other Toxoplasma genes become essential, because they mediate nutrient acquisition from the host or are involved in the trafficking of GRA23 to the PVM. To identify these genes, a genome-wide loss-of-function screen was performed in wild-type and Δgra17 parasites, which identified multiple genes that were synthetically sick/lethal with GRA17. Several of these genes were involved in the correct localization of GRAs, including GRA17/GRA23, to the PVM. One of the top hits, GRA72, was predicted to form a pore on the PVM, and its deletion led to the formation of enlarged "bubble vacuoles" with reduced PVM small molecule permeability, similar to what was previously observed for Δgra17 parasites. Furthermore, Δgra72 parasites had reduced in vitro growth and virulence in mice. These findings suggest that in the absence of GRA17, other genes become essential, likely because they play a role in the proper localization of GRA23 (and other GRAs) or because they determine host-derived nutrient acquisition at the PVM.

CRISPR Screens Identify Toxoplasma Genes That Determine Parasite Fitness in Interferon Gamma-Stimulated Human Cells.

Toxoplasma virulence depends on its ability to evade or survive the toxoplasmacidal mechanisms induced by interferon gamma (IFNγ). While many Toxoplasma genes involved in the evasion of the murine IFNγ response have been identified, genes required to survive the human IFNγ response are largely unknown. In this study, we used a genome-wide loss-of-function screen to identify Toxoplasma genes important for parasite fitness in IFNγ-stimulated primary human fibroblasts. We generated gene knockouts for the top six hits from the screen and confirmed their importance for parasite growth in IFNγ-stimulated human fibroblasts. Of these six genes, three have homology to GRA32, localize to dense granules, and coimmunoprecipitate with each other and GRA32, suggesting they might form a complex. Deletion of individual members of this complex leads to early parasite egress in IFNγ-stimulated cells. Thus, prevention of early egress is an important Toxoplasma fitness determinant in IFNγ-stimulated human cells. IMPORTANCE Toxoplasma infection causes serious complications in immunocompromised individuals and in the developing fetus. During infection, certain immune cells release a protein called interferon gamma that activates cells to destroy the parasite or inhibit its growth. While most Toxoplasma parasites are cleared by this immune response, some can survive by blocking or evading the IFNγ-induced restrictive environment. Many Toxoplasma genes that determine parasite survival in IFNγ-activated murine cells are known but parasite genes conferring fitness in IFNγ-activated human cells are largely unknown. Using a Toxoplasma adapted genome-wide loss-of-function screen, we identified many Toxoplasma genes that determine parasite fitness in IFNγ-activated human cells. The gene products of four top hits play a role in preventing early parasite egress in IFNγ-stimulated human cells. Understanding how IFNγ-stimulated human cells inhibit Toxoplasma growth and how Toxoplasma counteracts this, could lead to the development of novel therapeutics.

Effects of different levels of inosine-5'-monophosphate (5'-IMP) supplementation on the growth performance and meat quality of finishing pigs (75 to 100 kg).

This study aimed to assess the effects of dietary supplementation of inosine-5'-monophosphate (5'-IMP) on energy efficiency, growth performance, carcass characteristics, meat quality, oxidative status, and biochemical profile of blood plasma in finishing pigs. Fifty-four crossbred castrated male pigs were distributed in a randomized block design consisting of nine blocks, with six treatments per block and one animal per treatment per block. Experimental diets were as follows: positive control diet (PC, 3300 kcal ME/kg), negative control diet (NC, 3200 kcal ME/kg), and four diets prepared by supplementing the NC diet with 0.050%, 0.100%, 0.150%, or 0.200% 5'-IMP. Based on regression analysis, supplementation with 0.129% 5'-IMP increased average daily weight gain (1.30 kg). Backfat thickness, pH45minutes and redness of m. Longissimus Lumborum (LL) increased linearly with 5'-IMP supplementation level. Drip loss and pH at 24 h post-slaughter had a quadratic response to 5'-IMP supplementation. It is concluded that 5'-IMP supplementation positively influenced growth performance, carcass characteristics and LL meat quality in finishing barrows.

Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model". However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.

Loss of MUNC18-1 leads to retrograde transport defects in neurons.

Loss of the exocytic Sec1/MUNC18 protein MUNC18-1 or its target-SNARE partners SNAP25 and syntaxin-1 results in rapid, cell-autonomous and unexplained neurodegeneration, which is independent of their known role in synaptic vesicle exocytosis. cis-Golgi abnormalities are the earliest cellular phenotypes before degeneration occurs. Here, we investigated whether loss of MUNC18-1 causes defects in intracellular membrane transport pathways in primary murine neurons that may explain neurodegeneration. Electron, confocal and super resolution microscopy confirmed that loss of MUNC18-1 expression results in a smaller cis-Golgi. In addition, we now show that medial-Golgi and the trans-Golgi Network are also affected. However, stacking and cisternae ultrastructure of the Golgi were normal. Overall, ultrastructure of null mutant neurons was remarkably normal just hours before cell death occurred. By synchronizing protein trafficking by conditional cargo retention in the endoplasmic reticulum using selective hooks (RUSH) and immunocytochemistry, we show that anterograde Endoplasmic Reticulum-to-Golgi and Golgi exit of endogenous and exogenous proteins were normal. In contrast, loss of MUNC18-1 caused reduced retrograde Cholera Toxin B-subunit transport from the plasma membrane to the Golgi. In addition, MUNC18-1-deficiency resulted in abnormalities in retrograde TrkB trafficking in an antibody uptake assay. We conclude that MUNC18-1 deficient neurons have normal anterograde but reduced retrograde transport to the Golgi. The impairments in retrograde pathways suggest a role of MUNC18-1 in endosomal SNARE-dependent fusion and provide a plausible explanation for the observed Golgi abnormalities and cell death in MUNC18-1 deficient neurons.

Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages.

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages.

Toxoplasma Mechanisms for Delivery of Proteins and Uptake of Nutrients Across the Host-Pathogen Interface.

Many intracellular pathogens, including the protozoan parasite Toxoplasma gondii, live inside a vacuole that resides in the host cytosol. Vacuolar residence provides these pathogens with a defined niche for replication and protection from detection by host cytosolic pattern recognition receptors. However, the limiting membrane of the vacuole, which constitutes the host-pathogen interface, is also a barrier for pathogen effectors to reach the host cytosol and for the acquisition of host-derived nutrients. This review provides an update on the specialized secretion and trafficking systems used by Toxoplasma to overcome the barrier of the parasitophorous vacuole membrane and thereby allow the delivery of proteins into the host cell and the acquisition of host-derived nutrients.

Early Golgi Abnormalities and Neurodegeneration upon Loss of Presynaptic Proteins Munc18-1, Syntaxin-1, or SNAP-25.

The loss of presynaptic proteins Munc18-1, syntaxin-1, or SNAP-25 is known to produce cell death, but the underlying features have not been compared experimentally. Here, we investigated these features in cultured mouse CNS and DRG neurons. Side-by-side comparisons confirmed massive cell death, before synaptogenesis, within 1-4 DIV upon loss of t-SNAREs (syntaxin-1, SNAP-25) or Munc18-1, but not v-SNAREs (synaptobrevins/VAMP1/2/3 using tetanus neurotoxin (TeNT), also in TI-VAMP/VAMP7 knock-out (KO) neurons). A condensed cis-Golgi was the first abnormality observed upon Munc18-1 or SNAP-25 loss within 3 DIV. This phenotype was distinct from the Golgi fragmentation observed in apoptosis. Cell death was too rapid after syntaxin-1 loss to study Golgi abnormalities. Syntaxin-1 and Munc18-1 depend on each other for normal cellular levels. We observed that endogenous syntaxin-1 accumulates at the Golgi of Munc18-1 KO neurons. However, expression of a non-neuronal Munc18 isoform that does not bind syntaxin-1, Munc18-3, in Munc18-1 KO neurons prevented cell death and restored normal cis-Golgi morphology, but not synaptic transmission or syntaxin-1 targeting. Finally, we observed that DRG neurons are the only Munc18-1 KO neurons that do not degenerate in vivo or in vitro In these neurons, cis-Golgi abnormalities were less severe, with no changes in Golgi shape. Together, these data demonstrate that cell death upon Munc18-1, syntaxin-1, or SNAP-25 loss occurs via a degenerative pathway unrelated to the known synapse function of these proteins and involving early cis-Golgi abnormalities, distinct from apoptosis.SIGNIFICANCE STATEMENT This study provides new insights in a neurodegeneration pathway triggered by the absence of specific proteins involved in synaptic transmission (syntaxin-1, Munc18-1, SNAP-25), whereas other proteins involved in the same molecular process (synaptobrevins, Munc13-1/2) do not cause degeneration. Massive cell death occurs in cultured neurons upon depleting syntaxin-1, Munc18-1, and/or SNAP-25, well before synapse formation. This study characterizes several relevant cellular phenotypes, especially early cis-Golgi abnormalities, distinct from abnormalities observed during apoptosis, and rules out several other phenotypes as causal (defects in syntaxin-1 targeting and synaptic transmission). As proteins, such as syntaxin-1, Munc18-1, or SNAP-25, modulate α-synuclein neuropathy and/or are dysregulated in Alzheimer's disease, understanding this type of neurodegeneration may provide new links between synaptic defects and neurodegeneration in humans.

VEGF system expression by immunohistochemistry and real-time RT-PCR study on collared peccary placenta.

Vascular endothelial growth factor (VEGF) is known to induce endothelial cell proliferation, to promote cell migration, and to inhibit apoptosis, thus playing a central role in angiogenesis and in the regulation of vasculogenesis. The expression of the VEGF-ligand receptor system was studied in the placenta and uterus of the collared peccary in nonpregnant females in the luteal phase and throughout pregnancy (>35, 75, 115, and 135 days). The material was examined by immunohistochemistry and by real-time reverse transcription polymerase chain reaction. Intense positive immunolabeling was observed for VEGF and its receptors in the uterine epithelium, uterine glands, and trophoblast. The endothelial cells and smooth muscle cells in the maternal and fetal vessels, as well as the connective tissue and mesenchyme, had weak immunoreactivity during all periods of pregnancy. The regression analysis of the real-time polymerase chain reaction results demonstrated cubic behavior, showing a specific time-dependent profile during pregnancy, which increased over the last gestational period to VEGF and VEGFR-1. The relative expression of VEGFR-2 decreased in the middle-third of the pregnancy and increased in late pregnancy. In the collared peccary, the expression of the VEGF-ligand receptor system was similar to that in porcine and ruminant placentas, suggesting that an epitheliochorial placenta has the same physiological and interhemal barrier during vascular gestational development. The expression of VEGF among cells not related to the vascular system, such as those of the uterine epithelium, trophoblast, and uterine glands, suggests a distinct regulatory role for these cells in vasculogenesis and also a different role of VEGF pathway.

Utilização da vitamina D3 e seus metabólitos na alimentação de frangos de corte sobre parâmetros imunológicos e morfometria intestinal / Use of vitamin D3 and its metabolites in broiler feed on immunological parameters and intestinal morphometry

Os efeitos da suplementação da vitamina D3 e de seus diferentes metabólitos foram avaliados na resposta imune e na morfometria intestinal de frangos de corte. Foram utilizados 952 frangos de corte de um dia de idade, distribuídos em um delineamento inteiramente casualizado, com quatro tratamentos, sete repetições e 34 aves por unidade experimental. Os tratamentos foram constituídos por quatros diferentes fontes de vitamina D3: colecalciferol (D3), 25-hidroxicolecalciferol (25(OH)D3),1,25dihidroxicolecalciferol (1,25(OH)2D3) e 1α-hidroxicolecalciferol (1α(OH)D3). As diferentes fontes foram incluídas na dieta, fornecendo 2000 e 1600 UI de vitamina D, nas fases pré-inicial, inicial e de crescimento, respectivamente. O peso relativo do intestino delgado diferiu entre os tratamentos aos 7, 21 e 42 dias e o peso relativo do fígado somente aos 42 dias de idade. Os demais órgãos e parâmetros imunológicos avaliados (peso dos órgãos linfóides, reação de hipersensibilidade cutânea basofílica, avaliação da atividade de macrófagos, dosagem de nitrito e perfil heterofilo: linfócito foram similares entre os animais alimentados com os diferentes metabólitos. Houve efeito (P<0,05) dos diferentes metabólitos da vitamina D3 sobre o comprimento dos vilos de jejuno e íleo aos sete dias, diferindo entre os animais alimentados com 1,25(OH)2D3 e 1α(OH)D3 para jejuno e 1,25(OH)2D3 e vitamina D3 para íleo (P<0,05). Para as demais fases não foi observado influência (P>0,05) dos tratamentos. Os parâmetros imunológicos não foram afetados pelos diferentes metabólitos de vitamina D. Os diferentes metabólitos de vitamina D afetaram positivamente a morfometria intestinal na altura de vilo na fase inicial, sendo os melhores resultados obtido pelos animais alimentados com 1,25(OH)2D3, contudo os parâmetros imunológicos foram similares entre os metabólitos estudados.

The aim of this experiment was to evaluate the effect of different vitamin D metabolites on immune response and intestine morphometry of broiler chickens. We used 952 1-day-old chicks, male Cobb, distributed into a completely randomized design with four treatments, seven replications and 34 birds each. There were evaluated four different metabolites of vitamin D: D3, 25(OH)D3, 1,25(OH)2D3 and 1α(OH)D3, providing 2000 and 1600 IU/kg feed of vitamin D in initial and growth period, respectively. The different metabolites of vitamin D did not affect the relative weight of the organs except for the weight of the intestine and liver at 21 and 42 days. There was a significant effect (P<0.05) of the different metabolites of D3 vitamin on the villi length of jejunum and ileum at 7 days, between animals fed with 1,25(OH)2D3 and 1α(OH)D3 to the jejunum and 1,25(OH)2D3 and vitamin D3 to the ileum (P<0.05). There were no differences (P>0.05) for weight of lymphoid organs, cutaneous basophil hypersensitivity reaction, macrophage activity assessment, measurement of nitric oxide and heterophil profile: lymphocyte. The different sources of vitamin D affect the intestine morphometrics on the villi length in the initial phase, but the effect was not observed in other phases. The immunological parameters were not affected by metabolites of vitamin D.

Desenvolvimento corporal e testicular em machos de codornas de corte e de postura de 25 a 360 dias / Body and testicular development in males of meat- and egg-type quails of 25 to 360 days

Codornas do tipo carne e tipo ovos foram analisadas para determinar o desenvolvimento reprodutivo, a puberdade e o início da senilidade. Para tal, 288 codornas (144 codornas de corte e 144 de postura) foram acompanhadas desde a eclosão até os 360 dias de idade. As aves foram distribuídas por idade em 18 grupos, sendo 8 codornas/grupo/tipo de codorna. Após 35 dias as codornas foram mantidas em condições de fotoperíodo de dias longos (17luz: 7escuro). O peso vivo e os valores morfométricos e histológicos testiculares foram determinados em cada período. Os dados obtidos foram analisados para determinar a curva de crescimento e o comportamento dos parâmetros analisados. O modelo que mais se adequou aos dados foi o modelo não linear de Gompertz (Y=A exp [-B e (-kt)]). O peso vivo e as características testiculares macro e microscópicas apresentaram comportamento alométrico entre si, sendo que, aproximadamente aos 60 dias os machos apresentaram-se sexualmente desenvolvidos, e estabilizaram o peso corporal por volta dos 100 dias. O testículo direito é mais cranial que o esquerdo e diferem em relação a comprimento e largura, porém não foi observada diferenças (P>0,05) para peso testicular. As codornas de corte apresentaram peso corporal e peso testicular maiores que as codornas de postura, porém as codornas de postura apresentaram peso relativo testicular maior. Durante todo o período analisado os machos puderam ser considerados sexualmente aptos. Os reprodutores apresentaram características sexuais ativas até os 360 dias de idade, representadas pelo tamanho testicular e pela atividade celular nos túbulos seminíferos.

Meat- and egg-type quails were analyzed to determine reproductive development, puberty and the onset of senility. For this purpose, 288 quails (144 meat- and 144 egg-type quails) were followed from hatch to 360 days old. The birds were distributed by age in 18 groups, 8 quails/group/type of quail. After 35 days quails were maintained under long daylength conditions (17L: 7D). The live weight and the macro and microscopical testicular characteristics were determined in each period. The data were analyzed to determine the growth curve and the behavior of parameters. The model that best fits the data was the non-linear Gompertz model (Y = A exp [-B and (-kt)]). Live weight and testicular morphometric parameters had an allometric behavior among themselves, and at approximately 60 days the males had sexually development, and body weight stabilized at about 100 days. The right testicle was more cranial than the left, differing in the length and width, but no difference in testicular weight (P>0.05) was observed. The meat quails had a larger body weight and testicular weight than the egg quails, but the egg-quail had a higher percentage of testes in relation to body weight. During the whole adult period the males could be considered sexually fit. Breeders had active sex characteristics until 360 days of age, represented by testicular size and by cellular activity in the seminiferous tubules.

Effects of starter diet supplementation with arginine on broiler production performance and on small intestine morphometry / Efeito da suplementação de arginina na dieta de frangos na performance produtiva e na morfologia do intestino delgado

The effects of starter diet (days 1 to 21) supplemented with arginine (Arg) on the production performance and duodenum and jejunum mucosa morphometry of broilers were studied. Male Cobb broiler chickens (990) were randomly assigned to one of five treatments in a complete random design. Measurements of 33 chicks per treatment were made in six repetitions. The treatments consisted of a basal diet with 1.390% digestible Arg (no supplementation) and four dietary levels (1.490%, 1.590%, 1.690%, and 1.790%), providing a relationship with lysine of 1.103; 1.183; 1.262; 1.341 and 1.421%, respectively. From the age of 22 days on, all birds received conventional grower diet. The data were submitted to regression analysis by polynomial decomposition of the degrees of freedom in relation to the levels of Arg. The Arg supplementation increased (P<0.05) the live weight and the feed conversion ratio without increasing the feed intake of the birds. However, no effect was observed (P>0.05) in the growth phase (days 22 to 42) in the absence of the Arg supplementation. The supplementation of Arg over of NRC recommendation during the starter phase may be necessary for the expression of the maximal weight gain potential in birds. No effect (P<0.05) of Arg dietary supplementation was observed either on small intestine weight and length at any age. However, the duodenum villus:crypt ratio increased and the crypt depth decreased in the first week in response to increasing dietary Arg. It is concluded that broiler Arg dietary supplementation in the starter diet improved production performance and small intestine morphometry, especially in the first week.

O efeito na dieta inicial (1-21 dias) da suplementação de arginina (Arg) foi estudada sobre o desempenho e a morfologia da mucosa do jejuno em frangos de corte. Pintos machos Cobb (990) foram utilizados em um delineamento inteiramente casualizado com 5 tratamentos e 6 repetições com 33 aves cada. Os tratamentos consistiram em uma dieta basal com 1,390% de Arg digestível (sem suplementação) e 4 dietas com adição (1,490%; 1,590%; 1,690% e 1,790%), fornecendo uma relação de lisina de 1,103; 1,183; 1,262; 1,341 e 1,421%, respectivamente. A partir de 22 dias todas as aves receberam ração convencional. Os dados foram submetidos à análise de regressão pela decomposição polinomial dos graus de liberdade, referentes aos níveis de Arg A suplementação de Arg melhorou (P<0,05) o peso vivo e a conversão alimentar sem aumentar o consumo de ração. No entanto, não houve efeito (P>0,05) na fase de crescimento (22 a 42 dias) na ausência de suplementação de Arg. A suplementação acima do recomendado pelo NRC na fase inicial pode ser necessária para a expressão máxima do potencial de ganho de peso em aves. Não houve efeito da suplementação de Arg na dieta no peso e comprimento do intestino delgado em nenhuma idade. No entanto, a relação vilo:cripta no duodeno aumentou e a profundidade da cripta diminuiu na primeira semana em resposta ao incremento de Arg na dieta. Concluiu-se que em frangos de corte a suplementação de Arg na dieta inicial melhorou o desempenho e a morfometria do intestino delgado, especialmente na primeira semana.

Morfologia dos órgãos genitais masculinos e da cloaca da ema (Rhea americana americana) / Morphology of the male genital organs and cloaca of Rhea americana americana

As características morfológicas, macroscópicas e microscópicas, dos órgãos genitais masculinos e da cloaca foram analisados em 23 emas, quatro filhotes (duas semanas), sete jovens (de três a oito meses) e doze adultos (três anos), provenientes da Cooperativa Emas do Brasil, RS, e do CEMAS, Mossoró, RN. Os testículos da ema possuem formato alongado e localizam-se na cavidade celomática, na região intra-abdominal dorsal, com comprimento e larguras médias de 7,6±1,2cm e 2,6± 0,7cm nos adultos; 4,5±1,5cm e 0,9±0,4cm nos jovens; e 0,8±0,3cm, e 0,2±0,1cm nos filhotes. O testículo está envolto pela túnica albugínea e seu parênquima possui túbulos seminíferos irregulares, compostos por epitélio espermatogênico e por células de sustentação, e pelo tecido intersticial, com as células endócrinas intersticiais, tecido conjuntivo frouxo e vasos. Nos adultos observaram-se todas as células da linhagem espermatogênica, enquanto nos jovens com 3 meses, os testículos apresentaram túbulos seminíferos com luz reduzidas, espermatogônias e células de sustentação indiferenciadas. Os ductos eferentes possuem um epitélio cúbico ciliado, enquanto no ducto epididimário o epitélio é columnar. O epidídimo apresentou-se alongado e fusiforme junto a margem medial do testículo. O ducto deferentes apresentou trajeto sinuoso nos adultos, retilíneo nos jovens, convoluto na sua porção média, diminuindo seu formato sigmóide em sua porção caudal, próximo à cloaca. O epitélio é pseudoestratificado e reveste a luz irregular nos adultos e circular nos jovens, mantendo proximidade com o ureter. A cloaca dividiu-se em três segmentos: o coprodeu, o urodeo e o proctodeo. No urodeu os ductos deferentes desembocaram em papilas na parede ventro-lateral, próximo a inserção do falo fibroso. O falo é um órgão fibroso linfático, localizado na parede ventral, no assoalho da cloaca, e apresentou duas porções: uma rígida bifurcada e contorcida, e outra simples espiralada e flexível...

The rhea (Rhea americana americana) is a bird that belongs to the group of the Ratitas, order Rheiforme and family Rheidae. Macroscopic and microscopic morphology of the male genital organ (testes, epididymis, deferent ducts, and phallus) and the cloaca were analyzed in 23 emas, four chicken (2 weeks old), young (3 to 10 months old), and twelve adult ones (3 years old), from Cooperativa Emas do Brasil, RS and from CEMAS, Mossoró, RN. The testis of rhea had elongated shape and were located inside coelomatic cavity, in dorsal region of abdominal cavity, with medium length and width of 7.6±1.2cm and 2.6±0.7cm at adult animals; 4.5±1.5cm and 0.9±0.4cm at young animals; and 0.8±0.3cm, and 0.2±0.1cm at chicken. The testis were recovered by the tunica albuginea and its parenchyma had seminiferous tubules composed by spermatogenic epithelium and by sustentation cells, and also interstitial tissue, with interstitial endocrine cells, connective tissue and vessels. At the adult animals were observed all the cells from spermatogenic lineage, whilst at the youngs with 3 months the seminiferous tubules had a smale lumen with spermatogonia and undifferentiated sustentacular cells. The efferents ductus were composed by a cubic ciliated epithelium, while the epididimydis duct had a columnar epithelium. The epididymis was elongated and fusiform closely to medial testis board. The deferent duct had sinuous stretch at adult animals, rectilineae at young animal, convolute at its medium portion, decreasing its sigmoid shape at caudal portion, next to cloaca. The epithelium was pseudostratified ciliated, irregular lumen at adult animal, and circular at young animal, closely with urether. The cloaca was divided into three segments: coprodeum, urodeum and proctodeum. At urodeum the deferent ducts discharged into papillas at the ventral side wall, next to fibrous phallus's insertion. The phallus was a lymphatic fibrous organ, located at ventral wall, at the cloaca floor...

Growth and development of the placenta in the capybara (Hydrochaeris hydrochaeris).

BACKGROUND: The guinea pig is an attractive model for human pregnancy and placentation, mainly because of its haemomonochorial placental type, but is rather small in size. Therefore, to better understand the impact of body mass, we studied placental development in the capybara which has a body mass around 50 kg and a gestation period of around 150 days. We paid attention to the development of the lobulated arrangement of the placenta, the growth of the labyrinth in the course of gestation, the differentiation of the subplacenta, and the pattern of invasion by extraplacental trophoblast. METHODS: Material was collected from six animals at pregnancy stages ranging from the late limb bud stage to mid gestation. Methods included latex casts, standard histology, immunohistochemistry for cytokeratin, vimentin, alpha-smooth muscle actin, and proliferating cell nuclear antigen as well as transmission electron microscopy. RESULTS: At the limb bud stage, the placenta was a pad of trophoblast covered by a layer of mesoderm from which fetal vessels were beginning to penetrate at folds in the surface. By 70 days, the placenta comprised areas of labyrinth (lobes) separated by interlobular areas. Placental growth resulted predominantly from proliferation of cellular trophoblast situated in nests at the fetal side of the placenta and along internally directed projections on fetal mesenchyme. Additional proliferation was demonstrated for cellular trophoblast within the labyrinth.Already at the limb bud stage, there was a prominent subplacenta comprising cellular and syncytial trophoblast with mesenchyme and associated blood vessels. At 90 days, differentiation was complete and similar to that seen in other hystricognath rodents. Overlap of fetal vessels and maternal blood lacunae was confirmed by latex injection of the vessels. At all stages extraplacental trophoblast was associated with the maternal arterial supply and consisted of cellular trophoblast and syncytial streamers derived from the subplacenta. CONCLUSION: All important characteristics of placental development and organization in the capybara resembled those found in smaller hystricognath rodents including the guinea pig. These features apparently do not dependent on body size. Clearly, placentation in hystricognaths adheres to an extraordinarily stable pattern suggesting they can be used interchangeably as models of human placenta.

Veias do sistema porta-hepático em gansos domésticos / Veins from hepatic portal vein system in domestic geese

A distribuição intraparenquimal das veias porta-hepáticas foi estudada em 30 gansos domésticos. Latex Neoprene corado foi injetado pela veia isquiática e os animais forma fixados por imersão e injeção intramuscular com formol a 10 por cento e dissecados. O fígado esteve composto por um grande lobo hepático direito e por um lobo hepático esquerdo menor, os quais estiveram conectados por uma ponte de parênquima. O lobo direito do fígado teve exclusivamente vasos do sistema porta-hepático formados pela distribuição intraparenquimal da veia porta-hepática direita, enquanto que no lobo esquerdo estes originaram-se da veia porta-hepática direita e de pequenas veias porta-hepáticas esquerdas. A veia porta-hepática direita emitiu o ramo caudal direito, que emitiu um pequeno ramo caudolateral direito e um grande ramo caudomedial direito. Cranialmente esta veia emitiu os ramos craniais direito e ramos lateral direito. A porção transversa da veia porta-hepática direita cruzou para o lobo hepático esquerdo, emitindo de 1 a 6 pequenos ramos craniais e caudais para a região média do fígado. No lobo esquerdo, o ramo esquerdo da veia porta-hepática direita emitiu o ramo cranial esquerdo, o ramo lateral esquerdo e o ramo medial. De 1 a 6 veias porta-hepáticas esquerdas foram identificadas desembocando ou no ramo esquerdo da veia porta-hepática direita ou em sua porção transversa, oriundos do ventrículo gástrico e do pró-ventrículo. Em 40 por cento dos gansos uma veia porta-hepática própria oriunda da confluência de vasos venosos da face esquerda do ventrículo distribuiu-se na extremidade caudal do lobo esquerdo isoladamente.

The intraparenchymal distribution of the hepatic portal veins in 30 domestic geese were studied. Stained Neoprene latex was injected into the isquiatic vessels, and the animals were fixed in 10 percent formaldehyde by immersion and intramuscular injection. The liver of geese was composed of a large right and a smaller left hepatic lobe, connected by a parenchyma bridge. The right hepatic lobe had vessels exclusively from the hepatic portal system composed by intraparenchymal distribution of the right hepatic portal vein, while the vessels of the left hepatic lobe came from the right hepatic portal vein and from small left hepatic portal veins. The right hepatic portal vein emitted the right caudal branch, which emitted a small right caudolateral branch and a large right caudomedial branch. Cranially this vein emitted right cranial and right lateral branches. The tranverse portion of the right hepatic portal vein crossed to the left hepatic lobe, emitting 1 to 6 small cranial and caudal branches to the medial area of the liver. In the left hepatic lobe, the left branch from the right hepatic vein emitted the left cranial, left lateral and left median branches. One to six left hepatic portal veins were identified arising from the left branch or from the transverse portion of the right hepatic portal vein. These vessels arose from the gizzard and pro-ventricle. In 40 percent of geese one proper hepatic portal vein originated from venous vessels of the gizzard and was distributed into the caudal extremity of the left hepatic isolated lobe.

The materno-fetal interface in llama (Lama guanicoe glama)

Samples from 9 llamas (28 through 36 weeks of gestation) were collected and fixed in 4 percent buffered paraformaldehyde (light microscopy) and in 2.5 percent buffered glutaraldehyde (transmission and scanning electron microscopy). The material was processed in paraplast and slides (5mm) were stained with HE, PAS, Masson-Trichrome, acid phosphatase and Perl's. The uteroferrin was immunolocalized. The results show that llama placenta is chorioallantoic, diffuse, folded and epitheliochorial, and the fetus is covered with an epidermal membrane. The trophoblast cells have variable morphology: cubic, rounded and triangular cells, with cytoplasm containing PAS-positive granules. Binucleated cells with large cytoplasm and rounded nuclei, as well as giant trophoblastic cells with multiple nuclei were also observed. Numerous blood vessels were observed beneath the cells of the uterine epithelium and around the chorionic subdivided branches. Glandular activity was shown by PAS, Perl's, and acid phosphatase positive reactions in the cytoplasm and glandular lumen, and by immunolocalization of the uteroferrin in the glandular epithelium. The uterine glands open in spaces formed by the areoles, which are filled by PAS-positive material. The llama fetus was covered by the epidermal membrane, composed of stratified epithelium, with up to seven layers of mono-, bi- or trinucleated cells. The high level of maternal and fetal vascularization surfaces indicates an intense exchange of substances across both surfaces. The metabolic activity shown in the uterine glands suggests an adaptation of the gestation to the high altitudes of the natural habitat of this species.

Fragmentos da placenta de 9 animais (28-36 semanas de gestação), provenientes do Instituto Veterinario de Investigaciones Tropicales y de Altura (IVITA), Cusco-Peru, e da Universidad del Altiplano (UNA), Puno-Peru, foram colhidos e fixados em paraformoldeído 4 por cento em PBS para microscopia de luz e em glutaraldeido em 2,5 por cento PBS para microscopia eletrônica de transmissão e de varredura. O material incluido em paraplast cortado com 5 mm foi processado para HE, PAS, Tricrômio de Masson, fosfatase ácida e Perl's e para imuno-histoquimica da uteroferrina. Os resultados mostraram que a placenta da lhama é corialantóide, difusa, pregueada e epiteliocorial e o feto está recoberto pela membrana epidermal. O trofoblasto possui morfologia variada: células cúbicas, arredondadas ou triangulares, com citoplasma contendo grânulos PAS+. Células binucleadas com citoplasma aumentado e núcleo arredondado e células trofoblásticas aumentados com múltiplos núcleos, também foram observadas. Grande quantidade de vasos sanguíneos foi observada entre as células do epitélio uterino e ao redor das projeções coriônicas, as quais estavam subdivididas. A atividade glandular foi demonstrada pelas reações de PAS, Perl's e fosfatase ácida positivas e pela imunolocalização da uteroferrina na luz e epitélio glandular. As glândulas uterinas abrem-se nos espaços formados pelas aréolas, as quais estavam preenchidas por material PAS+. Os fetos das lhamas estavam recobertos pela membrana epidermal, constituída por um epitélio estratificado composto por mais de 7 camadas de células mono, bi ou trinucleadas. A alta vascularização das superfícies materna e fetais indica intensa capacidade de trocas de substâncias entre as duas superfícies, e a atividade metabólica mostrada pelas glândulas uterinas sugere adaptação da gestação às altitudes elevadas do habitat natural desta espécie.