A promiscuous T cell epitope-based HIV vaccine providing redundant population coverage of the HLA class II elicits broad, polyfunctional T cell responses in nonhuman primates

Over the last few decades, several emerging or reemerging viral diseases with no readily available vaccines have ravaged the world. A platform to fastly generate vaccines inducing potent and durable neutralizing antibody and T cell responses is sorely needed. Bioinformatically identified epitope-based vaccines can focus on immunodominant T cell epitopes and induce more potent immune responses than a whole antigen vaccine and may be deployed more rapidly and less costly than whole-gene vaccines. Increasing evidence has shown the importance of the CD4+ T cell response in protection against HIV and other viral infections. The previously described DNA vaccine HIVBr18 encodes 18 conserved, promiscuous epitopes binding to multiple HLA-DR-binding HIV epitopes amply recognized by HIV-1-infected patients. HIVBr18 elicited broad, polyfunctional, and durable CD4+ and CD8+ T cell responses in BALB/c and mice transgenic to HLA class II alleles, showing cross-species promiscuity. To fully delineate the promiscuity of the HLA class II vaccine epitopes, we assessed their binding to 34 human class II (HLA-DR, DQ, and -DP) molecules, and immunized nonhuman primates. Results ascertained redundant 100% coverage of the human population for multiple peptides. We then immunized Rhesus macaques with HIVBr18 under in vivo electroporation. The immunization induced strong, predominantly polyfunctional CD4+ T cell responses in all animals to 13 out of the 18 epitopes; T cells from each animal recognized 7–11 epitopes. Our results provide a preliminary proof of concept that immunization with a vaccine encoding epitopes with high and redundant coverage of the human population can elicit potent T cell responses to multiple epitopes, across species and MHC barriers. This approach may facilitate the rapid deployment of immunogens eliciting cellular immunity against emerging infectious diseases, such as COVID-19.

Rhesus macaques self-curing from a schistosome infection can display complete immunity to challenge.

The rhesus macaque provides a unique model of acquired immunity against schistosomes, which afflict >200 million people worldwide. By monitoring bloodstream levels of parasite-gut-derived antigen, we show that from week 10 onwards an established infection with Schistosoma mansoni is cleared in an exponential manner, eliciting resistance to reinfection. Secondary challenge at week 42 demonstrates that protection is strong in all animals and complete in some. Antibody profiles suggest that antigens mediating protection are the released products of developing schistosomula. In culture they are killed by addition of rhesus plasma, collected from week 8 post-infection onwards, and even more efficiently with post-challenge plasma. Furthermore, cultured schistosomula lose chromatin activating marks at the transcription start site of genes related to worm development and show decreased expression of genes related to lysosomes and lytic vacuoles involved with autophagy. Overall, our results indicate that enhanced antibody responses against the challenge migrating larvae mediate the naturally acquired protective immunity and will inform the route to an effective vaccine.

Rhesus macaques self-curing from a schistosome infection can display complete immunity to challenge

The rhesus macaque provides a unique model of acquired immunity against schistosomes, which afflict >200 million people worldwide. By monitoring bloodstream levels of parasite-gut-derived antigen, we show that from week 10 onwards an established infection with Schistosoma mansoni is cleared in an exponential manner, eliciting resistance to reinfection. Secondary challenge at week 42 demonstrates that protection is strong in all animals and complete in some. Antibody profiles suggest that antigens mediating protection are the released products of developing schistosomula. In culture they are killed by addition of rhesus plasma, collected from week 8 post-infection onwards, and even more efficiently with post-challenge plasma. Furthermore, cultured schistosomula lose chromatin activating marks at the transcription start site of genes related to worm development and show decreased expression of genes related to lysosomes and lytic vacuoles involved with autophagy. Overall, our results indicate that enhanced antibody responses against the challenge migrating larvae mediate the naturally acquired protective immunity and will inform the route to an effective vaccine.

Involvement of von Willebrand factor and botrocetin in the thrombocytopenia induced by Bothrops jararaca snake venom

Patients bitten by snakes consistently manifest a bleeding tendency, in which thrombocytopenia, consumption coagulopathy, mucous bleeding, and, more rarely, thrombotic microangiopathy, are observed. Von Willebrand factor (VWF) is required for primary hemostasis, and some venom proteins, such as botrocetin (a C-type lectin-like protein) and snake venom metalloproteinases (SVMP), disturb the normal interaction between platelets and VWF, possibly contributing to snakebite-induced bleedings. To understand the relationship among plasma VWF, platelets, botrocetin and SVMP from Bothrops jararaca snake venom (BjV) in the development of thrombocytopenia, we used (a) Wistar rats injected s.c. with BjV preincubated with anti-botrocetin antibodies (ABA) and/or Na2-EDTA (a SVMP inhibitor), and (b) VWF knockout mice (Vwf-/-) injected with BjV. Under all conditions, BjV induced a rapid and intense thrombocytopenia. In rats, BjV alone reduced the levels of VWF:Ag, VWF:CB, high molecular weight multimers of VWF, ADAMTS13 activity, and factor VIII. Moreover, VWF:Ag levels in rats that received BjV preincubated with Na2-EDTA and/or ABA tended to recover faster. In mice, BjV caused thrombocytopenia in both Vwf-/- and C57BL/6 (background control) strains, and VWF:Ag levels tended to decrease in C57BL/6, demonstrating that thrombocytopenia was independent of the presence of plasma VWF. These findings showed that botrocetin present in BjV failed to affect the extent or the time course of thrombocytopenia induced by envenomation, but it contributed to decrease the levels and function of plasma VWF. Thus, VWF alterations during B. jararaca envenomation are an ancillary event, and not the main mechanism leading to decreased platelet counts.

Early exposure to environmental enrichment protects male rats against neuropathic pain development after nerve injury.

Because environmental elements modify chronic pain development and endogenous mechanisms of pain control are still a great therapeutic source, we investigated the effects of an early exposure to environmental enrichment (EE) in a translational model of neuropathic pain. Young male rats born and bred in an enriched environment, which did not count on running wheel, underwent chronic constriction injury (CCI) of sciatic nerve. EE abolished neuropathic pain behavior 14 days after CCI. Opioid receptors' antagonism reversed EE-analgesic effect. ß-endorphin and met-enkephalin serum levels were increased only in EE-CCI group. Blockade of glucocorticoid receptors did not alter EE-analgesic effect, although corticosterone circulating levels were increased in EE animals. In the spinal cord, EE controlled CCI-induced serotonin increase. In DRG, EE blunted the expression of ATF-3 after CCI. Surprisingly, EE-CCI group showed a remarkable preservation of sciatic nerve fibers compared to NE-CCI group. This work demonstrated global effects induced by an EE protocol that explain, in part, the protective role of EE upon chronic noxious stimulation, reinforcing the importance of endogenous mechanisms in the prevention of chronic pain development.

Early exposure to environmental enrichment protects male rats against neuropathic pain development after nerve injury

Because environmental elements modify chronic pain development and endogenous mechanisms of pain control are still a great therapeutic source, we investigated the effects of an early exposure to environmental enrichment (EE) in a translational model of neuropathic pain. Young male rats born and bred in an enriched environment, which did not count on running wheel, underwent chronic constriction injury (CCI) of sciatic nerve. EE abolished neuropathic pain behavior 14?days after CCI. Opioid receptors' antagonism reversed EE-analgesic effect. ß-endorphin and met-enkephalin serum levels were increased only in EE-CCI group. Blockade of glucocorticoid receptors did not alter EE-analgesic effect, although corticosterone circulating levels were increased in EE animals. In the spinal cord, EE controlled CCI-induced serotonin increase. In DRG, EE blunted the expression of ATF-3 after CCI. Surprisingly, EE-CCI group showed a remarkable preservation of sciatic nerve fibers compared to NE-CCI group. This work demonstrated global effects induced by an EE protocol that explain, in part, the protective role of EE upon chronic noxious stimulation, reinforcing the importance of endogenous mechanisms in the prevention of chronic pain development.

Early exposure to environmental enrichment protects male rats against neuropathic pain development after nerve injury

Because environmental elements modify chronic pain development and endogenous mechanisms of pain control are still a great therapeutic source, we investigated the effects of an early exposure to environmental enrichment (EE) in a translational model of neuropathic pain. Young male rats born and bred in an enriched environment, which did not count on running wheel, underwent chronic constriction injury (CCI) of sciatic nerve. EE abolished neuropathic pain behavior 14?days after CCI. Opioid receptors' antagonism reversed EE-analgesic effect. ß-endorphin and met-enkephalin serum levels were increased only in EE-CCI group. Blockade of glucocorticoid receptors did not alter EE-analgesic effect, although corticosterone circulating levels were increased in EE animals. In the spinal cord, EE controlled CCI-induced serotonin increase. In DRG, EE blunted the expression of ATF-3 after CCI. Surprisingly, EE-CCI group showed a remarkable preservation of sciatic nerve fibers compared to NE-CCI group. This work demonstrated global effects induced by an EE protocol that explain, in part, the protective role of EE upon chronic noxious stimulation, reinforcing the importance of endogenous mechanisms in the prevention of chronic pain development.

Comparative study of platelet aggregation and secretion induced by Bothrops jararaca snake venom and thrombin.

Victims of Bothrops jararaca snakebites manifest bleedings, blood incoagulability, platelet dysfunction, and thrombocytopenia, and the latter has been directly implicated in the genesis of hemorrhagic diathesis. We addressed herein the direct effects of B. jararaca venom (BjV) on ex vivo platelet aggregation and granule secretion in washed human and mouse platelets. BjV directly aggregated platelets, but the extent of platelet aggregation was lower in human than mouse platelets. On the other hand, BjV (24.4 µg/mL) and thrombin (0.1 U/mL) induced a similar extent of ATP and platelet factor 4 (PF4) secretion in both species. BjV-induced platelet aggregation was independent of the platelet dense body content, as in pearl mouse (Ap3b1-/-) platelets, whose dense bodies are deficient in adenine nucleotides and serotonin, the extent of platelet aggregation was superior to that induced in BALB/c or C57BL/6 mice. BjV-induced ß-hexosaminidase secretion in human platelets was less intense than that evoked by thrombin, and the contrary was observed in mouse platelets. Irreversible inactivation of platelet cyclooxygenase 1 by acetylsalicylic acid did not reduce BjV-induced platelet aggregation. BjV exerted no cytotoxic activity in human and mouse platelets, as evaluated by lactate dehydrogenase loss. Eptifibatide, which inhibits the binding of fibrinogen to platelet glycoprotein complex GPIIb-IIIa, differently blocked BjV-induced platelet aggregation in mice and humans. BjV-induced platelet aggregation did not depend on snake venom serine proteinases nor metalloproteinases in mice, whilst serine proteinases were rather important for platelet aggregation in humans. Our results show that BjV induces direct activation, aggregation, and secretion in human and mouse platelets, but it exerts diverse responses in them, which should be considered in comparative studies to understand pathophysiological events during Bothrops envenomation.

Distinct environmental enrichment protocols reduce anxiety but differentially modulate pain sensitivity in rats.

Environmental enrichment (EE) can alter anxiety as well as perception of nociceptive stimuli, suggesting a relationship between well-being and analgesia. Considering that, we aimed to evaluate the influence of different EE types on anxiety and peripheral pain sensitivity of male Wistar rats. Animals were submitted to two different types of EE: On the first one, animals were housed in standard size cages after weaning and received three different objects, one type per week, on a regular basis (simple EE - sEE). On the second one, animals were born in an already enriched environment and, after five weeks, were housed in larger cages and received five different objects, three of each type per week, on a regular basis (improved EE - iEE). Control group remained in standard cages and did not receive objects. Within 7 weeks of life, anxiety and thermal sensibility were evaluated using elevated plus maze and tail flick tests, respectively. Mechanical hyperalgesia was analyzed in the presence of acute and chronic noxious stimuli by paw pressure test. Both EE protocols tested were effective in diminish anxiety but they did not alter thermal sensibility. On the other hand, sEE protocol did not alter acute and chronic induced mechanical hypersensitivity, whereas iEE completely abolished such pain behavior, even without exercise wheel as part of the enrichment. Our results show that specific parameters (anxiety and pain sensitivity) can be differentially modulated depending on EE protocol used, making possible the implementation of welfare to experimental animals in pain research.

Imunização de base com antígeno do gênero bothrops em equinos soroprodutores visando uma resposta imunológica

Os equinos são animais de grande porte, com isso apresenta grande volume sanguíneo, sendo assim sempre foi o animal de escolha para realização dos procedimentos de hiperimunização, além de se tratar de um animal de fácil manejo e dócil. Os acidentes ofídicos podem ocorrer em qualquer espécie animal, inclusive nos seres humanos. O gênero de serpentes no Brasil que mais ocasionam esses acidentes são conhecidas como Bothrops. O cavalo é utilizado para ser imunizado com o veneno da serpente, com o objetivo de criar anticorpos específicos contra esse veneno. O presente trabalho teve como intuito realizar a mensuração de anticorpos em nove equinos, que foram submetidos à imunização de base com o antígeno do gênero Bothrops. Os adjuvantes utilizados para o estudo foram Emulsão múltipla incompleta (EMI) e o Tampão fosfato salina. Os animais foram sujeitos a três imunizações e em seguida foram efetuadas seis sangrias exploradoras após isso foi mensurado a titulação de cada animal, para saber qual individuo obteve uma titulação adequada e se está apto ou não para entrar no serviço definitivo de Botrópico.

Avaliação da transferência passiva de imunoglobulinas específicas do gênero Bothrops de éguas hiperimunizadas para o neonato

A placenta da égua epiteliocorial, impede a passagem de macromoléculas como as imunoglobulinas IgG’s para o feto, fazendo com que o recém-nascido venha ao mundo extremamente susceptível aos microrganismos exógenos, dependendo exclusivamente do colostro, rico em imunoglobulinas da mãe, que lhe dará proteção passivamente durante as primeiras horas de vida. Esse trabalho objetivou a avaliação da transferência de imunoglobulinas especificas do gênero Bothrops de éguas hiperimunizadas durante o sexto mês de gestação para o neonato, mediante a utilização do teste de ELISA (enzyme-linked immuno sorbent), verificando a correlação da concentração de imunoglobulina específica do soro materno para o neonato. Para isso foram utilizadas cinco éguas e cinco neonatos. A primeira amostra de sangue foi coletada das éguas sete dias antes de parirem. Os partos foram acompanhados e as amostras dos neonatos seguiram os seguintes tempos: T0 antes da ingestão do colostro, T1 após 24 horas da ingestão do colostro, T2 48 horas após a ingestão do colostro e T3 30 dias após o nascimento. Foram processadas pela Seção de Processamento de Plasma Hiperimune-Instituto Butantan. Observou-se que três éguas obtiveram a transferência de imunoglobulinas específicas para o neonato, mantendo o nível de imunoglobulinas constante em 12h, 48h e 30 dias após nascimento. Uma égua não obteve o valor necessário para ser considerada hiperimunizada, por isso não houve transferência para o neonato. E outra égua obteve uma titulação considerada para a hiperimunização porém, o seu neonato não teve valor de imunoglobulinas específicas encontradas no soro em todos os momentos.

Parental behavior and anxiety in isogenic and outbred mice given access to two types of nesting materials

The objective was to investigate if providing two types of nesting materials could modulate parental behavior and anxiety in laboratory mice. For that, 54 full-sib BALB/cJ and 50 randomly mated Swiss Webster mouse pairs were employed in a completely randomized designwith a 2×2 factorial arrangement (two genetic groups and with/ without access to nesting materials). Eight pieces of disposable polypropylene caps/ hairnets and 3g of cotton were provided as nesting materials to half the cages from each genetic group. Maternal and paternal behaviors were recorded on the third reproductive cycle, twice a week, for three weeks, using scan sampling. The behaviors were recorded every 10s for a period of 10min (totaling 60 records) each day. Dams (N=40) were tested in the elevated plus maze (EPM) on the fifth reproductive cycle, 7–10 days postpartum. Their 21-days-old weanlings (N=208) were tested in the same apparatus. Total number of closed arm entries was used as measurement of motor activity, the percentage of time spent on open arms as measurement of anxiety and head dipping time as measurement of exploratory behavior. The provision of nesting materials increased the frequency of dams licking pups (1.72±0.20 vs. 1.10±0.21, P=0.0342) and of sires resting in contact with pups (25.0±1.5 vs. 18.9±1.5, P=0.0050), while simultaneously decreased the frequency of sires in non-contact rest with pups (6.4±1.1 vs. 10.8±1.2, P=0.0074). Swiss Webster dams were recorded nursing their pups in the arched-back posture more frequently than BALB/cJ dams (9.63±0.89 vs. 7.13±0.86, P=0.0187) and Swiss Webster sires showed a higher frequency of nest building than BALB/cJ sires (0.80±0.16 vs. 0.31±0.15, P=0.0281). Motor activity was higher (8.00±0.43 vs. 2.83±0.41, P<0.0001) and anxiety was lower (3.2±28.4 vs. 2.4±3.1%, P<0.0001) in Swiss Webster than in BALB/cJ dams. Anxiety (26.6±3.2 vs. 37.9±3.5%, P=0.0168) was also lower in Swiss Webster than in BALB/cJ weanlings. The availability of nesting materials increased the time dams spent in head dipping (10.9±2.0 vs. 4.6±1.9, P=0.0087), but weanling behaviors in the EPM were unaffected. Slight differences in parental behavior and contrasting patterns of anxiety and motor activity were found between genetic groups. The provision of nesting materials promoted an intensification of favorable parent-offspring interactions and enhanced exploratory behavior of dams.

Distinct environmental enrichment protocols reduce anxiety but differentially modulate pain sensitivity in rats

Environmental enrichment (EE) can alter anxiety as well as perception of nociceptive stimuli, suggesting a relationship between well-being and analgesia. Considering that, we aimed to evaluate the influence of different EE types on anxiety and peripheral pain sensitivity of male Wistar rats. Animals were submitted to two different types of EE: On the first one, animals were housed in standard size cages after weaning and received three different objects, one type per week, on a regular basis (simple EE – sEE). On the second one, animals were born in an already enriched environment and, after five weeks, were housed in larger cages and received five different objects, three of each type per week, on a regular basis (improved EE – iEE). Control group remained in standard cages and did not receive objects. Within 7 weeks of life, anxiety and thermal sensibility were evaluated using elevated plus maze and tail flick tests, respectively. Mechanical hyperalgesia was analyzed in the presence of acute and chronic noxious stimuli by paw pressure test. Both EE protocols tested were effective in diminish anxiety but they did not alter thermal sensibility. On the other hand, sEE protocol did not alter acute and chronic induced mechanical hypersensitivity, whereas iEE completely abolished such pain behavior, even without exercise wheel as part of the enrichment. Our results show that specific parameters (anxiety and pain sensitivity) can be differentially modulated depending on EE protocol used, making possible the implementation of welfare to experimental animals in pain research.

Comparative study of platelet aggregation and secretion induced by Bothrops jararaca snake venom and thrombin

Victims of Bothrops jararaca snakebites manifest bleedings, blood incoagulability, platelet dysfunction, and thrombocytopenia, and the latter has been directly implicated in the genesis of hemorrhagic diathesis. We addressed herein the direct effects of B. jararaca venom (BjV) on ex vivo platelet aggregation and granule secretion in washed human and mouse platelets. BjV directly aggregated platelets, but the extent of platelet aggregation was lower in human than mouse platelets. On the other hand, BjV (24.4 mu g/mL) and thrombin (0.1 U/mL) induced a similar extent of ATP and platelet factor 4 (PF4) secretion in both species. BjV-induced platelet aggregation was independent of the platelet dense body content, as in pearl mouse (Ap3b1(-/-))platelets, whose dense bodies are deficient in adenine nucleotides and serotonin, the extent of platelet aggregation was superior to that induced in BALB/c or C57BL/6 mice. BjV-induced beta-hexosaminidase secretion in human platelets was less intense than that evoked by thrombin, and the contrary was observed in mouse platelets. Irreversible inactivation of platelet cyclooxygenase 1 by acetylsalicylic acid did not reduce BjV-induced platelet aggregation. BjV exerted no cytotoxic activity in human and mouse platelets, as evaluated by lactate dehydrogenase loss. Eptifibatide, which inhibits the binding of fibrinogen to platelet glycoprotein complex GPIIb-IIIa, differently blocked BjV-induced platelet aggregation in mice and humans. BjV-induced platelet aggregation did not depend on snake venom serine proteinases nor metalloproteinases in mice, whilst serine proteinases were rather important for platelet aggregation in humans. Our results show that BjV induces direct activation, aggregation, and secretion in human and mouse platelets, but it exerts diverse responses in them, which should be considered in comparative studies to understand pathophysiological events during Bothrops envenomation.

Parental behavior and anxiety in isogenic and outbred mice given access to two types of nesting materials

The objective was to investigate if providing two types of nesting materials could modulate parental behavior and anxiety in laboratory mice. For that, 54 full-sib BALB/cJ and 50 randomly mated Swiss Webster mouse pairs were employed in a completely randomized designwith a 2×2 factorial arrangement (two genetic groups and with/ without access to nesting materials). Eight pieces of disposable polypropylene caps/ hairnets and 3g of cotton were provided as nesting materials to half the cages from each genetic group. Maternal and paternal behaviors were recorded on the third reproductive cycle, twice a week, for three weeks, using scan sampling. The behaviors were recorded every 10s for a period of 10min (totaling 60 records) each day. Dams (N=40) were tested in the elevated plus maze (EPM) on the fifth reproductive cycle, 7–10 days postpartum. Their 21-days-old weanlings (N=208) were tested in the same apparatus. Total number of closed arm entries was used as measurement of motor activity, the percentage of time spent on open arms as measurement of anxiety and head dipping time as measurement of exploratory behavior. The provision of nesting materials increased the frequency of dams licking pups (1.72±0.20 vs. 1.10±0.21, P=0.0342) and of sires resting in contact with pups (25.0±1.5 vs. 18.9±1.5, P=0.0050), while simultaneously decreased the frequency of sires in non-contact rest with pups (6.4±1.1 vs. 10.8±1.2, P=0.0074). Swiss Webster dams were recorded nursing their pups in the arched-back posture more frequently than BALB/cJ dams (9.63±0.89 vs. 7.13±0.86, P=0.0187) and Swiss Webster sires showed a higher frequency of nest building than BALB/cJ sires (0.80±0.16 vs. 0.31±0.15, P=0.0281). Motor activity was higher (8.00±0.43 vs. 2.83±0.41, P<0.0001) and anxiety was lower (3.2±28.4 vs. 2.4±3.1%, P<0.0001) in Swiss Webster than in BALB/cJ dams. Anxiety (26.6±3.2 vs. 37.9±3.5%, P=0.0168) was also lower in Swiss Webster than in BALB/cJ weanlings. The availability of nesting materials increased the time dams spent in head dipping (10.9±2.0 vs. 4.6±1.9, P=0.0087), but weanling behaviors in the EPM were unaffected. Slight differences in parental behavior and contrasting patterns of anxiety and motor activity were found between genetic groups. The provision of nesting materials promoted an intensification of favorable parent-offspring interactions and enhanced exploratory behavior of dams.

Comparative study of platelet aggregation and secretion induced by Bothrops jararaca snake venom and thrombin

Victims of Bothrops jararaca snakebites manifest bleedings, blood incoagulability, platelet dysfunction, and thrombocytopenia, and the latter has been directly implicated in the genesis of hemorrhagic diathesis. We addressed herein the direct effects of B. jararaca venom (BjV) on ex vivo platelet aggregation and granule secretion in washed human and mouse platelets. BjV directly aggregated platelets, but the extent of platelet aggregation was lower in human than mouse platelets. On the other hand, BjV (24.4 mu g/mL) and thrombin (0.1 U/mL) induced a similar extent of ATP and platelet factor 4 (PF4) secretion in both species. BjV-induced platelet aggregation was independent of the platelet dense body content, as in pearl mouse (Ap3b1(-/-))platelets, whose dense bodies are deficient in adenine nucleotides and serotonin, the extent of platelet aggregation was superior to that induced in BALB/c or C57BL/6 mice. BjV-induced beta-hexosaminidase secretion in human platelets was less intense than that evoked by thrombin, and the contrary was observed in mouse platelets. Irreversible inactivation of platelet cyclooxygenase 1 by acetylsalicylic acid did not reduce BjV-induced platelet aggregation. BjV exerted no cytotoxic activity in human and mouse platelets, as evaluated by lactate dehydrogenase loss. Eptifibatide, which inhibits the binding of fibrinogen to platelet glycoprotein complex GPIIb-IIIa, differently blocked BjV-induced platelet aggregation in mice and humans. BjV-induced platelet aggregation did not depend on snake venom serine proteinases nor metalloproteinases in mice, whilst serine proteinases were rather important for platelet aggregation in humans. Our results show that BjV induces direct activation, aggregation, and secretion in human and mouse platelets, but it exerts diverse responses in them, which should be considered in comparative studies to understand pathophysiological events during Bothrops envenomation.

The gingival vein as a minimally traumatic site for multiple blood sampling in guinea pigs and hamsters.

Laboratory animals are still necessary in scientific investigation and vaccine testing, but while novel methodological approaches are not available for their replacement, the search for new, humane, easy, and painless methods is necessary to diminish their stress and pain. When multiple blood samples are to be collected from hamsters and guinea pigs, the number of available venipuncture sites-which are greatly diminished in these species in comparison with other rodents due to the absence of a long tail-, harasses animal caregivers and researchers. Thus, this study aimed to evaluate if gingival vein puncture could be used as an additional route to obtain multiple blood samples from anesthetized hamsters and guinea pigs in such a way that animal behavior, well-being or hematological parameters would not be altered. Thus, twelve anesthetized Syrian golden hamsters and English guinea pigs were randomly allocated in two groups: a control group, whose blood samples were not collected, and an experimental group in which blood samples (200 microliters) were collected by gingival vein puncture at weekly intervals over six weeks. Clinical assessment, body weight gain and complete blood cell count were evaluated weekly, and control and experimental animals were euthanized at week seven, when the mentolabial region was processed to histological analyses. Multiple blood sampling from the gingival vein evoked no clinical manifestations or alteration in the behavioral repertoire, nor a statistically significant difference in weight gain in both species. Guinea pigs showed no alteration in red blood cell, leukocyte or platelet parameters over time. Hamsters developed a characteristic pattern of age-related physiological changes, which were considered normal. Histological analyses showed no difference in morphological structures in the interdental gingiva, confirming that multiple blood sampling is barely traumatic. Thus, these results evidence that blood collection from multiple gingival vein puncture is minimally invasive and traumatic to hamsters and guinea pigs, and that it can be accomplished during at least six weeks.

Diet density in rearing and reproductive phases influences carcass composition, pregnancy rate and litter performance of primiparous rabbit does

The objective was to evaluate the effect of the interaction of diet density in the rearing phasexdiet density in the reproductive phase on carcass composition, pregnancy rate, and litter performance of primiparous rabbit does. The experiment followed a 2x2x2 factorial (2 seasons, 2 diet densities in the rearing phase and 2 diet densities in reproductive phase, that is, from mating to weaning of the first litter). The reference diet (RD) contained 184 g/kg of crude protein (CP), 165 g/kg of acid detergent fibre (ADF) and 10.5 MJ/kg of digestible energy (DE). The low-density diet (LD) had 147 g/kg of CP, 24 g/kg of ADF and 8.4 MJ/kg of DE. The treatments were applied from 70 d of age until weaning of the first litter at 35 d of age. Ninety-six females from the Botucatu Genetic Group (24 females/experimental group) were mated at 142 d of age. On day 12 of gestation, 23 does were slaughtered to evaluate weights of carcass, organs and dissectible fat, and embryo implantation rate. No effects of diet density in the rearing or in the reproductive phases were detected on feed intake of does during the reproductive phase. Does fed LD during the rearing phase showed lower body weight at mating (3574 47 vs. 3866 43 g, P=0.0001) and during most of the reproductive phase, but they lost less weight in the peripartum. Perirenal fat was lighter in these does (72.8 +/- 10.0 vs. 102.1 +/- 9.6 g, P=0.048) and they showed a lower pregnancy rate (76.1 vs. 91.7%, P=0.045). The does fed RD in the reproductive phase were heavier during this phase (4055 +/- 40 g vs. 3887 +/- 41 g, P=0.0044). The does fed LD in rearing phase and RD in the reproductive phase showed larger litters at weaning, due to decreased kit mortality, than those fed RD in both phases (6.16 +/- 0.47 vs. 3.93 +/- 0.71, P=0.0361). Litters were lighter at weaning when LD was fed in the reproductive phase (3582 +/- 201 vs. 4733 +/- 187, P<0.0001). Feeding a low density diet during the rearing phase and a reference diet during the reproductive phase is the best alternative to improve reproductive performance at the first parity.

The gingival vein as a minimally traumatic site for multiple blood sampling in guinea pigs and hamsters

Laboratory animals are still necessary in scientific investigation and vaccine testing, but while novel methodological approaches are not available for their replacement, the search for new, humane, easy, and painless methods is necessary to diminish their stress and pain. When multiple blood samples are to be collected from hamsters and guinea pigs, the number of available venipuncture sites-which are greatly diminished in these species in comparison with other rodents due to the absence of a long tail-, harasses animal caregivers and researchers. Thus, this study aimed to evaluate if gingival vein puncture could be used as an additional route to obtain multiple blood samples from anesthetized hamsters and guinea pigs in such a way that animal behavior, well-being or hematological parameters would not be altered. Thus, twelve anesthetized Syrian golden hamsters and English guinea pigs were randomly allocated in two groups: a control group, whose blood samples were not collected, and an experimental group in which blood samples (200 microliters) were collected by gingival vein puncture at weekly intervals over six weeks. Clinical assessment, body weight gain and complete blood cell count were evaluated weekly, and control and experimental animals were euthanized at week seven, when the mentolabial region was processed to histological analyses. Multiple blood sampling from the gingival vein evoked no clinical manifestations or alteration in the behavioral repertoire, nor a statistically significant difference in weight gain in both species. Guinea pigs showed no alteration in red blood cell, leukocyte or platelet parameters over time. Hamsters developed a characteristic pattern of age-related physiological changes, which were considered normal. Histological analyses showed no difference in morphological structures in the interdental gingiva, confirming that multiple blood sampling is barely traumatic. Thus, these results evidence that blood collection from multiple gingival vein puncture is minimally invasive and traumatic to hamsters and guinea pigs, and that it can be accomplished during at least six weeks.