Protocol for behavioral tests using chemogenetically manipulated mice.

Behavioral analyses using mice chemogenetically manipulated by designer receptors exclusively activated by designer drugs (DREADDs) are powerful tools to elucidate neural functions. Here, we describe the detailed protocols for stereotaxic surgery, adeno-associated virus (AAV)-mediated introduction to Gq-DREADDs in mice, and for behavioral testing and analyses related to anxiety, risk assessment, and burying behaviors. A series of these tests are useful in evaluating animal anxiety and their defensive response patterns to potential threats. For complete details on the use and execution of this protocol, please refer to Horii-Hayashi et al. (2021).

Pain behaviors before and after treatment of oral disease in cats using video assessment: a prospective, blinded, randomized clinical trial.

BACKGROUND: Specific behaviors associated with pain in cats with oral disease have not been consistently studied. The aim of this exploratory study was to identify pain-induced behaviors in cats before and after treatment of oral disease using video assessment. Twenty-four cats (6 ± 3.3 years old; 4.9 ± 1.7 kg) were included in a prospective, blinded, randomized clinical trial. Cats were equally divided into minimal (G1: minimal dental treatment) or severe (G2: multiple dental extractions) oral disease groups. After acclimation at day 0, they underwent oral examination, radiographs, scaling, and dental extractions under general anesthesia (anesthetic protocol: acepromazine, hydromorphone, propofol, isoflurane, meloxicam, and local anesthetic blocks; day 1), and were discharged at day 6. Cats were filmed remotely for 10 min using a wide-angle glass lens camera before surgery (baseline) and throughout the study at different time points (36 h of video recording). The videos consisted of four parts namely general, playing, feeding and post-feeding behaviors. A board-certified behaviorist evaluated the duration/frequency of different behaviors based on an ethogram, which were analyzed using linear mixed models and a generalized linear model, respectively (p < 0.05). RESULTS: In comparison with baseline, duration of "not pawing the face" was significantly shorter at day 3 in G2. These cats spent significantly longer time "standing" and "laying" at days 3 and 6, respectively; G1 spent significantly less time "walking" and "standing" at days 3 and 4, respectively and significantly longer time "immobile" at day 3. Duration of "no/slow tail movement" was significantly longer in G2 than G1 at day 5. Duration of "pawing the ribbon" (playing) was significantly shorter in G2 than G1 at day 1. Feeding and post-feeding behaviors with soft food were not significantly different between groups or over time. Frequency of "difficulty grasping dry food" was significantly higher in G2 than G1 up to day 6. Frequency of post-feeding "head shaking" was significantly higher in both groups at day 6 when compared with baseline. CONCLUSIONS: This study identified pain-induced behaviors in cats undergoing treatment of oral disease. These behaviors may be used to differentiate painful versus pain-free cats in clinical practice.

Establishment of brain ischemia model in tree shrew.

BACKGROUND: Tree shrew, as a kind of small and inexpensive animal between insectivores and primates with the general anatomy being similar to human, could be considered as developed animal model for brain ischemia (BI) study. However, there is no neural behavior scores criterion from tree shrew with BI up to now. METHODS: To produce BI model of tree shrew, a novel systematic neurobehavioral assessment scale, named as neural behavior scores (NBS) including aggressive behavior, seeking behavior, gait, startle reflex, high jump and warped-tail phenomenon was firstly established and used in this study. Moreover, magnetic resonance imaging (MRI) was performed on the first day after the operation to detect the imaging changes caused by ischemia. Then TTC, HE staining and immunofluorescent staining for GFAP and NeuN, were performed 24 h after surgery respectively. RESULTS: NBS in BI group were significantly higher than that of sham operation group at 1d, 3d, 5d and 7d after ischemia. Meanwhile, compared with the sham operation group, the T2 images demonstrated significant higher signal and local brain swelling after cerebral ischemia in tree shrews. The staining of TTC and HE showed apparent infarction and necrosis of the cerebral region, and most of neurons exhibited a shrink. CONCLUSION: We have successfully established the BI model of tree shrew, confirmed by NBS (a new developed method), MRI, HE staining, TTC staining and immunofluorescence staining. It is the first time to report a novel neurobehavioral assessment scale for BI in tree shrew.

Bioacústica como método de avaliação do comportamento em pastejo de novilhas Girolando / Bioacoustics as a tool for evaluating grazing behavior of Girolando heifers

Objetivou-se validar o método de análise de dados bioacústicos na descrição do comportamento em pastejo de novilhas em área de integração lavoura-pecuária (iLP) e floresta (iLPF). Foram utilizadas oito novilhas Girolando (¾Holandês x »Gir) com média de 25±6,8 meses de idade e 268±83kg de peso vivo (PV), distribuídas em delineamento crossover 2x2 (dois sistemas x dois períodos de avaliação). A coleta de dados para análise bioacústica foi realizada utilizando-se gravadores MP3. Esses dados foram analisados pelo software Audacity® para identificação das atividades de pastejo, ruminação e ócio. Concomitantemente, o comportamento também foi avaliado pelo método de observação visual, realizado no período de 8 até 16 horas, em intervalos de 15 minutos. Não houve diferença (P>0,05) entre médias dos tempos (minutos) de pastejo, ruminação e ócio registrados pelos métodos de bioacústica e avaliação visual, sendo, respectivamente, 127 vs. 137; 122 vs. 113 e 238 vs. 216, no sistema iLP, e 134 vs. 103; 130 vs. 165 e 233 vs. 203, no sistema iLPF. Por meio desses resultados, foi possível concluir que o método da bioacústica é uma importante ferramenta para avaliar o comportamento de pastejo em diferentes sistemas de produção a pasto, com a vantagem de possibilitar o registro de atividades durante o período noturno, além de maior praticidade e precisão do que o método observacional.(AU)

Aiming to validate the analysis of bioacoustic data for describing grazing behavior of heifers in integrated crop, livestock (ICL) and forestry (ICLF) systems. Eight 25±6.8 month-old Girolando (¾Holstein × »Gir) heifers with 268±83kg of live weight (LW) were distributed in 2x2 crossover design (two systems x two evaluation periods). Data collection for bioacoustic analysis was done with MP3 recorders. These data were analyzed by Audacity® software for identification of the activities of grazing, rumination, and resting. Concomitantly, behavior was also analyzed by the method of visual observation from 8AM to 4PM in intervals of 15 minutes. There was no difference (P>0.05) between the methods of bioacustic and visual observation considering the means of time (minutes) of grazing, rumination, and resting, respectively: 127 vs. 137; 122 vs. 113; and 238 vs. 216 in ICL system and; 134 vs. 103; 130 vs. 165 and 233 vs. 203 in ICLF system. In conclusion, the bioacustic method is a tool that can be useful for evaluating bovine grazing behavior within different grazing systems, with the advantage of allowing report of activities during the night with higher practicality and precision than the visual observation method.(AU)

Evasive mating behaviour by female nurse sharks, Ginglymostoma cirratum (Bonnaterre, 1788), in an equatorial insular breeding ground

Mating events and aggregations of vulnerable nurse sharks Ginglymostoma cirratum were recorded in the insular protected area of Fernando de Noronha (FEN), Brazil, between April and August 2015. Female sharks were observed clustering in groups of up to 14 individuals in shallow water adjacent to the shore. Several evasive mating behaviours in the presence of males were noticed, including shoreward movement, positioning ventral side up at the sea surface with emerged pectoral and pelvic fins, body rolling and caudal fin thrashing. Fresh bite marks indicative of male courtship and coupling attempts were visible in female's pectoral and caudal fins. Altogether, the observed behaviours match previous reports of non-cooperative female nurse sharks during mating opportunities. An extended mating season coupled with a persistent use of inshore habitats result in nurse sharks being particularly vulnerable to human pressure during a most sensitive stage of their life cycle. The effective conservation of nurse shark populations from the western South Atlantic may thus depend on the protection of critical habitats where this species aggregates to reproduce. Explicitly addressing environmental requirements by vulnerable species in local management strategies is indispensable to ensure that human pressure, including ecotourism development, does not collide with stipulated conservation aims.(AU)

Agregações e eventos reprodutivos do vulnerável tubarão-lixa, Ginglymosytoma cirratum , foram registrados na área insular protegida do Arquipélago de Fernando de Noronha (FEN), Brasil, entre Abril e Agosto de 2015. Grupos constituídos por até 14 fêmeas foram observados em águas rasas adjacentes à linha costeira. Perante a presença de machos, diversos comportamentos evasivos por parte das fêmeas foram identificados, incluíndo movimentação para águas ainda mais rasas, posicionamento na superfície do mar com a face ventral orientada para cima e emersão das nadadeiras peitorais e pélvicas, enrolamento do corpo e batimento da nadadeira caudal na superfície do mar. Foram identificadas marcas recentes de mordida nas nadadeiras peitorais e caudais das fêmeas, sugerindo interações de acasalamento com machos. No geral, os comportamentos observados coincidem com os comportamentos exibidos por fêmeas não aquiescentes à cópula reportados no hemisfério norte. Em FEN, a duração relativamente longa do período de acasalamento, juntamente com a utilização persistente dos habitats mais costeiros, promovem a vulnerabilidade do tubarão lixa às pressões antrópicas durante uma das fases mais sensíveis do seu ciclo de vida. A conservação efetiva das populações de tubarão-lixa do Oceano Atlântico Sul ocidental poderá, assim, depender da proteção dos habitats críticos de agregação onde esta espécie se reproduz. Por conseguinte, a abordagem explícita dos requisitos ambientais associados a espécies vulneráveis no âmbito dos planos de manejo locais deve ser considerada como indispensável para assegurar que as pressões antrópicas, incluíndo o desenvolvimento ecoturístico, não comprometerão os objetivos conservacionistas estipulados.(AU)

Comportamento de poedeiras criadas em diferentes densidades populacionais de alojamento / Behavior of laying hens raised in different population density accommodations / Comportamiento de ponedoras creadas en diferentes densidades poblacionales de alojamiento

Objetivou-se avaliar os parâmetros fisiológicos e comportamentais de poedeiras de ovos vermelhos em diferentes densidades de alojamento. Utilizaram-se 150 poedeiras Dekalb Brown com 30 semanas de idade, durante 84 dias (quatro ciclos de 21 dias). As aves foram alojadas em galpão convencional com gaiolas metálicas de arame galvanizado, com duas subdivisões de 50 x 45 x 40 cm. Adotou-se o delineamento experimental inteiramente casualizado com três tratamentos e cinco repetições. As densidades avaliadas foram: oito (562,5 cm²/ave); 10 (450,0 cm²/ave) e 12 aves/gaiola (375,0 cm²/ave). Foram avaliados os parâmetros fisiológicos (frequência respiratória e temperaturas da pele, retal, crista, barbela e canela) e comportamentais (comendo, bebendo, sentada, postura, parada, movimentos de conforto, investigando pernas, bicagens - agressiva e não agressiva) em dois períodos distintos (manhã e tarde), considerando 30 minutos para cada período de avaliação comportamental. Conclui-se que a criação de poedeiras em menor densidade (562,5 cm2/ave) permite a maior dissipação de calor por meio da crista. O alojamento das aves na densidade de oito aves/gaiola (562,5 cm²/ave) mostrou-se mais adequado às condições fisiológicas de poedeiras criadas em sistema de gaiolas.

The present paper aimed to evaluate the physiological and behavioral parameters of red-egg laying hens in different housing densities. A total of 150 Dekalb Brown hens with 30 weeks of age, for 84 days (four 21-day cycles) were used. The birds were housed in conventional sheds with galvanized wire cages, with two 50 x 45 x 40 cm subdivisions. A completely randomized design with three treatments and five replications was adopted. The evaluated densities were eight (562.5 cm²/bird); 10 (450.0 cm²/bird) and 12 birds/cage (375.0 cm²/bird). The physiological (respiratory rate and temperature of the skin, rectal, crest, wattles and shin) and behavior parameters (eating, drinking, sitting, holding the position, stop, comfort movements, investigating feathers, aggressive and not aggressive pecking) were analyzed in two different periods (morning and afternoon), given 30 minutes for each behavioral evaluation period. It could be concluded that the breeding of laying hens in a lesser density (562.5 cm2/bird) enables further dissipation of heat through the crest. The housing density of birds in eight birds/cage (562.5 cm²/bird) was the most appropriate for the physiological conditions of laying hens in cage system.

El objetivo fuera evaluar los parámetros fisiológicos y de comportamiento de gallinas ponedoras de huevos rojos en diferentes densidades de vivienda. Se utilizaron 150 ponedoras Dekalb Brown con 30 semanas de edad, durante 84 días (cuatro ciclos de 21 días). Las aves fueron alojadas en galpón convencional con jaulas de alambre galvanizado, con dos subdivisiones de 50 x 45 x 40 cm. Se adoptó un diseño completamente al azar con tres tratamientos y cinco repeticiones. Las densidades evaluadas fueron: ocho (562,5 cm²/ave); 10 (450,0 cm²/ave) y 12 aves/jaula (375,0 cm²/ave). Se evaluaron los parámetros fisiológicos (frecuencia respiratoria y temperaturas de la piel, rectal, cresta, barbillas y canela) y el comportamiento (comiendo, bebiendo, sentada, realizando la postura, parada, movimientos de confort, investigando plumas, picotazos - agresivos y no agresivos) en dos períodos diferentes (mañana y tarde), dado 30 minutos para cada período de evaluación conductual. Se concluye que la creación de ponedoras en densidad menor (562,5 cm2/ave) permite mayor disipación de calor a través de la cresta. El alojamiento de las aves en menor densidad de ocho aves/jaula (562,5 cm2/ave) fuera más apropiado a las condiciones fisiológicas de las gallinas ponedoras creadas en sistema de jaulas.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Mutilating predation in the Cheirodontinae Odontostilbe pequira (Characiformes: Characidae)

We observed individuals of Odontostilbe pequira, a small characid, approaching and biting individuals of larger-bodied fishes of other species. This observation was made in two clear water headwater streams of the Cuiabá basin, Paraguay River system, located in Nobres, Mato Grosso State, Brazil, which led us to investigate the behavioral interactions of these fish. We characterized behavioral interactions between species by direct underwater observations using snorkelling and video recordings. Additionally, we proceeded diet analyses of O. pequira, obtaining intestinal coefficient and the index of alimentary importance. During underwater observations we checked the relative frequency of attacks by O. pequira on larger fish species. Odontostilbe pequira attacked individually or in large groups, and the anostomid Leporinus friderici was the preferred target prey species, while Prochilodus lineatus was apparently avoided. Our study sustains that O. pequira is omnivorous, with a diet that varies seasonally. It feeds mainly on plants, but also on animal prey, including the scales of small fishes, and, possibly, the mucus and epidermis of larger fish species. We suggest the term "mutilating predation" to describe the latter relationship.

Observamos indivíduos de Odontostilbe pequira, um caracídeo pequeno, abordando e mordendo peixes maiores de outras espécies. Essa observação foi feita em dois riachos de cabeceira com águas cristalinas na bacia do rio Cuiabá, sistema do rio Paraguai, localizados em Nobres, Mato Grosso, Brasil, o que nos levou a investigar a interação comportamental desses peixes. Nós o fizemos através de observações subaquáticas diretas usando mergulho livre e vídeo. Adicionalmente, realizamos a análise da dieta de O. pequira, obtendo seu coeficiente intestinal e índice de importância alimentar. Nas observações subaquáticas avaliamos a frequência relativa dos ataques de O. pequira sobre peixes maiores. Odontostilbe pequira ataca individualmente ou em grupos grandes, o anostomídeo Leporinus friderici foi a presa preferida, enquanto Prochilodus lineatus foi evitado. Este estudo sustenta que O. pequira é um peixe onívoro cuja dieta varia sazonalmente. Ele come principalmente plantas, mas também presas animais, como escamas de peixes pequenos e, possivelmente, muco e epiderme de peixes maiores. Sugerimos o termo "predação mutilante" para descrever essa relação.

Impacts of early life exposure to estrogen on subsequent breeding behavior and reproductive success in zebrafish.

Impacts of exposure to environmental estrogens on reproductive development are well documented, but recently wider concern has been raised due to evidence that such exposures can disrupt normal patterns of reproductive behavior, dominance, and parentage, with potential population level implications. It is fundamental therefore to understand any such effects for effective risk assessment. This study investigated the impact of a transient exposure to ethinylestradiol (EE(2)) during early life (from 20-60 days post fertilization), including at a dosing level within the environmental range, on the subsequent reproductive behavior and success in both male and female zebrafish (Danio rerio) in competitive breeding scenarios. There were no obvious effects of the early life EE(2) exposures on the subsequent gonadal phenotypes in either mature males or females. In fact, reproductive success in males exposed to 2.76 ng EE(2)/L was increased in competitive spawning scenarios. In contrast, exposure of females to EE(2) (9.86 ng/L) during early life reduced their subsequent reproductive success in competitive spawning scenarios. Mate choice experiments suggested this was a consequence of the females' diminished courting behavior toward males, rather than any male preference for unexposed females. Reproductive capability of females is generally considered a key determinant in population demographics and dynamics, and therefore the effect of exposure to EE(2) on female reproductive success may have significant implications for exposed fish populations.

Rasgos del temperamento de los perros domésticos (Canis familiaris): evaluaciones conductuales / Temperament traits of domestic dogs (Canis familiaris): Behavioural tests

Los rasgos del temperamento se definen como tendencias conductuales estables entre situaciones similares y a lo largo del tiempo. En el presente trabajo se revisan las pruebas conductuales diseñadas para la evaluación de rasgos aislados del temperamento en los perros domésticos. Para cada dimensión se describen los estímulos empleados, las respuestas evaluadas y el correlato fisiológico de las mismas. Los rasgos más estudiados fueron la temerosidad, la agresividad y la sociabilidad. Sin embargo, sólo la primera cuenta con correlatos fisiológicos bien establecidos. Finalmente, las evaluaciones conductuales resultan de suma importancia para la selección de los perros para las distintas funciones que cumplen en la sociedad humana: detección de drogas, compañía, guardia, rescate de personas, etc.

A temperament trait is defined as a stable behavioral tendency along time and among similar situations. This study intends to analyze the behavioral tests designed for the evaluation of isolated temperamental traits of domestic dogs. The type of stimulus used, the behaviors evaluated and the physiological substrates are described for each dimension. Fearfulness, aggressiveness and sociability were the most studied traits. Nevertheless, only the first one has well established physiological bases. Finally, behavioral tests are of great relevance for selecting dogs for the different roles that they perform in human society, such as drug detection, company, security, human rescue, etc.

Effect of weaning age and commingling after the nursery phase of pigs in a wean-to-finish facility on growth, and humoral and behavioral indicators of well-being.

Pigs from one farrowing group in which gilts were bred to farrow pigs that would be either 14 or 21 d of age at weaning, were divided into older and younger age groups (108 pigs per group) and penned 12 pigs per pen in a wean-to-finish facility. At the end of the nursery phase, half the pigs in each age group were removed, rerandomized, and commingled for the finishing phase. The other half remained in their original pens. Pigs were fed common Phase 1 (d 0 to 14) and Phase 2 (d 14 to 35) nursery diets, and a common 4-phase program diet during growing/finishing, with transitions at 45, 68, and 90 kg of BW. The study ended when the lightest weight block averaged 107 kg. Blood was obtained on d 0, 2, 10, 27, 37, 44, and 65 after weaning to determine leukocyte concentrations. In addition, behavior was monitored during the nursery period at weaning (d 0), on d 7, 14, and 27 after weaning, and during the growing/finishing phase on d 35 (after commingling following the nursery phase), 38, 44, and 65 after weaning. Older pigs were heavier (P < 0.001) throughout the nursery period, and the BW difference between younger and older pigs increased from 2 to 6.5 kg at the start and end of the nursery period, respectively. Older pigs had a greater concentration of white blood cells (P < 0.05) and lymphocytes (P < 0.10) on d 0, 2, and 10 after weaning than younger pigs. Younger pigs spent less (P < 0.05) time resting on the day of weaning, and more (P < 0.05) time active during the overall nursery phase. During Phase 3 and in the overall finishing phase, younger pigs had greater (P < 0.01) ADG and G:F than older pigs. Moreover, during Phase 3, ADFI (as fed) decreased (P < 0.05) when older pigs were commingled compared with older pigs that were not commingled. There was no difference in ADFI of younger pigs, regardless of commingling (interaction; P < 0.10). Results of this study indicate that weaning age affects growth performance in a wean-to-finish facility, as well as behavioral and immunological responses to weaning and commingling after the nursery phase. Management strategies should be further explored to optimize these benefits without the detrimental effects on health observed during the nursery period in this study.