Isabel Martinele

Efeito da monensina e do óleo de soja sobre os protozoários ciliados do rúmen e correlação dos protozoários com parâmetros da fermentação ruminal e digestivos

Four lactating dairy cows with ruminal cannula and 155 DIM were allotted to a 4 × 4 Latin square and fed twice daily corn silage and concentrate (55:45, %DM) to evaluate the effect of monensin and soybean oil in the diet of lactating cows on the counting of ciliate protozoa beyond establishing correlations between number of protozoa with some parameters of digestion and ruminal fermentation. The treatments consisted of the inclusion of 33 ppm of sodium monensin or soybean oil at 4% DM, as following: control diet, without oil or monensin - CT; diet with monensin - MN; diet with soybean oil - OL; diet with the combination of 33 ppm of monensin and 4% of soybean oil - OM. Occurrence of 11 ciliate genera was observed, being Entodinium the predominant in all treatments. No significant soybean oil × monensin interaction was observed on protozoa number and genera. The genera Entodinium, Dasytricha, Eremoplastron and Isotricha reduced in the diets with oil, while monensin reduced the counting of Dasytricha, Eremoplastron and Epidinium. The total number of ciliate protozoa and cellulolytics protozoa was reduced by soybean oil and monensin effects, showing a defaunatory additive effect when combined the oil and the monensin. The ciliate cellulolytics was reduced by linoleic acid intake and was related positively with NDF ruminal digestibility and ruminal ammonia. The total number of protozoa was correlated with the molar proportion of propionate in the ruminal liquid, suggesting that monensin and soybean oil, due their defaunatory effect, can reduce the loss of methane in the rumen.

Effect of an energy-deficient diet on populations of ciliate protozoans in bovine rumen

Foram utilizados 10 novilhos mesticos com cânula ruminal, distribuidos em dois grupos: no grupo controle (C; n=4) receberam dieta balanceada para ganho diario de 900g; no grupo tratado com carencia pronunciada de energia (CP; n=6), receberam dieta com 30% a menos do nivel de mantenca em energia. Apos 140 dias sob esses regimes de alimentacao, foram coletadas amostras do fluido ruminal e urina, para realizacao de provas bioquimicas e funcionais, antes e as 1, 3, 6 e 9 horas apos o fornecimento do alimento. A carencia energetica resultou em diminuicao significativa na quantidade dos protozoarios Entodinium, Eodinium, Isotricha, Dasytricha, Eremoplastron, Eudiplodinium, Metadinium, Charonina, Ostracodinium e Epidinium. Nao houve efeito da hora de coleta sobre o total de ciliados nos grupos C e CP. Maior numero de formas em divisao binaria foi registrado no grupo C, na sexta e nona horas pos-alimentacao (P<0,019). Observaram-se altas correlacoes positivas entre a contagem total de protozoarios e a fermentacao de glicose, amonia e o indice de excrecao urinaria de alantoina e negativa entre a contagem total de protozoarios e a reducao do azul de metileno, e correlacao media entre a contagem total de protozoarios e os acidos graxos volateis totais. A determinacao da populacao de protozoarios do rumen e um metodo simples de avaliacao, alem de que particularmente auxilia o diagnostico clinico da funcao ruminal.

Methods for Taxonomic Studies of Rumen Ciliates (Alveolata: Ciliophora): A Brief Review

This review presents the principal methods used in taxonomic studies of rumen ciliates: live observation, Lugol staining, fixation and staining with methyl-green formalin saline (MFS) solution, protargol staining, silver carbonate impregnation, scanning electron microscopy and molecular techniques. Mastering these techniques is essential for successful research on the taxonomy of rumen ciliates. No single technique reveals all of the characteristics required for a complete description of a rumen ciliate; therefore, it is necessary to combine the use of these techniques as appropriate to the rumen ciliate group under study. Tables are provided to summarize: 1) morphological methods more appropriate for revealing morphological structures of interest, 2) morphological methods indicated for each group of rumen ciliates, and 3) main primers used for PCR amplification of the 18S rDNA of rumen ciliates.

Diet botanical composition and rumen protozoa of sheep in Brazilian Semi-Arid area

This study aimed to determine the influence of seasonal variations on diet botanical composition and rumen protozoa of five rumen fistulated Santa Ines crossbred sheep kept in natural Caatinga vegetation pasture, Pernambuco State, Brazil. Samples were collected in July 2005 and January 2006, the respective rainy and dry seasons. Botanic composition of animal diet was determined by analysis of ruminal extrusa. The rainy season diet was composed mainly by herbaceous and grass species (88%), with Herissantia tiubae making up 51.7%. The dry season diet consisted of shrub species (67.4%), mainly Capparis yco (26.3%) and Bauhinia cheilantha (22.3%). Under such conditions variations in protozoa populations were observed, with the higher total concentration (p<0.05) during the rainy season. Populations of Entodinium genus predominated in both seasons, although decreasing (p<0.05) during the dry season, as did the Epidinium and Eremoplastron genera. The Dasytricha, Diploplastron, Eudiplodinium and Isotricha genera had the higher concentrations (p<0.05) in the dry season.

Molecular Characterization and Biochemical and Histopathological Aspects of the Parasitism of Haemoproteus spp. in Southern Caracaras (Caracara plancus)

Abstract:  Haemoproteid species have a wide global distribution, and they have been described in falcon species in several parts of the world. However, few studies in South America have focused on these birds. Haemoproteus spp. infections have been reported as the causative agents of serious histopathological changes, which can lead to the death of the host. Thus, this study aimed to molecularly and phylogenetically characterize Haemoproteus spp. in Caracara plancus, to characterize aspects of parasitism through clinical analysis and biochemical parameters, and to describe the histopathology of infection. To examine these aspects, 5 southern caracaras were examined clinically, and blood samples were collected. Blood smears were subsequently utilized in parasitemia calculations, PCR amplification, and serum biochemical investigations. Histological sections of the liver, kidneys, spleen, and heart were examined to check for possible histopathological changes. The birds showed clinical signs such as pallor and prostration that are consistent with Haemoproteus spp. infection. Moreover, the examination of the blood smears revealed 0.07% parasitemia in young gametocytes only. The PCR and sequencing results confirmed that the parasites belonged to Haemoproteus spp. The activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymes, albumin, total serum proteins, and enzymatic urea were first described in C. plancus and serve as reference for future studies of bird species parasitized by Haemoproteus spp. Histopathology results showed signs of injury that were consistent with haemosporidian infection in the tissues of the analyzed organs. The present study is preliminary, and additional studies of Haemoproteus spp. infections in other bird species are needed to better understand the relationship between parasites and hosts, because despite the low parasitemia recorded, biochemical and histopathological changes in various organs were observed.

Abundance and diversity of rumen protozoa in lambs fed Gliricidia sepium silage

The objective of this study was to evaluate changes in ruminal protozoa in lambs after partial replacement of feed concentrates in their diets with Gliricidia sepium silage. Twenty-four male Santa Ines lambs with an average initial weight of 14.5 kg were used. The experimental design was completely randomized, with four treatments and six replications. Treatments (given as a percentage of dry matter) were as follows: control - corn silage (600 g kg-1 as fed) + concentrate (400 g kg-1 as fed); GS133 - corn silage (600 g kg-1 as fed) + G. sepium silage (133 g kg-1as fed) + concentrate (267 g kg-1 as fed); GS267 - corn silage (600 g kg-1 as fed) + G. sepium silage (267g kg-1as fed) + concentrate (133 g kg-1 as fed); and GS400 - corn silage (600 g kg-1 as fed) + G. sepium silage (400 g kg-1as fed). Samples of rumen contents were obtained at slaughter, and analysis revealed the presence of nine genera of rumen protozoa that were present in all animals, with the exception of Enoploplastronand Eremoplastron. There were no significant differences in the average total numbers of rumen ciliates or in the composition of species between lambs. Inclusion of up to 400 g kg-1 (as fed) G. sepium silage in the diet of lambs does not affect the diversity or density of rumen protozoa.

Quantitative and differential analysis of ciliate protozoa in rumen content samples filtered before and after fixation

The objective of this study was to assess whether the straining of rumen content samples influences the estimation of protozoal density. Ninety rumen samples were obtained from 30 cattle (three samples per animal). The samples were subjected to one of three treatments at the moment of collection: 1) fixation informalin without straining(control treatment), 2) straining before fixation informalin, or 3)straining after fixation informalin.To test the hypothesis of the variation in the protozoa composition in the samples, multivariate analyses with non-metric multidimensional scaling (NMDS) were carried out. The diversity and density of rumen protozoa were negatively affected by straining before fixation.In the pre-filtered sample, the number of ciliates from the genus Entodinium was reduced, and no individuals from the Diploplastron, Elytroplastron and Eudiplodinium genera were detected; these effects were not observed in the other two treatments. Straining after fixation did not interfere with the diversity of the ruminal community, but the abundance of protozoa was greater thanin the control treatment and significantly greater than in the samples filtered before fixation. These factors suggest that post-fixation straining is the recommended technique to analyze rumen protozoa.

Rumen ciliates in Brazilian sheep ( Ovis aries ), with new records and redescription of Entodinium contractum (Entodiniomorphida: Ophryoscolecidae)

The species composition, prevalence, and average relative abundance of rumen ciliates were analyzed in 16 Brazilian sheep. 28 species of ciliates were identified belonging to 3 families: Isotrichidae, Ophryoscolecidae, and Parentodiniidae. Among these ciliates, Entodinium alces, Metadinium esalqum, and M. rotundatum were found for the first time in sheep, and other 12 species for the first time in Brazilian sheep. Different morphotypes of parentodiniid ciliates were identified in 7 of the 16 sheep analyzed and this is the second report of this family in ruminants. The species Entodinium contractum was redescribed based on the new data on the general morphological features; for the first time, we described the oral infraciliature, which in this species is of the Entodinium-type. In this species, it is noteworthy that the prominent vestibular polybrachykinety can be used as an important identifying characteristic.

Molecular Phylogeny of the Family Ophryscolecidae (Ciliophora, Litostomatea) Inferred from 18S rDNA Sequences.

The 18S rDNA was used to infer oral ciliature patterns of evolution within the family Ophryoscolecidae, with the addition of five new sequences of ciliates from the genus Ostracodinium. Our data confirmed the monophyly of the subfamilies Entodiniinae and Ophryoscolecinae, but more analysis would be required for the definition of the status of the subfamily Diplodiniinae. The oral infraciliature patterns reflect evolutionary divergence in the family Ophryscolecidae, observing monophyly on Entodinium‐type, Diplodinium‐type, Ostracodinium‐type, Epidinium‐type, and Ophryoscolex‐type. The ancestral infraciliature of Entodinium‐type cannot be proven, however, the position of Entodinium‐type showed closer of Diplodinium‐type than Ophryoscolex‐type, corroborating previous studies using morphological characters. The observed inconsistencies reflect the need to increase the number of 18S rDNA sequences to family Ophryoscolecidae and investigate the evolution of this group using other molecular markers.

Predation and cannibalism among ciliate protozoans (Ciliophora: Entodiniomorphida: Ophryoscolecidae) in the rumen of sheep (Ovis aries)

The objective of the present study was to record the occurrence of predation and cannibalism among ciliate protozoans in the rumen of sheep. The analyzed samples consisted of ruminal contents of five fistulated Santa Inez crossbred sheep, maintained in a natural caatinga (scrubland) pasture in the municipality of Sertânia, Pernambuco. Six samples of ruminal content were obtained from each sheep (total of 30 samples) and fixed in formalin at 18.5% (v/v). One 1 ml aliquot was obtained from each sample of ruminal content and received tree drops of a lugol solution for staining and identification of protozoa ciliates. These aliquots were analyzed in slides and coverslips, at randomly determined fields. An instance of predator-prey interaction was observed among ciliates of Ophryoscolecidae Stein, 1859, where Elytroplastron bubali (Dogiel, 1928) preyed on Enoploplastron triloricatum (Dogiel, 1925) and on species of Epidinium Crawley, 1923 and Entodinium Stein, 1859. Cases of cannibalism were observed among specimens of E. bubali, which consist of the first record this type of interaction in this species. This is, moreover, the first record of occurrence of E. bubali in the rumen of sheep in Brazil.