Supplying sows energy on the expected day of farrowing improves farrowing kinetics and newborn piglet performance in the first 24 h after birth.

The farrowing process is one of the most energy-demanding activities for the modern hyperprolific sow. This study evaluated the effects of supply of energy on the expected date of farrowing on the farrowing kinetics and piglets' performance during the first 24 h after birth. A total of 80 sows were used. The sows and their respective litters were considered as the experimental unit. On the expected day of farrowing, the sows were allocated to one of the following groups: sows that did not have access to feed from farrowing induction until the end of the farrowing process (CON, n = 40); sows fed 500 g of energetic supplement, which consisted of 250 g of the basal lactation diet plus 250 g of cane sugar, 18 h after farrowing induction (SUP, n = 40). The farrowing duration, farrowing assistance, birth interval, number of total born, stillborn and mummified piglets were recorded for each sow. Piglets were weighed individually at birth and 24 h later. The interval from birth to first suckle was evaluated individually for each piglet in 16 randomly selected litters (eight litters per treatment group). Blood glucose concentrations of six sows were measured shortly after expulsion of the first piglet. Farrowing duration, farrowing assistance and stillborn rate tended to be greater (P = 0.06, P = 0.09 and P = 0.07, respectively) in sows from the CON group compared to sows from the SUP group. However, there was no difference (P > 0.05) between the groups for birth interval. Colostrum intake was greater (P < 0.05) for piglets from the SUP group compared to piglets from the CON group. Additionally, BW gain of the piglets suckling the SUP group was greater (P < 0.05) than those suckling the CON group at 24 h after birth. The blood glucose concentrations during the expulsive stage of farrowing were greater (P < 0.05) in the SUP group than for sows from the CON group. In conclusion, supplying modern hyperprolific sows energy on the expected day of farrowing is a valuable nutritional intervention to improve the farrowing kinetics and piglets' performance in early life.

Effects of glycerol, equilibration time and antioxidants on post-thaw functional integrity of bovine spermatozoa directly obtained from epididymis.

This work aimed to evaluate the effect of stabilisation times, glycerol concentration, and the catalase and superoxide dismutase supplementation of diluent on parameters of frozen-thawed spermatozoa from epididymis of Nelore bulls: Experiment 1: spermatozoa diluted in Tris-egg yolk with glycerol (3%, 5% or 7%) and stabilisation times (0, 2 or 4 hr at 5°C); Experiment 2: Tris-egg yolk only, Tris-egg yolk with catalase (CAT, 50 or 100 U ml-1 ) or superoxide dismutase (SOD, 50 or 100 U ml-1 ). Frozen-thawed spermatozoa were evaluated for kinetic parameters, plasma membrane and acrosome integrity, mitochondrial activity and IVF capacity. ALH and BCF were affected (p < .05) by glycerol at 3% after 4-hr equilibration time and 7% after 2-hr equilibration time. Glycerol 3% had lower (p < .05) iPM and iAc after 4 hr. Glycerol 5% had greater (p < .05) hPMM after 4 hr and iAc after 2 hr than at 0 hr. SOD 100 U ml-1 had lower (p < .05) linearity and wobble compared to control group. No was observed differences to fertilisation rate (p < .05) among groups. In conclusion, glycerol 5% in Tris-egg yolk extender for 4 hr is suitable for the preservation of sperm kinetics and membrane integrity. CAT (50 and 100 U ml-1 ) or SOD (50-100 U ml-1 ) had no beneficial effects on sperm kinetics, plasma and acrosomal membrane integrity, mitochondrial activity or the capacity for IVF of frozen-thawed spermatozoa from epididymis of Nelore bulls.

High resveratrol or quercetin concentrations reduce the oscillation index of frozen goat semen / Altas concentrações de resveratrol ou quercetina reduzem o índice de oscilação do sêmen congelado caprino

The aim of this study was to evaluate the effect of different concentrations of trans-resveratrol or quercetin on the ability of goat sperm to withstand being frozen. Six pools of semen obtained from six male goats were processed with different concentrations of resveratrol or quercetin (Experiment 1: 0, 15, 25, 50, 75 or 100µM resveratrol; Experiment 2: 0, 15, 25, 50, 75 or 100µM quercetin) and frozen. After thawing, the semen was evaluated for sperm kinematics, plasma membrane and acrosome integrity, morphology and oxidative stress following 0 and 1h of incubation. Immediately after thawing (0h), wobble (oscillation index) in the groups treated with 100µM of quercetin or resveratrol was lower (P<0.05) than in those treated with 0 and 25µM resveratrol and 0µM quercetin, respectively. After 1h of incubation, the total motility in treatments with 15, 50 and 75µM quercetin, as well as the plasma membrane integrity in all quercetin concentrations were lower (P<0.05) than at 0h. In opposition, the linearity of semen samples treated with 100µM quercetin and the straightness of those treated with 75 and 100µM quercetin were lower (P<0.05) at 0h than at 1h after thawing. Thus, it can be concluded that resveratrol and quercetin at high concentrations (100µM) transiently reduce the wobble of goat sperm submitted to frozen storage, and that quercetin (75 and 100µM) increases the linearity and straightness over time, which can be favorable for fertility.(AU)

O objetivo deste estudo foi avaliar o efeito de diferentes concentrações de transresveratrol ou quercetina sobre a capacidade dos espermatozoides caprinos de resistirem à congelação. Seis pools de sêmen, obtidos de seis reprodutores caprinos, foram processados com diferentes concentrações de resveratrol ou quercetina (Experimento 1: 0, 15, 25, 50, 75 ou 100µM de resveratrol; Experimento 2: 0, 15, 25, 50, 75 ou 100µM de quercetina) e congelados. Após o descongelamento, o sêmen foi avaliado quanto à cinética espermática, à integridade das membranas plasmática e acrossomal, à morfologia e ao estresse oxidativo nos tempos zero e uma hora de incubação. Imediatamente após a descongelação (zero hora), o wobble (índice de oscilação) nos grupos tratados com 100µM de quercetina ou de resveratrol foi menor (P<0,05) do que nos tratados com 0 e 25µM de resveratrol e com 0µM de quercetina, respectivamente. Após uma hora de incubação, a motilidade total dos tratamentos com 15, 50 e 75µM de quercetina, assim como a integridade de membrana plasmática em todas as concentrações de quercetina, foi menor (P<0,05) do que à zero hora. Em oposição, a linearidade das amostras de sêmen tratadas com 100µM de quercetina e a retilinearidade daquelas tratadas com 75µM e 100µM de quercetina foram menores (P<0,05) à zero hora do que à uma hora após descongelação. Assim, pode-se concluir que o resveratrol e a quercetina, em concentrações elevadas (100µM), reduzem, transitoriamente, o índice de oscilação de espermatozoides caprinos submetidos à congelação e que a quercetina (75 e 100µM) aumenta a linearidade e a retilinearidade ao longo do tempo, o que pode ser favorável à fertilidade.(AU)

Ultrastructure evaluation of goat spermatozoa after freezing in a skim milk-based extender with Trolox supplementation.

The aim of this work was to evaluate the in vitro effect of adding Trolox in freezing extender for goat semen. Ejaculates from five bucks were evaluated, and when approved, the samples were pooled, diluted according to experimental groups [Trolox 0 (control), 30, 60 and 120 nmol ml(-1) ] and frozen in an automated system. Thawed samples (37 °C/30 s) were evaluated for plasma membrane (PMi) and acrosome integrity (Aci), mitochondrial membrane potential (MMP) and sperm kinematics by CASA system. Spermatozoa ultrastructure was evaluated in fresh and post-thawed semen. No significant difference (P > 0.05) was observed among control and Trolox groups in the analyses of PMi, Aci, MMP and CASA in goat spermatozoa after thawing. Samples of 60 and 120 nmol ml(-1) Trolox groups had a higher percentage of cells that had intact plasma membranes in spermatozoa head than in the other groups, although they did not differ (P > 0.05) before being frozen. A higher percentage (P < 0.05) of spermatozoa with intact mitochondria was observed in fresh semen, control and Trolox 60 nmol ml(-1) groups than in the other groups. Addition of Trolox to skim milk extender at 60 nmol ml(-1) ultrastructurally preserves the plasma membrane and mitochondrial sheath integrity in goat spermatozoa after cryopreservation.

Testicular and epididymal ltrasonography in Santa Inês lambs raised in Brazil

The objective of this work was to study, through ultrasonographic evaluation, changes in testes and epididymides of clinically healthy, peripubertal and pubertal Santa Inês lambs raised in Brazil. Periodic e valuations of weight, biometric characteristics (scrotal circumference, width and length) and ultrasound examinatio ns of the testes and epididymides of 20 lambs were performed between 84 and 280 days old at intervals of 28 days. Scans were performed in the sagittal, transverse, frontal and oblique planes to evaluate the echotexture of the testicular parenchyma and mediastinum and the tail epididymis as well as the thickness and width of the mediastinum testis. The testicular parenchyma demonstrated a homogeneous echogenicity patter n ranging from low to moderate. The echogenicity of testicular parenchyma increased in direct proportion to animal age, being higher in pubertal lambs when compared to prepubertal at the same age. The mediastinum testis was observed in 100% of the evaluated animals, regardless of the scan plane used, and was classified as diffuse or moderately or highly echogenic. Echogenicity and the thickness of the mediastinum testis increased in direct proportion to animal age. The epididymal tail was presented in hypoechoic relation to the testicular parenchyma. Based on these results, it was concluded that ultrasound is useful tool for selection and morphophysiological evaluation of Santa Inês lambs on peripubertal and pubertal phases, when used in combination with other methods such as semen evaluation.

Efeito da temperatura de descongelação na integridade de espermatozoides criopreservados de cães / The effect of the thawing temperature on the integrity of canine cryopreserved sperm

Avaliou-se a influência da temperatura de descongelação na integridade de espermatozoides criopreservados de cães. Foram utilizados reprodutores das raças Basset Hound (n=3) e Rottweiler (n=3), submetidos a colheitas de sêmen por manipulação peniana. As amostras de sêmen foram descongeladas a 37ºC/1min (G1) ou 70ºC/6s (G2) e avaliadas quanto à motilidade progressiva, vigor e integridade do acrossoma após 0, 30 e 60 minutos de incubação (37ºC), e ultraestrutura espermática imediatamente após a descongelação. Em todos os tempos de incubação, a motilidade progressiva dos espermatozoides descongelados a 70ºC por 6s (74,6%) foi mais alta (P<0,05) que a dos descongelados a 37ºC por 1min (64,6%). O vigor espermático não diferiu (P>0,05) entre os grupos, e o porcentual de gametas com acrossomas íntegros foi maior (P<0,05) nos espermatozoides do G1 do que no G2. Lesões ultraestruturais foram identificadas nos espermatozoides descongelados de ambos os grupos, em maior quantidade nos gametas do G2. Conclui-se que amostras congeladas de sêmen de cães devam ser descongeladas a 37ºC por 1min.

Aiming to evaluate the influence of the thawing temperature on the viability of canine cryopreserved sperm, Basset Hound (n=3) and Rottweiler (n=3) dogs were used, submitted to semen collected through manual manipulation. Semen samples were thawed at 37ºC during 1min (G1) or at 70ºC during 6s (G2), and evaluated for progressive motility, vigor and acrosome integrity, after 0, 30 e 60 minutes of incubation (37ºC), and sperm ultrastructure immediately after thawing. In all incubation times, the average of progressive motility was higher (P<0.05) in samples from G2 Group (74.6%) than from G1 (64.6%). Sperm vigor had no difference (P>0.05) between groups, and the percentage of gametes with intact acrosome was higher (P<0.05) on sperm cells from G1 than from G2. Ultrastructural changes were identified on dog sperm from both groups, and were observed in higher quantity in gametes from G2 Group. It can be concluded that samples of frozen dog sperm must be thawed at 37°C for 1min.

Efeito da adição de glutationa peroxidase e cisteína ao diluidor de congelação do sêmen equino / Effect of glutathione peroxidase and cysteine addition in an equine frozen semen medium

Foram utilizados ejaculados (n=25) de garanhões para avaliar o efeito de glutationa peroxidase (GPx) e cisteína na viabilidade de espermatozoides congelados. O sêmen foi diluído em Botu Crio, com antioxidantes, e foram formados os grupos: G1, Controle; G2, 1U GPx ; G3, 5U GPx; G4, 0,5mM cisteína; G5, 1mM cisteína. Depois foi envasado em palhetas (0,5mL) e congelado. Após descongelação, 37°C por 30 segundos, alíquotas foram analisadas quanto à integridade de membrana plasmática (IMP) e acrossoma (IAc), potencial de membrana mitocondrial (PMM) e cinética, nos tempos zero (T0) e 60 minutos (T60). GPx 5U e cisteína 0,5mM determinaram maior (P<0,05) IAc em T0 do que em T60. Cisteína 1mM resultou em maior (P<0,05) IAc em T60 do que GPx 1 e 5U e cisteína 0,5mM. O PMM de um garanhão no T60 foi mais alto (P<0,05) do que o de dois garanhões. VCL e VAP foram maiores (P<0,05) no T0 do que no T60 do grupo controle, e um garanhão apresentou, em geral, valores cinéticos mais altos (P<0,05) do que os demais. Conclui-se que a adição de glutationa peroxidase, nas concentrações de 1U e 5U, e de cisteína, nas concentrações de 0,5mM e 1mM, não interferem na integridade de espermatozoides criopreservados de equinos, mas preservam os parâmetros cinéticos de VCL e VAP após 60 minutos de incubação. Ressalta-se, ainda, que o garanhão tem uma forte influência nas características espermáticas pós-congelação.

Ejaculates (n=25) of horses were used to assess the effect of glutathione peroxidase (GPx) and cysteine on the viability of frozen sperm cells. Semen was extended at Botu Crio with antioxidants, and divided in groups: G1, control; G2, 1 U GPx; G3, 5U GPx; G4, 0.5mM cysteine and G5, 1mM cysteine, packed in 0.5mL straws, and frozen. After thawing (37° C for 30 seconds) samples were analyzed for plasma membrane (IMP) and acrosome integrity (IAc), mitochondrial membrane potential (MMP) and kinematic, at zero (T0) and 60 minutes after (T60). GPx 5U and cysteine 0.5mM increased (P<0.05) IAc at T0, when compared to T60. Cysteine 1mM resulted in a higher (P<0.05) IAc on T60, than GPx 1 and 5U, and cysteine 0.5mM. The PMM from a stallion on T60 was higher (P<0.05) than those of two stallions. In sperm kinematic, VCL and VAP were higher (P<0.05) at T0 compared to T60 for the control group, and one stallion showed larger (P<0.05) kinematic values than other animals. It is concluded that the addition of glutathione peroxidase at concentrations 1U and 5U, and cysteine, at concentrations of 0.5mM and 1mM, does not interfere with the integrity of cryopreserved equine sperm, but preserves the kinetic parameters VCL and VAP after 60 minutes of incubation. It should be noted also that the stallion has a strong influence on sperm characteristics post-freezing.

Heme modulates smooth muscle cell proliferation and migration via NADPH oxidase: a counter-regulatory role for heme oxygenase system.

Accumulation of vascular smooth muscle cells (VSMC) in response to inflammatory stimuli is a key event in atherogenesis, which commonly occurs in sinuous vessels with turbulent blood flow what leads to hemolysis and consequent free heme accumulation, a known pro-oxidant and pro-inflammatory molecule. In this work, we investigated the effects of free heme on VSMC, and the molecular mechanisms underlying this process. Free heme induces a concentration-dependent migration and proliferation of VSMC which depends on the production of reactive oxygen species (ROS) derived from NADPH oxidase (NADPHox) activity. Additionally, heme activates redox-sensitive proliferation-related signaling routes, such as mitogen activated protein kinase (MAPK) and NF-κB, and induces heme oxygenase-1 (HO-1) expression. NADPHox-dependent proliferative effect of heme seems to be endogenously modulated by HO since the pretreatment of VSMC with HO inhibitors potentiates heme-induced proliferation and, in parallel, increases ROS production. These effects were no longer observed in the presence of heme metabolites, carbon monoxide and biliverdin. The data indicate that VSMC proliferation induced by heme is endogenously modulated by a critical counter-regulatory crosstalk between NADPHox and HO systems.

In vitro evaluation of ram sperm frozen with glycerol, ethylene glycol or acetamide.

The aim was to assess the in vitro effect of glycerol, ethylene glycol or acetamide on frozen-thawed ram spermatozoa. Aliquots of each sixteen ejaculates from four rams of the Morada Nova breed were diluted in Tris-egg yolk with glycerol (5%), ethylene glycol (3% or 5%) or acetamide (3% or 5%) and frozen at -196°C. After thawing, progressive sperm motility was greater (P<0.05) in cryopreservation with glycerol 5% and ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Acrosome integrity was greater (P<0.05) with ethylene glycol 5% than acetamide (3% or 5%). The percentage of sperm without oxidative stress was greater (P<0.05) with ethylene glycol (3% or 5%) than with acetamide (3% or 5%). Plasma membrane integrity was greater with glycerol 5% (P<0.05) than with the other cryoprotectants. Thus, it is concluded that glycerol 5% and ethylene glycol 3% or 5% protect ram sperm against the harmful effects of freezing and that glycerol 5% offers greater protection to sperm plasma membrane.

Effect of antioxidants resveratrol and quercetin on in vitro evaluation of frozen ram sperm.

The objective was to assess the effects of the antioxidants resveratrol and quercetin on frozen-thawed ram sperm. Semen samples (which exceeded minimum standards) from four mature crossbreed Santa Inês rams were pooled and aliquots of each pool were diluted in Tris-egg yolk-glycerol, with the addition of 0, 5, 10, 15, and 20 µg/mL of resveratrol and quercetin in Experiment 1 and Experiment 2, respectively. In Experiment 1, the proportion of sperm with a high mitochondrial membrane potential was greater (P < 0.02) in the control group than in resveratrol 20 µg/mL group. In Experiment 2, the proportion of sperm with high mitochondrial membrane potential was greater in the control group (P < 0.0001) than in the other experimental groups, and greater in the quercetin 5 µg/mL group (P < 0.05) than in the other quercetin-treated groups. Thus, addition of 5 to 20 µg/mL of either resveratrol or quercetin to the Tris-egg yolk-glycerol extender reduced sperm mitochondrial membrane potential.

Interferência da condição climática na integridade de espermatozoides ovinos submetidos à criopreservação / Climate condition interference on ram spermatozoa integrity submitted to cryopreservation

Avaliou-se a integridade de espermatozoides ovinos colhidos e criopreservados em diferentes situações climáticas na região semiárida do estado da Paraíba. As colheitas de sêmen foram realizadas em duas épocas do ano, período seco e período chuvoso, utilizando cinco machos adultos da raça Santa Inês, com histórico favorável de fertilidade. Os ejaculados foram avaliados quanto à motilidade, vigor, concentração e morfologia espermática, após a formação do pool e diluição em Tris-gema, na concentração final de 240x10(6) espermatozoides/mL. Motilidade total e vigor espermático não diferiram entre as épocas seca e chuvosa. Valores de integridade do acrossoma e da membrana plasmática, e o potencial de membrana mitocondrial foram mais baixos (P<0,05) na época com menor índice pluviométrico. Conclui-se que a reprodução de ovinos criados na região do semiárido paraibano sofre ação da condição climática e sugere-se que a criopreservação de espermatozoides ovinos seja realizada no período de maior índice pluviométrico na região.

Ram spermatozoa integrity collected and cryopreserved in different climatic situations in the semiarid region of Paraíba state was evaluated. Semen samples were collected in two periods: a dry and a rainy season, using five mature Santa Inês males, with favorable historical fertility. Ejaculates were evaluated for motility, vigor, concentration and morphology, after a pool formation and dilution in Tris-yolk egg, at 240x10(6)/mL final concentration. Motility and sperm vigor did not differ between dry and rainy seasons. Acrosome integrity, plasma membrane and mitochondrial membrane potential were lower (P>0.05) at the time with less rainfall. We concluded that sheep reproduction raised in semi-arid Paraiba region suffers climatic condition influence and the lowest rainfall period is deleterious to the sperm cells integrity subjected to cryopreservation process. It is suggested that ram spermatozoa cryopreservation be done in period of greatest rainfall in this region.

Effects of antioxidants and duration of pre-freezing equilibration on frozen-thawed ram semen.

The objective was to evaluate the effects of various antioxidants and duration of pre-freezing equilibration on cryopreservation of ram semen. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control), or supplemented with reduced glutathione (GSH: 0.5, 1.0, and 2.0 mM), superoxide dismutase (SOD: 5, 10, and 20 U/mL), or catalase (CAT: 5, 10, and 20 U/mL), and cryopreserved, immediately after thermal equilibrium was reached at 5 °C (0 h), or 12 or 24 h after equilibration. Total antioxidant capacity was determined in the in natura extenders and after addition of semen samples for various durations of processing (fresh/dilute, throughout refrigeration, and post-thaw). Plasma membrane (PI-CFDA), acrosome integrity (FITC-PNA), and mitochondrial membrane potential (JC-1) were determined in fresh/diluted, refrigerated, and post-thaw samples. Post-thaw sperm motility was assessed with a computerized analysis system (CASA). There were no significant differences in acrosome damage or mitochondrial membrane potential after refrigeration and freeze-thaw, regardless of antioxidant addition. Sperm plasma membrane integrity was worse (P < 0.05) with cryopreservation immediately after equilibration (average 20.1 ± 8.3; mean ± SD) than after 12 h of equilibration (average 42.5 ± 10.9); however, the addition of SOD and CAT (10 and 20 U/mL) resulted in no significant difference between post-equilibration intervals of 0 and 12 h. Total antioxidant activity was not different (P > 0.05) among treatments after sperm addition or throughout the refrigeration and post-thaw. In conclusion, adding GSH, SOD or CAT did not increase the total antioxidant capacity of semen, nor did it enhance the quality of the post-thaw sperm. However, maintenance of ram semen at 5 °C for 12 h prior to cryopreservation reduced membrane damage of frozen-thawed sperm.

In vitro and in vivo evaluation of ram sperm frozen in tris egg-yolk and supplemented with superoxide dismutase and reduced glutathione.

The aim of the present study was to evaluate the in vitro and in vivo effect of the addition of superoxide dismutase (SOD) and reduced glutathione (GSH) to ram semen freezing extender. Significant differences (p < 0.05) were detected between groups regarding total motility (TM), straightness (STR) and wobble (WOB), for which the GSH 7 mM group had lesser TM and better STR than the other groups and the GSH 5 and 7 mM groups had higher wobble values than the control, SOD 25 and 100 U/ml groups. The ultrastructural analysis revealed that the acrosome was better preserved after freezing in the SOD 100 U/ml and GSH 2 and 5 mM (p < 0.05) groups than the other groups, whereas mitochondria in both the control group and the 7 mM GSH group suffered the greatest damage. The plasma membrane remained preserved after freezing, regardless of the group. For in vivo fertilization, the SOD group achieved better results than the GSH group (p > 0.05). It can therefore be concluded that the addition of SOD 100 U/ml and GSH 2 and 5 mM preserves the acrosome integrity of frozen ram spermatozoa, while the addition of SOD 100 U/ml to Tris egg-yolk extender offers protection to the membranes of sperm cells after thawing.

Localization of HSP single-copy genes by inexpensive, permanent non-fluorescent in situ hybridization on meiotic chromosomes of the grasshopper Schistocerca pallens (Acrididae).

There have been many studies on Schistocerca gregaria and Locusta migratoria, which are important grasshopper pests in many parts of the world. However, the main pest grasshopper species in Brazil, S. pallens, Rhammatocerus schistocercoides and Stiphra robusta, are very poorly characterized genetically. We adapted a permanent in situ hybridization method to extend the genetic characterization of S. pallens by mapping the single-copy genes Hsp70, Hsp83, Hsp27, and Ubi on meiotic chromosomes. Hsp70 was mapped on the L2 chromosome, in which 82% of the signals were observed. Hsp83 was mapped on a medium-sized chromosome, on which 81% of the signals were observed, tentatively identified as M7. The hybridization signals for the Hsp27 gene were detected on the L1 chromosome at a frequency of 58%. The main hybridization site of the Ubi probe was on the L2 chromosome, with 73% of the signals. All mapped genes also presented secondary hybridization signals, always at frequencies below 30%. These are the first single-copy genes mapped for S. pallens and also for the Acrididae family. Since the Acrididae generally present very similar karyotypes, these data are useful as new landmarks for chromosome identification and as a tool for phylogenetic studies on the genus Schistocerca and for comparison with other insects.

Localization of HSP single-copy genes by inexpensive, permanent non-fluorescent in situ hybridization on meiotic chromosomes of the grasshopper Schistocerca pallens (Acrididae)

There have been many studies on Schistocerca gregaria and Locusta migratoria, which are important grasshopper pests in many parts of the world. However, the main pest grasshopper species in Brazil, S. pallens, Rhammatocerus schistocercoides and Stiphra robusta, are very poorly characterized genetically. We adapted a permanent in situ hybridization method to extend the genetic characterization of S. pallens by mapping the single-copy genes Hsp70, Hsp83, Hsp27, and Ubi on meiotic chromosomes. Hsp70 was mapped on the L2 chromosome, in which 82% of the signals were observed. Hsp83 was mapped on a medium-sized chromosome, on which 81% of the signals were observed, tentatively identified as M7. The hybridization signals for the Hsp27 gene were detected on the L1 chromosome at a frequency of 58%. The main hybridization site of the Ubi probe was on the L2 chromosome, with 73% of the signals. All mapped genes also presented secondary hybridization signals, always at frequencies below 30%. These are the first single-copy genes mapped for S. pallens and also for the Acrididae family. Since the Acrididae generally present very similar karyotypes, these data are useful as new landmarks for chromosome identification and as a tool for phylogenetic studies on the genus Schistocerca and for comparison with other insects.

Bioactive peptides in ovine and caprine cheeselike systems prepared with proteases from Cynara cardunculus.

The potential angiotensin-converting enzyme (ACE)-inhibitory and antioxidant activities of peptides in water-soluble extracts, obtained from raw and sterilized ovine and caprine cheeselike systems coagulated with enzymes from the plant Cynara cardunculus, were assessed. Prior to the assay, the 3,000-Da permeate from 45-d-old cheeselike systems was fractionated by tandem chromatographic techniques. Several peaks were obtained in each chromatogram, but only some were associated with ACE-inhibitory or antioxidant activity or both. Peptides Tyr-Gln-Glu-Pro, Val-Pro-Lys-Val-Lys, and Tyr-Gln-Glu-Pro-Val-Leu-Gly-Pro-* from beta-casein, as well as Arg-Pro-Lys and Arg-Pro-Lys-His-Pro-Ile-Lys-His-* from alpha(s1)-casein exhibited ACE-inhibitory activity. Peptides released upon cleavage of the peptide bond Leu190-Tyr191 (either in ovine or caprine beta-casein), and corresponding to the beta-casein sequence Tyr-Gln-Glu-Pro-*, possessed antioxidant activity.

Partial identification of water-soluble peptides released at early stages of proteolysis in sterilized ovine cheese-like systems: influence of type of coagulant and starter.

Cheese-like systems were manufactured from sterilized ovine milk, using crude aqueous extracts of Cynara cardunculus or cardosin A isolated therefrom as clotting agent. The effect of adding a commercial starter culture was also assessed. The impact of the type of coagulant used during the initial 24 h of proteolysis was evaluated via separation of peptides in the water-soluble extracts by reverse-phase HPLC, followed by partial sequencing via Edman degradation. Cardosin A accounted for most events of primary proteolysis. The major cleavage sites were Phe105-Met106 in kappa-casein, and Leu127-Thr128, Ser142-Trp143, Leu165-Ser166, and Leu190-Tyr191 in beta-casein. The starter culture did not play an active role during the initial stages of ripening.

Impairment of inflammatory response in adult rats submitted to maternal undernutrition during early lactation: role of insulin and glucocorticoid.

OBJECTIVE: Early nutritional environment may program permanent metabolic alterations, predisposing to later diseases. We have investigated the interference of maternal malnutrition during lactation with the development of acute inflammation in adult rats. MATERIALS AND METHODS: Adult rats, offspring of dams fed with either protein-free diet (UN group) or 22% protein diet (C group) during the first 10 days of lactation, were submitted to pleurisy with carrageenan (500 microg/cavity). Pleural edema, neutrophil migration and ICAM expression, were evaluated 4 h after and correlated with alterations in plasma insulin and corticosterone. Leukocyte-endothelium interactions were evaluated by intravital microscopy 1 h after inflammation. RESULTS: Compared to controls, UN rats showed a decrease in pleural edema formation (50%), neutrophil migration (50%), endothelial ICAM-1 expression on pulmonary tissue, and impairment in leukocyte adhesion (50%) and migration (80%) through endothelium. Circulating insulin was lower (42%) and corticosterone was higher (34%) in UN, compared to controls. Pre-treatment of UN with insulin (5 IU/day) or RU486 (20 mg/kg/day), inhibitor of glucocorticoid receptor, restored leukocyte functions and ICAM-1 expression. CONCLUSION: Postnatal maternal malnutrition, programming for permanent alterations in insulin and glucocorticoid secretion in progeny, that were unable to properly mount an inflammatory response, probably predisposes to chronic diseases in adult life.

Secondary triatomine species in dwellings and other nearby structures in municipalities under epidemiological surveillance in the state of Paraná, Brazil.

OBJECTIVE: Since data are scarce regarding secondary triatomine species in the Brazilian state of Paraná, this study investigated infestations in inhabited and abandoned houses and in various other nearby structures in rural areas of that state. METHODS: Triatomines were manually captured in inhabited and uninhabited houses and other nearby structures in nine areas (eight municipalities and one district) of Paraná from June 1996 to February 2000. Testing for Trypanosoma cruzi infection was performed, as were also precipitin tests to determine the triatomines' food sources. RESULTS: While Triatoma infestans was not found in any of the nine areas of Paraná that were studied, three secondary triatomine species were detected: Triatoma sordida, Panstrongylus megistus, and Rhodnius neglectus. T. sordida was the most common species found, comprising 575 of the 658 triatomines captured (87.4%). The second-most common was P. megistus, with 82 specimens (12.5%). Of the various categories of structures investigated, uninhabited houses was the most frequently infested category (19/62, or 30.6%), followed by chicken coops (24/350, or 6.9%). The primary food source of the triatomines was the blood of birds. Nevertheless, in the municipality with the highest density of triatomines, the food sources included domestic animals and even humans. We found that 13.4% of the T. sordida and 13.5% of the P. megistus were infected with Trypanosoma cruzi. CONCLUSIONS: These results demonstrate the need to maintain entomological surveillance measures in the studied areas. This is especially important since Brazil and other countries of Latin America have affirmed the need to interrupt the vector-borne transmission of Chagas' disease.