Effects of supplemental dietary vitamin C on quality of semen from Nile tilapia (Oreochromis niloticus) breeders.

The objective of this study was to evaluate the effects of vitamin C on growth and quality of semen from Oreochromis niloticus breeders. One hundred and sixty males were fed with different levels of vitamin C (0, 261, 599 and 942 mg/kg diet). The higher weight values were recorded for 599 (166 g) and 942 (175 g) mg of vitamin C/kg diet. Sperm motility, vigour and concentration were higher with 599 and 942 mg of vitamin C/kg diet. The semen volume, gonadosomatic index and plasma protein data from the last week showed a direct relationship with increasing levels of vitamin C. No changes were observed in the hepatosomatic index and blood glucose. The haematocrit and erythrocyte showed higher values estimated by equations derived at 850 and 638 mg vitamin C/kg diet, respectively. The leucocytes were inversely proportional to the increasing levels of vitamin C. After 100 days of feeding, animals fed the diet containing 942 mg vitamin C/kg diet had higher sperm motility, linearity, curvilinear velocity, straight line velocity and average path velocity (p < .05). Higher values of beat cross-frequency were observed in broodfish fed diets containing 942 and 599 mg vitamin C/kg. The different vitamin C levels did not cause differences in straightness, lateral head displacement and sperm morphology. For Nile tilapia males on intensive rearing and handling conditions, vitamin C levels between 599 and 942 mg/kg may be used for a better performance and quality of semen.

Análise histomorfométrica do útero na espécie canina do nascimento aos seis meses de idade / Histomorphometric analysis of the uterus in dogs from birth to six months of age

Objetivou-se caracterizar para a espécie canina o desenvolvimento histológico uterino do nascimento aos seis meses de idade. Foram analisados úteros (n=32) de animais com idades entre um e 180 dias pós-nascimento (DPN), distribuídos em oito grupos: G1-1 DPN (1 Yorkshire Terrier, 1 Poodle, 2 Sem Raça Definida (SRD)/peso médio (Pm)=190g), G2-15 DPN (2 Yorkshire Terrier e 2 SRD/Pm=354g), G3-30 DPN (1 Rottweiler, 1 Poodle, 2 SRD/Pm=985g), G4-45 DPN (1 Poodle, 3 SRD/Pm=1,1kg), G5-60 DPN (1 Yorkshire Terrier, 1 Poodle e 2 SRD/Pm=1,4kg), G6-90 DPN (4 SRD/Pm=2,8kg), G7-120 DPN (1 Poodle e 3 SRD/Pm=6,6kg) e G8-180 DPN (1 Rottweiler, 1 Poodle e 2 SRD/Pm=11kg). A análise histológica constituiu de mensurações das espessuras (µm) da parede uterina, miométrio, endométrio, assim como diâmetro (µm) e número de glândulas endometriais. A análise estatística demonstrou estabilidade e homogeneidade nas estruturas avaliadas com coeficientes de variação baixos (<10%). Observou-se: útero com um DPN: epitélio com células cúbicas simples, miométrio rudimentar, presença de perimétrio e ausência de glândulas endometriais; aos 15 DPN: desenvolvimento de estrutura primordial de formação das glândulas endometriais; dos 30 aos 45 DPN: endométrio e glândulas endometriais simples; aos 60 DPN: glândulas endometriais em lâmina própria com ramificações e discreto pregueamento endometrial; de 90 a 180 DPN: todas as estruturas uterinas apresentaram histoarquitetura de um animal maduro. Todas as variáveis analisadas apresentaram correlação positiva com a idade pós-nascimento (R2≥72,2%). Conclui-se que o útero da cadela desenvolve-se continuamente do nascimento à 180 dias de vida e que apresenta-se desprovido de glândulas endometriais ao nascimento. As primeiras glândulas endometriais são observadas aos 15 dias de vida e apresenta conformação histológica de um animal adulto após 60 dias do nascimento.

This study aimed to characterize canine species' uterine histological development from birth to six months of age. Uteri (n = 32) of animals aged between one and 180 days postnatal (PND), distributed into eight groups were analyzed: G1-1 PND (1 Yorkshire Terrier, Poodle 1, 2 Mixed Breed (MB)/Medium weight (Mw) = 190g), G2-15 PND (2 Yorkshire Terrier and 2 MB/Mw = 354g), G3-30 PND (1 Rottweiler, 1 Poodle, 2 MB/Mw = 985g), G4-45 PND (1 Poodle, 3 MB/Mw = 1.1kg), G5-60 PND (1 Yorkshire Terrier, Poodle 1 and 2 MB/Mw = 1.4kg), G6-90 PND (4 MB/Mw = 2.8kg), G7-120 PND (1 Poodle and 3 MB/Mw = 6.6kg) and G8-180 PND (1 Rottweiler, Poodle 1 and 2 MB/Mw = 11kg). Histological examination consisted of thickness measurement (µm) of the uterine wall, myometrium, endometrium, as well as diameter (µm) and number of endometrial glands. Statistical analysis showed stability and uniformity in the evaluated structures with low coefficients of variation (<10%). We observed: uterus with one PND: simple cubic epithelium cells, rudimentary myometrium, perimetrium presence and absence of endometrial glands; at 15 PND: development of primordial structure formation of endometrial glands; from 30 to 45 PND: simple endometrium and endometrial glands; PND 60: endometrial glands in the lamina propria with branches and discreet endometrial pleating; 90-180 PND: uterine all structures presented histoarchitecture of a mature animal. All variables were positively correlated with postnatal age (R2 ≥ 72.2%). It is concluded that the uterus of the bitch continuously evolves from birth to 180 days old and is presented devoid of endometrial glands at birth. The first endometrial glands are observed at 15 days of life and present histological conformation to an adult 60 days after birth.

Production of oocytes of Nile tilapia (Oreochromis niloticus) for in vitro fertilization via hormonal treatments.

Only a few studies have described hormonal treatments for induction of synchronicity and gamete collection in Nile tilapia (Oreochromis niloticus), both important for assortative matings in breeding programmes and essential for polyploidy technologies. In this study, we compared the effectiveness of carp pituitary extract (CPE), Nile tilapia pituitary extract (TPE), human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone (GnRH) protocols on the induction of spawning and egg production in Nile tilapia. Among the hormonal treatments analysed, only hCG was effective for producing viable gametes for in vitro fertilization. To verify the viability of this hormonal treatment, hCG was tested using different doses (1000, 2000, 3000, 4000 and 5000 IU/kg) in a large number of females (208 animals) from two Nile tilapia lines. The results indicated that hCG doses between 1000 and 5000 IU/kg could be used to induce final oocyte maturation in Nile tilapia with collection of stripped oocytes. This is the first study to report differential reproductive responses to hormonal treatment between tilapia lines: line 1 was more efficient at producing eggs and post-hatching larvae after hCG induction than line 2. In conclusion, we demonstrated that the hCG protocol may be applied on a large scale to induce final oocyte maturation in Nile tilapia. The development of a protocol for in vitro fertilization in Nile tilapia may aid in breeding programmes and biotechnological assays for the development of genetically modified lines of Nile tilapia.

Efeitos da eletroacupuntura, aquapuntura e farmacopuntura em cadelas anestesiadas com isofluorano e submetidas à ovário-histerectomia / Effects of electroacupuncture, aquapuncture and farmacopuntura in dogs anesthetized with isoflurane and submitted to ovariohysterectomy

Avaliaram-se os efeitos analgésicos da eletroacupuntura, aquapuntura e farmacopuntura com morfina nos acupontos VB41 e TA5 de 24 cadelas hígidas submetidas à ovário-histerectomia. Os animais foram distribuídos em quatro grupos (G) de igual número - GDest, GMorf, GElet e GC - e anestesiados com acepromazina, propofol e isofluorano. Após a estabilização do plano anestésico, os animais do GDest receberam 0,5mL de água destilada em cada acuponto; os do GMorf receberam 0,1mg/kg de morfina distribuído nos quatro acupontos; os do GElet foram submetidos à eletroacupuntura; e os do GC, acupuntura em pontos sham. Os animais do GC receberam, após o término do procedimento cirúrgico e antes do início da avaliação pelas escalas de dor, 2,0mg/kg de tramadol. Foram avaliadas: frequências cardíaca e respiratória, temperatura retal e glicemia. A dor foi avaliada por duas escalas, uma de analogia numérica e outra contagem variável, por três observadores. A avaliação iniciou-se imediatamente após a extubação e foi realizada a cada 15 minutos, durante duas horas. Não houve diferença entre os tratamentos em todas as variáveis. Pode-se concluir que eletroacupuntura, aquapuntura e farmacopuntura com morfina resultaram em analgesia similar ao tramadol no pós-operatório imediato de cadelas submetidas à ovário-histerectomia eletiva.

In order to evaluate the analgesic effects of electro-acupuncture, aquapuncture and morphine pharmacopuncture on VB41and TA5 acupoints, 24 healthy bitches were anesthetized with acepromazine, propofol and isoflurane and underwent ovariohysterectomy. The animals were equally divided into four groups: GDest, GMorf, GElet and CG. After anesthetic stabilization, GDest animals received pure water (0.5ml per acupoint); GMorf received morphine (0.1mg/kg); electroacupuncture was performed in GElet animals, and CG animals were submitted to acupuncture on Sham points. The CG animals received 2.0mg/kg of tramadol after the end of surgery and before the postoperative evaluation through pain scales. Data recorded were: heart and respiratory rate, rectal temperature and blood glucose. Pain was assessed using two different scales, one using numerical analogy and another using variable counting, both performed by three different professionals. Evaluation started immediately after the animal was extubated and was performed every 15 minutes, for 2 hours. There was no statistical difference between groups in all variables studied. It is concluded that electro-acupuncture, aquapuncture and morphine pharmacopuncture produce analgesia similar to tramadol in the postoperative period of bitches undergoing elective ovariohysterectomy.

High embryonic recovery rates with in vivo and ex vivo techniques in the bitch.

The embryonic collection techniques in dogs present a vast methodological variation and low recovery rates. The objectives were to compare and describe two techniques as to the recovery of canine embryos, on the 12th day after the first mating or artificial insemination. Embryos were recovered through uterine horn flushing in vivo, before performing the ovariohysterectomy (OHE) (Group 1; n = 9) or ex vivo, immediately after the OHE (Group 2; n = 9). In total, 43 and 47 embryonic structures were recovered in Groups 1 and 2, respectively. There was no significant difference (p > 0.05) between groups on recovery rates (72.8% and 81.0%, respectively). We inferred that both in vivo and ex vivo techniques allow a high rate of embryonic recovery; in the collection technique prior to the OHE, it is essential to carefully handle the reproductive system during the trans-surgical period and that the 12th day (D12) after the first mating/artificial insemination is an efficient option for the high recovery rate of morulae and blastocysts.

Técnicas de acesso cirúrgico vascular destinadas à monitoração invasiva em cães / Técnicas de acceso quirúrgico vascular para el monitoreo invasivo en perros / Surgical vascular access techniques for the dog invasive monitoring

A monitoração é essencial durante os procedimentos anestésicos, e pode ser realizada por métodos invasivos ou não-invasivos. A monitoração invasiva é obtida mediante a colocação de instrumentos no interior do corpo do animal, principalmente em vasos sanguíneos. Desse modo, objetivou-se com este estudo apresentar técnicas de abordagem cirúrgica vascular destinadas a monitoração invasiva em cães, descrever os acessos dos principais vasos sanguíneos, como as artérias femoral, safena medial, carótida comum, metatársica e sublingual e das veias jugular externa, femoral e safena medial e revisar os materiais necessários para a canulação destes vasos, bem como, as principais complicações dessas técnicas de abordagem cirúrgica.

Monitoring is essencial during anhestesic procedures, and can be performed by invasive or non-invasive methods. Invasive monitoring is anchieved with the insert of instruments inside the animal body, mainly in blood vessels. Thus, it was aimed with this study to present techniques of vascular surgical approach for invasive monitoring of dogs, describe the access of the main blood vessels, as the femoral artery, medial saphenous artery, carotid artery, metatarsal artery and sublingual artery, and the extern jugular vein, femoral and saphenous veins. Besides, the materials needed for vessel canulation are revised, and also the main complications of these surgical approach techniques.

El monitoreo es esencial durante los procedimientos anestésicos, y puede realizarse por métodos invasivos o no-invasivos. El monitoreo invasivo se logra mediante la colocación de instrumentos en el interior del cuerpo del animal, principalmente en vasos sanguíneos. Así, se buscó con este estudio presentar técnicas de abordaje quirúrgica vascular destinadas al monitoreo invasivo en perros, describir los accesos de los principales vasos sanguíneos, como las arterias femoral, safena medial, carótida común, metatársica y sublingual y de las venas yugular externa, femoral y safena medial, también revisar los materiales necesarios para canulación de estos vasos, así como las principales complicaciones de esas técnicas de abordaje quirúrgica.

A reliable and specific enzyme-linked immunosorbent assay for the capture of IgM from human chagasic sera using fixed epimastigotes of Trypanosoma cruzi.

A rapid, sensitive, specific, and reliable enzyme-linked immunosorbent assay (ELISA) is proposed for determination of the levels of anti-Trypanosoma cruzi IgM in acute chagasic sera (ACD). The efficiency of this ELISA as a diagnostic method was compared with that of parasite DNA detection by polymerase chain reaction (PCR) and that of indirect immunofluorescence (iIF) anti-T. cruzi IgM detection. We tested whether this ELISA using fixed epimastigotes (epi) could detect anti-T. cruzi IgM in serum samples from two groups of children with acute Chagas' disease from a hyperendemic area in Bolivia. In a comparison of the ELISA method with other techniques, 95% and 71% of the results correlated with PCR and iIF findings, respectively. At the serum dilution applied (1:250), rheumatoid factor (RF) did not influence the results, and samples from patients carrying leishmaniasis or mixed Leishmania and T. cruzi infection could also be excluded from ACD. Highly specific and reliable results were obtained, a great number of the sera could be tested in only one assay, and a quantitative index of reactivity (IR) could be calculated without serial titration. Using test samples in triplicate, the method provides a useful tool for the detection of early acute-phase T. cruzi infection in humans.

Acute Chagas' disease: plasma levels of alpha-2-macroglobulin and C-reactive protein in children under 13 years in a high endemic area of Bolivia.

We measured the levels of two human acute phase proteins (APP), alpha-2-macroglobulin (A2M) and C-reactive protein (CRP), in sera of 56 healthy and 84 acute chagasic children aged from 1 to 13 years old, from a highly endemic area in Bolivia. In such areas, children are continuously exposed to vectors and the frequency of acute cases increases with age. Quantitation of A2M and CRP were performed using sandwich ELISAs, that were shown to be sensitive, reproducible and suited for studying many samples rapidly. A2M levels observed were higher in healthy younger children, decreasing with age until children reached their teens, and their distribution suggested a relationship between A2M concentration and age that could be consistently expressed by a power function. The same does not occur with CRP levels. Concentrations of A2M were age-adjusted to allow comparison using sera collected from children with different ages. Both A2M and CRP were significantly increased in acute chagasic children. Since parasites are commonly present in blood and tissues during the acute phase of Chagas' disease, it is possible that the high levels of A2M may act as inhibitors of a high load of proteinases, derived either from the parasites, from host cell damage or from both.

Acute-phase proteins and serologic profiles of chagasic children from an endemic area in Bolivia.

Chagas' disease represents a major public health problem in Latin America. In endemic areas, it is important to detect acute and even asymptomatic infections in children so that specific therapy can be started immediately. We studied 203 sera from children from the region of Cochabamba, Bolivia. A high percentage of seropositive individuals was found in the three villages studies. Levels of alpha-2 macroglobulin (A2M) and C-reactive protein (CRP) increased in a significant number of children with acute Chagas' disease. The combined analysis of serologic and biochemical parameters can define the different stages of acute infection by Trypanosoma cruzi: 1) an early stage, with an increase only in specific immunoglobulin M (IgM) levels; 2) intermediate stages, with high specific IgM and IgG levels and/or high anti-galactose (anti-Gal) levels and increased A2M and/or CRP levels; and 3) a late acute stage, with low IgM levels but high A2M, CRP, anti-Gal, and specific IgG levels. The detection of high IgG levels alone is indicative of the chronic/indeterminate stage of Chagas' disease. We also show serologic differences between seropositive asymptomatic villagers and symptomatic patients undergoing medical care; asymptomatic cases presented higher levels of A2M and lower levels of specific antibodies.

Heterogeneity in the synthesis of alpha-macroglobulins in outbred Swiss albino mice acutely infected with Trypanosoma cruzi.

Alpha-Macroglobulins (AM) are protease inhibitors with important roles in inflammation and in immunomodulation that behave as acute-phase proteins in many experimental models. In the present work the levels of AM in the plasma of outbred Swiss albino mice acutely infected with Trypanosoma cruzi were studied. The results showed that increased levels of AM were present in the majority of the infected mice and that AM levels increased independently of the rise in parasitaemia. There was a high degree of heterogeneity in the intensity of the modulation of AM levels as well as in the kinetics of AM synthesis. This heterogeneity was related neither with the intensity of infection nor with the sex of the host. No correlation between AM levels and survival to the acute phase could be observed in the outbred mice. The consequence of such a heterogeneity is unclear, although AM as immunoregulatory molecules could play a role in the development of the symptoms of the chronic phase of Chagas' disease.

An inhibition ELISA to determine alpha macroglobulin levels in mouse plasma.

A sensitive method for quantifying mouse plasma alpha-macroglobulins (AM) using an inhibition ELISA is described. AM are important plasma proteinase inhibitors that possibly act also as immunomodulatory molecules. The standard protocol developed in our experiments involves coating well with 10 micrograms/ml A2M in carbonate buffer, followed by incubation with a 1:1 (v/v) mixture of the plasma to be tested (diluted 1/1000) and goat anti-AM (diluted 1/1250). This is followed by further incubation, first with the enzyme-conjugated antibody and with the substrate prior to the reading of absorbance levels of the reaction products. Standard curve samples must be included in each plate, employing known amounts of the purified Murine Alpha-2-Macroglobulin (MuA2M) used for coating, with concentrations ranging from 0.001 to 10 micrograms/ml. Using test samples in triplicates and a 6-point standard curve in a single ELISA plate, 25 plasma samples can be tested accurately. The method offers an useful tool for establishing AM levels in small samples of mouse plasma.

Heterogeneity in the plasma levels of two acute-phase proteins in mice from inbred strains infected with Trypanosoma cruzi.

We analyzed the variations observed in the plasma levels of both alpha-macroglobulins (AM) and serum amyloid P (SAP) in mice from three different inbred strains (C3H, Balb/C and C57black/6) acutely infected with Trypanosoma cruzi. SAP levels increased in C57black/6 and Balb/C mice but not C3H mice. AM levels increased in all C3H mice but not in C57black/6 mice and rose slightly in only 43% of the Balb/C mice. AM and SAP levels are differently modulated in patterns that may be strain-determined.

The second messenger cyclic 3',5'-adenosine monophosphate in pathogenic microorganisms with special reference to protozoa.

There is a growing body of information on signal transduction components in microorganisms. Elements of the cyclic 3',5'-adenosine monophosphate signaling system and molecules similar to hormones and receptors have been identified in the majority of prokaryotes and unicellular eukaryotes that have been studied. The presence of ligand- and receptor-like molecules in parasitic microorganisms raises the possibility that these molecules may interact with host communication systems. Adrenergic control of proliferation, differentiation, and infectivity has been described in the flagellate protozoan Trypanosoma cruzi, the etiological agent of Chagas' disease. Interactions between host and parasitic systems could also be antibody mediated, with antigenic cross-reactivity between components of their cAMP-dependent systems. In this review, we discuss these possibilities and summarize the existing data in this area.

Improved enzyme-linked immunoadsorbent assay (ELISA) for the study of Trypanosoma cruzi-host cell interaction in vitro.

We herein present an improved assay for detecting the presence of Trypanosoma cruzi in infected cultures. Using chagasic human sera (CHS), we were able to detect T. cruzi infection in primary cultures of both peritoneal macrophages and heart muscle cells (MHC). To avoid elevated background levels--hitherto observed in all experiments especially in those using HMC--CHS were preincubated with uninfected cells in monolayers or suspensions prior to being used for detection of T. cruzi in infected monolayers. Preincubation with cell suspensions gave better results than with monolayers, reducing background by up to three times and increasing sensitivity by to twenty times. In addition, the continuous fibroblastic cell line L929 was shown to be suitable for preadsorption of CHS. These results indicate that the high background levels observed in previous reports may be due to the presence of human autoantibodies that recognize surface and/or extracellular matrix components in cell monolayers. We therefore propose a modified procedure that increases the performance of the ELISA method, making it an useful tool even in cultures that would otherwise be expected to present low levels of infection or high levels of background.

Sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs.

The sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs were identified on the basis of electrophoretic mobility, enzymatic degradation with specific mucopolysaccharidases and by the type of degradation products formed. The results obtained indicated that chondroitin sulfate and heparan sulfate were the main glycosaminoglycans found, that most of the labeled glycosaminoglycans were found in the pericellular pool, and that no marked differences were observed in the sulfated glycosaminoglycan composition of the smooth muscle cells obtained from different organs. 'Liver connective tissue cells', isolated from pathological livers (which had been shown to possess biochemical and physiological features typical of smooth muscle cells) showed a pattern of glycosaminoglycan synthesis similar to that of the smooth muscle cells.