Fernanda Assaife de Mello

Dynamic expression of tgf-β2, tgf-β3 and inhibin βA during muscle growth resumption and satellite cell differentiation in rainbow trout (Oncorhynchus mykiss).

Members of the TGF-β superfamily are involved in numerous cell functions; however, except for myostatin, their roles in the regulation of muscle growth in fish are completely unknown. We measured tgf-β1, tgf-β2, tgf-β3, inhibin βA (inh) and follistatin (fst) gene expression during muscle growth recovery following a fasting period. We observed that tgf-β1a and tgf-β2 expression were quickly down-regulated after refeeding and that tgf-β3 reached its highest level of expression 7days post-refeeding, mirroring myogenin expression. Inh βA1 mRNA levels decreased sharply after refeeding, in contrast to fst b2 expression, which peaked at day 2. No significant modification of expression was observed for tgf-β1a, tgf-β1b, tgf-β1c and tgf-β6 during refeeding. In vitro, tgf-β2 and inh βA1 expression decreased during the differentiation of satellite cells, whereas tgf-β3 expression increased following the same pattern as myogenin. Surprisingly, fst b1 and fst b2 expression decreased during differentiation, whereas no variation was observed in fst a1 and fst a2 expression levels. In vitro analyses also indicated that IGF1 treatment up-regulated tgf-β3, inh βA1 and myogenin expression, and that MSTN treatment increased fst b1 and fst b2 expression. In conclusion, we showed that the expression of tgf-β2, tgf-β3 and inh βA1 is dynamically regulated during muscle growth resumption and satellite cell differentiation, strongly suggesting that these genes have a role in the regulation of muscle growth.

Caracterização da mastite subclínica em caprinos produzidos em sistema orgânico no Rio Grande do Sul

Milk and derivates are recognized as vehicle of different pathogens for humans. These micoorganisms may manifest as post-milking contamination or as in infections in the animal itself, particularly in mastitis. The aim of present study was characterizes the occurrence of mastitis and management aspects in dairy-goats raised in three farms of organic milk. The correlation between mastitis and goat husbandry was also investigated. The clinical exam of the mammary glands of 64 dairy-goats did not reveal the presence of clinical mastitis. Nevertheless, the California Mastitis Test (CMT) identified 54 (22.7%) reactive mammary halves (+ or ++). From 238 milk samples collected, 37 (15.6%) were positive for bacterial isolates. Coincident positive results for bacterial isolate tests and CMT were observed in only eight samples, which indicate a sensitivity index of 21.6% for the caprine subclinical mastitis diagnosis test. Coagulase-negative staphylococci (CNS) were identified as the most frequent pathogen in milk samples (83.8%). The in vitro sensitivity test revealed CNS strains resistant to cotrimoxazole (50%), ampicillin (48.1%), nitrofurantoine (7.7%), cefaclor (7.14%), and oxacilline (3.85%). Cefalotine, gentamicin, neomycin, streptomycin, and tetracycline were the most effective drugs. No correlation was observed between the occurrence of subclinical mastitis and race, lactation period, drinking water quality in farms, or milking system as adopted in the present study.

Growth curve by Gompertz nonlinear regression model in female and males in tambaqui (Colossoma macropomum)

Was evaluated the pattern of growth among females and males of tambaqui by Gompertz nonlinear regression model. Five traits of economic importance were measured on 145 animals during the three years, totaling 981 morphometric data analyzed. Different curves were adjusted between males and females for body weight, height and head length and only one curve was adjusted to the width and body length. The asymptotic weight (a) and relative growth rate to maturity (k) were different between sexes in animals with ± 5 kg; slaughter weight practiced by a specific niche market, very profitable. However, there was no difference between males and females up to ± 2 kg; slaughter weight established to supply the bigger consumer market. Females showed weight greater than males (± 280 g), which are more suitable for fish farming purposes defined for the niche market to larger animals. In general, males had lower maximum growth rate (8.66 g / day) than females (9.34 g / day), however, reached faster than females, 476 and 486 days growth rate, respectively. The height and length body are the traits that contributed most to the weight at 516 days (P <0.001).

Identification of TGF-β, inhibin βA and follistatin paralogs in the rainbow trout genome

Since their initial discovery, TGF-β superfamily members have been considered multifunctional growth and differentiation factors in many cell types. Various studies have clearly demonstrated the key roles of specific TGF-β members in muscle growth, including myostatin and inhibin as well as genes, such as follistatin. By binding to TGF-β members, follistatin prevents TGF-β from binding to its receptors and thus neutralizes its activity. Here, we report the identification of the gene sequences of four TGF-β isoforms and three paralogs of TGF-β1, which we called TGF-β1a, TGF-β1b and TGF-β1c, four sequences of inhibin βA paralogs; and two sequences of follistatin paralogs from rainbow trout. A phylogenetic analysis clearly indicated the existence of four monophyletic clades, corresponding to TGF-β1, -β2, -β3 and -β6. Based on their sequence identity TGF-β1a and -β1c are grouped together, whereas TGF-β1b appears more divergent even though it is grouped within the TGF-β1 clade. Alignments and phylogenetic analyses showed that the protein sequences of TGF-β, inhibin βA and follistatin are extremely well conserved (>90%) relative to each other; however, their regulation and expression patterns are different. TGF-β2 and -β3 showed the most abundant expression in muscle and were the main TGF-β members expressed in this tissue. Follistatin and inhibin βA paralogs were expressed in all tissues examined but with different patterns. Our identification of multiple copies of TGF-β, inhibin βA and follistatin with different expression patterns suggests non-redundant functions for these paralogs in rainbow trout.

Association of the polymorphism g.8514C>T in the osteopontin gene (SPP1) with milk yield in the dairy cattle breed Girolando

Source/description: The genomic DNA of 434 Girolando animals was amplified for the fourth intron of the osteopontin gene (SPP1) (HUGO nomenclature: secreted phosphoprotein 1) and then digested with restriction enzyme BsrI to detect the alleles of this SNP by PCR-RFLP. Three genotypes were identified in the population, TT, CT and CC (52.53%, 38.71% and 8.76%), respectively, and these were used for an association study with data on 305 day milk yield (M305) and predicted transmitting ability for milk yield (PTAM). Recorded milk data were from the Dairy Control Service of the Brazilian Association of Breeders Girolando managed by Embrapa Dairy Cattle. The predicted values of transmitting ability for milk (PTAM) for bulls and their daughters were taken from the national breed genetic evaluation programme in 2009 by Embrapa; these values are adjusted in relation to the genetic basis (7.59 kg), defined as the mean genetic 646 cows born in 2000.

The effect of cryoprotectant agents on DNA methylation patterns and progeny development in the spermatozoa of Colossoma macropomum

DNA methylation patterns are inherited from parents and are imperative for proper embryonic development; however, alterations in these patterns can compromise fertilization and development into a fully functioning adult animal because DNA methylation is part of a complex program of gene transcription. In this study, we investigated the impact of cryoprotectant agents (CPAs) on DNA methylation patterns in spermatozoa and the consequences on embryonic development and the survival rate of progeny. Global methylation was assessed by enzymatic reactions in Colossoma macropomum spermatozoa that were cryopreserved using dimethylsulfoxide, dimethylformamide, methanol, ethyl glycol and glycerol as CPAs. Fertilization was carried out to evaluate survival rates and abnormalities in embryonic development upon treatment with each of the CPAs. Fresh semen served as the control. Our results indicated that, compared to the control group, spermatozoa cryopreservation decreased the fertilization rate and delayed embryonic development from the midblastula stage. Furthermore, spermatozoa cryopreserved in all CPAs had lower methylation levels and exhibited more delays and abnormalities during embryonic development than did fresh semen. Methanol resulted in fertilization, hatching rates and embryonic development that were closer to the control but had lower methylation levels. In conclusion, ours results show significant alterations on spermatozoa DNA methylation patterns caused by CPAs that are used in the semen cryopreservation process. DNA methylation pattern alterations affected the viability of progeny (r=0.48); however, these effects can be minimized by choosing the CPA that will compose the freezing solution.

Morphological and morphometric analysis of skeletal muscle between male and female young adult Colossoma macropomum (Characiformes: Serrasalmidae)

This study aimed to evaluate muscle organization in tambaqui in order to describe the muscle growth process. We analyzed the morphometric pattern of fibers from white muscle of young-adults (300 days) by smaller diameter. The organization of white muscle exhibited a typical morphological pattern found in other fish species. Heavier animals showed higher frequency of larger diameter fibers (>50 μm ) and smaller animals had higher frequency of smaller diameter fibers ( 50 μm ) than males. However, there was no difference between body weight and sex (P =0.8). Our results suggest that muscle growth is by hypertrophy and hyperplasia due to a mosaic appearance from different diameters fibers, which is characteristic of large size fish species.

Análise de diversidade genética do gene da osteopontina em bovinos da raça girolando

The objective was to obtain the indices of genetic diversity for the SNP (single nucleotide polymorphism) of the 4 intron osteopontin gene (OPN) for 434 animals (87 bulls and 347 cows) participants in the Teste de Progenie da raca girolando (Girolando Progeny Test) in Brazil. For amplification, primers used were described for the Holstein breed, and differentiation of alleles C/T SNP that was obtained by PCR-RFLP. Genotype frequencies of TT (52.53%), CT (38.71%) and CC (8.76%) and allele frequencies of T (71.9%) and C (28.1%) indicate that the population is in Hardy-Weinberg principle (HWP). Although the OPN gene locus is in HWP, the higher frequency of allele T of SNP in these animals may suggest a setting-trend of allele T in the race. No difference was observed between the group of bulls and cows (FST = -0.018), supporting the estimate of population balance. Considering the values estimated by the FIS (0.043), it is likely that high numbers of individuals homozygous for the T allele observed in the population occur because of possible inheritance of this allele coming from the zebu breed, rather than inbreeding. Thus, to better characterize the OPN gene polymorphism, assessments in a larger number of animals must be performed, since only animals that participated in the Progeny Test were assessed.