Arlindo A. Moura

Efeito do estresse climático sobre os parâmetros produtivos e fisiológicos de ovinos Santa Inês mantidos em confinamento na região litorânea do Nordeste do Brasil

A study was conducted to determine the effect of environmental stress on physiological criteria of feedlot sheep. Treatments consisted of two different housing conditions (shade and no shade) and two levels of concentrate in the diet: high concentrate (70% of concentrate and 30% of roughage-70C:30R); low concentrate (30% of concentrate and 70% of roughage-30C:70R). Animals raised under shade and fed a high concentrate diet (70C:30R) had greater dry mater intake (1258 g/animal/day) and crude protein (0.8% of body weight (BW) and 18 g/BW0.75), as well as weight gain (247 g/animal/day). Air temperature in the afternoon was 30.6:C, under shade, and 32.1:C, under sun and values of temperature humidity index(THI), 82.3 and 81.1, respectively. Animals fed more concentrate (70C:30R) had also higher respiratory frequency (RF), either raised under shade (87 mov/min) or under the sun (71 mov/min.). Rectal temperature (RT) was higher in the afternoon (39.1:C) than in the morning (38.9:C), but animals raised under shade and fed diets with 70% concentrate (70C:30R) had the highest value of RT (39.3:C). Regardless of type of housing conditions, the animals that received more concentrate (70C:30R) had greater rectal temperature (39.2:C) than those fed a diet with less concentrate (30C:70R) (38.8:C). Therefore, the type of diet influenced the response of the animals to housing conditions. Moreover, Santa Ines sheep are susceptible to environmental stress because had lower performance when raised under no shade.

Proteomics of cauda epididymal fluid from mature Holstein bulls.

The proteome of cauda epididymal fluid (CEF) from Holstein bulls was defined. Fluid was collected from the vas deferens, subjected to 2-D SDS-PAGE and spots identified by CapLC-MS/MS and MALDI-ToF/ToF. Because albumin accounted for 21.1% of all spot intensities in the gels examined by PDQuest, samples were subjected to albumin depletion and then analyzed again as before. Original CEF gels had 114 ± 3 spots, including as the most abundant: albumin, epididymal secretory protein E1, prostaglandin d-synthase and gelsolin. Epididymal fluid also expressed: clusterin, transferrin, N-acetyl-β-glucosaminidase, cauxin, glutathione peroxidase, acidic seminal fluid protein (aSFP), aldehyde reductase, α-l-fucosidase, α-1-β-glycoprotein, apolipoprotein A-1, β actin, calmodulin, cathepsin D, cystatin E/M, enolase, galectin 3-binding protein, leucine amino-peptidase and nucleobindin. Albumin depletion decreased that very spot to 10% of its original intensity and the resulting gels had, on average, 137 ± 4 spots. Spots identified as dipeptidyl-peptidase 7, angiotensin-converting enzyme, arylsulfatase A, aspartylglucosaminidase, serine protease inhibitors, new isoforms of calmodulin, cystatin E/M and a 17-kDa nucleobindin appeared only in depleted maps. This study is the first to report nucleobindin and aSFP as epididymal components. We suggest that CEF proteins act to facilitate membrane remodeling, transport of lipophilic substances, protect sperm and prevent premature acrosome reaction.

Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams ☆

The present study is focused on the proteome of reproductive tract fluids from tropically-adapted Santa Ines rams. Seminal plasma, cauda epididymal (CEF) and vesicular gland fluid (VGF) proteins were analyzed by 2-D electrophoresis and mass spectrometry. Seminal plasma maps contained 302 ± 16 spots, within the 4-7 pH range. From these maps, 73 spots were identified, corresponding to 41 proteins. Ram Seminal Vesicle Proteins (RSVP) 14 and 22kDa and bodhesins 1 and 2 represented the most abundant seminal components. Other seminal proteins included clusterin, angiotensin-converting enzyme, matrix metalloproteinase-2, tissue-inhibitor of metalloproteinase-2, plasma glutamate carboxypeptidase, albumin, lactoferrin, alpha enolase, peroxiredoxin, leucine aminopeptidase, β-galactosidase, among others. Later, seminal plasma gels were run within narrow pH intervals (3.9-5.1; 4.7-5.9; 5.5-6.7), allowing the additional identification of 21 proteins not detected in 4-7 pH maps. Major proteins of CEF and VGF were albumin and transferrin, and RSVPs, respectively. Western blots confirmed that RSVPs were mainly present in VGF while bodhesins, in VGF and CEF. Based on RT-PCR, RSVP and bodhesin genes were primarily expressed in the vesicular glands. In summary, the reproductive tract fluids of Brazilian hairy rams contain several categories of proteins, with potential roles in sperm protection, capacitation, acrosome reaction and sperm-oocyte interaction.

Binding patterns of bovine seminal plasma proteins A1/A2, 30 kDa and osteopontin on ejaculated sperm before and after incubation with isthmic and ampullary oviductal fluid ,

Previous studies from our laboratory have reported empirical associations between bovine seminal plasma protein(s) (BSP) A1/A2 and 30 kDa and osteopontin (OPN) in accessory sex gland fluid and bull fertility. These BSP and OPN are believed to bind to sperm at ejaculation and to remain bound until sperm reach the oviduct. The objective of the present study was to evaluate the topographical distribution of BSP A1/A2, 30 kDa and OPN binding on: (1) bovine ejaculated sperm; (2) ejaculated sperm incubated with isthmic oviductal fluid (ODF); (3) ejaculated sperm+isthmic ODF incubated in ampullary ODF. From each of these media, aliquots of sperm for BSP and OPN were processed for immunocytochemistry and analysis by laser scanning confocal microscopy. Isthmic and ampullary ODF was collected from indwelling catheters and used as pools from three cows in the non-luteal phase of the estrous cycle. Anti-BSP A1/A2 was detected bound to the midpiece, post-equatorial and equatorial segments and acrosome of sperm after ejaculation and after incubation with isthmic and ampullary ODF. The BSP A1/A2 fluorescence was more concentrated on the midpiece and increased as acrosome-intact sperm came in contact with ODF. As compared with acrosome-intact sperm, non-intact acrosome intact sperm had 39 and 68% reductions of acrosome fluorescence and 36% and 90% increases of post-equatorial fluorescence after contact with isthmic and ampullary ODF (P<0.05). Anti-BSP 30 kDa was more intense on the midpiece than on post-equatorial, equatorial and acrosome regions of sperm after ejaculation and contact with ODF. However, equatorial fluorescence was 141% and 89% more intense and acrosome stainning was 80% and 76% less (P<0.05) in non-intact acrosome sperm than in acrosome intact cells, during all ODF incubations. Anti-OPN was identified on the acrosome of ejaculated sperm, but with less fluorescence (P<0.05) on the post-equatorial segment and midpiece. Incubation of sperm with isthmic ODF increased fluorescence on post-equatorial segment (P<0.05). There were 72% and 78% reductions (P<0.05) of acrosome fluorescence and intensification (P<0.05) in equatorial fluorescence in non-intact acrosome sperm as compared with acrosome intact cells incubated with isthmic and ampullary ODF. In summary, interactions of BSP A1/A2 and 30 kDa and osteopontin with the sperm membrane undergo modifications dictated by the oviductal fluid. The BSP are thought to modulate cholesterol and phospholipid movement from the sperm membrane and help sperm binding to the oviductal epithelium. Furthermore, our model suggests that OPN participates in sperm-oocyte interaction, affecting fertilization and early embryonic development.

Desempenho reprodutivo de vacas Nelore no Norte e Nordeste do Brasil

The objective of this study was to estimate genetic and phenotypic parameters for reproductive traits of Nelore cows raised in the North and Northeast regions of Brazil. The traits studied were age at first calving (AFC), calving interval (CI), post-partum interval (PPI), number of services per conception (NSC) and gestation length (GL). The statistical analyses were done using the SAS program (Statistical Analysis System) and variance components were estimated by Restricted Maximum Likelihood method using the MTDFREML software. Means and heritability estimates for AFC, CI, PPI, NSC and GL were 45.14±10.83 months; 465.55±128.49 days; 165.76±110.29 days; 1.34±0.67 services and 295.03±5.85 days and 0.21±0.05; 0.05±0.02; 0.32±0.11; 0.05±0.04 e 0.12±0.04, respectively. The repeatability for CI, PPI, NSC and GL were, respectively, 0.05; 0.63; 0.09 and 0.22. The results suggest that despite the small genetic variability for the reproductive traits, AFC and GL must be included in selection programs. The high heritability estimate for PPI is probably associated to the structure of the data, in its majority coming from the same herd. The repeatability estimate for CI suggests that female culling based on first calving interval is not accurate and there is a risk of culling animals with probable good reproductive efficiency.

The Effects of Insulin and Follicle-Simulating Hormone (FSH) During In Vitro Development of Ovarian Goat Preantral Follicles and the Relative mRNA Expression for Insulin and FSH Receptors and Cytochrome P450 Aromatase in Cultured Follicles

ABSTRACT The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 μg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles. Low concentration of insulin with follicle-stimulating hormone is essential for promoting oocyte meiosis resumption in goat preantral follicles.

Análise do desempenho produtivo de diversos grupos genéticos Holandês x Gir no Brasil.

Total milk yield per lactation (PL), milk yield in 305 days (PL305) and lactation length (DL) were estimated based on records of the Brazilian Association of Girolando Breeders (3,574 lactations). Milk production was recorded monthly and 10.8% of the herds were raised under an extensive feeding system, 67.9% under a semi-intensive feeding system and 21.3% under an intensive feeding system. Data were analyzed through the least square method by GLM procedure (SAS, 1996). Preliminary analysis indicated a high interaction between genetic group and feeding system. Thus, the performance of genetic groups was evaluated within each feeding system. Genetic effects of additive difference between (g) Holstein and Gir breeds, dominance (d) and additive x additive epistatic interactions (gg) were also studied. It was observed that, in some cases, the effects of epistatic interactions should be taken into account. Moreover, increasing the proportion of Holstein-Friesian genes did not have any significant effect on milk yield when herds were raised under poor management conditions. However, as such conditions improved, crosses with higher proportion of Holstein tended to show better performance.

Efeitos da Insulação Escrotal sobre a Biometria Testicular e Parâmetros Seminais em Carneiros da Raça Santa Inês Criados no Estado do Ceará

A study was conducted to determine the effect of heat stress on semen criteria and testis size in Santa Ines hairy rams, in the State of Ceara, Northeast of Brazil. The scrotum of eight adult rams was insulated for seven days. Animals were evaluated twice before insulation and, after insulation, at 12 different periods until 118 days. Scrotal insulation did not affect semen volume, but pH was increased after eight days, returning to normal values after 15 days. Sperm concentration was reduced eight days after insulation was removed and animals became azzospermic between 33 and 50 days. After 79 days, sperm concentration returned to values similar to those observed before treatment. Scrotal circumference (26.4 cm) decreased to 22.4 cm eight days after insulation and to 21 cm at 21 days, but returned to 24.9 cm after 50 days. Moreover, sperm motility and vigor were reduced right after insulation was removed and returned to normal profiles only after 90 days. Before treatment, there were 1.7% of sperm cells with primary defects and 9.9% with secondary defects. Right after insulation, sperm cells with primary defects increased to 3.6% and those with secondary defects, to 43.4%, and after eight days, these values increased to 8.4 and 60.4%, respectively. From 15 to 60 days, sperm cells with primary defects varied from 27.3 to 16.8%, while those with secondary defects showed only small variations (39.9 to 39%). At the end of the experiment (118 days after insulation), primary defects were reduced to 0.7%, but the percentage of cells with secondary defects was still high (24.4 %). Therefore, heat stress caused temporary interruption of sperm production in the ram and sperm motility and secondary defects seemed to be the most sensitive criteria.

Sperm Protamine-Status Correlates to the Fertility of Breeding Bulls

ABSTRACT During fertilization, spermatozoa make essential contributions to embryo development by providing oocyte activating factors, centrosomal components, and paternal chromosomes. Protamines are essential for proper packaging of sperm DNA; however, in contrast to the studies of oocyte-related female infertility, the influence of sperm chromatin structure on male infertility has not been evaluated extensively. The objective of this study was to determine the sperm chromatin content of bull spermatozoa by evaluating DNA fragmentation, chromatin maturity/protamination, PRM1 protein status, and nuclear shape in spermatozoa from bulls with different fertility. Relationships between protamine 1 (PRM1) and the chromatin integrity were ascertained in spermatozoa from Holstein bulls with varied (high vs. low) but acceptable fertility. Sperm DNA fragmentation and chromatin maturity (protamination) were tested using Halomax assay and toluidine blue staining, respectively. The PRM1 content was assayed using Western blotting and in-gel densitometry, flow cytometry, and immunocytochemistry. Fragmentation of DNA was increased and chromatin maturity significantly reduced in spermatozoa from low-fertility bulls compared to those from high-fertility bulls. Field fertility scores of the bulls were negatively correlated with the percentage of spermatozoa displaying reduced protamination and fragmented DNA using toluidine blue and Halomax, respectively. Bull fertility was also positively correlated with PRM1 content by Western blotting and flow cytometry. However, detection of PRM1 content by Western blotting alone was not predictive of bull fertility. In immunocytochemistry, abnormal spermatozoa showed either a lack of PRM1 or scattered localization in the apical/acrosomal region of the nuclei. The nuclear shape was distorted in spermatozoa from low-fertility bulls. In conclusion, we showed that inadequate amount and localization of PRM1 were associated with defects in sperm chromatin structure, coinciding with reduced fertility in bulls. These findings are highly significant because they reveal molecular and morphological phenotypes of mammalian spermatozoa that influence fertility.

Thymol and eugenol derivatives as potential antileishmanial agents

In Northeastern Brazil visceral leishmaniasis is endemic with lethal cases among humans and dogs. Treatment is toxic and 5-10% of humans die despite treatment. The aim of this work was to survey natural active compounds to find new molecules with high activity and low toxicity against Leishmania infantum chagasi. The compounds thymol and eugenol were chosen to be starting compounds to synthesize acetyl and benzoyl derivatives and to test their antileishmanial activity in vitro and in vivo against L. i. chagasi. A screening assay using luciferase-expressing promastigotes was used to measure the growth inhibition of promastigotes, and an ELISA in situ was performed to evaluate the growth inhibition of amastigote. For the in vivo assay, thymol and eugenol derivatives were given IP to BALB/c mice at 100mg/kg/day for 30 days. The thymol derivatives demonstrated the greater activity than the eugenol derivatives, and benzoyl-thymol was the best inhibitor (8.67 ± 0.28 μg/mL). All compounds demonstrated similar activity against amastigotes, and acetyl-thymol was more active than thymol and the positive control drug amphotericin B. Immunohistochemistry demonstrated the presence of Leishmania amastigote only in the spleen but not the liver of mice treated with acetyl-thymol. Thus, these synthesized derivatives demonstrated anti-leishmanial activity both in vitro and in vivo. These may constitute useful compounds to generate new agents for treatment of leishmaniasis.