Milk ejection in mice LG/J x SM/J.

In mammals, milk provision is crucial to offspring survival and growth from birth to weaning. Milk deficiency early in life may cause death or changes in the progeny metabolism that later may lead to obesity and metabolic disorders. This study investigates milk ejection (ME) the first day after birth (D1) in F(2) females from the intercross of LG/J and SM/J inbred mice strains. The absence of milk in F(3) pups' stomach at D1 is directly associated with their survival (p < 0.001) and growth pattern (p < 0.001) in the early stages of life. Furthermore, late growth pattern is also affected by this lack of nutrients at D1 because pups that survive this absence, mostly males, are heavier at weaning (p < 0.001) which, after necropsy, is shown to be due to significant higher total fat deposition (p < 0.01). We performed QTL analysis for ME at D1 in these F(2) females. Maternal performance of ME revealed a complex genetic architecture which even though it contains only a single QTL (accounting for 8 % of the variation in ME), it is totally context-dependent on the genetic background. We discovered many regions involved in epistatic interactions that together with the single QTL explain 19 % of the genetic variation for this trait. Milk ejection is an important component of maternal care, and understanding the mechanisms modulating its variation, along with other maternal features, may help to disentangle the complexity that is the mother/offspring relationship.

Teste de preferência entre enriquecimentos ambientais para promoção do bem-estar de fêmeas LG/J / Environmental enrichment preferences for female LG/j mice

A experimentação animal permite estudos avançados na pesquisa básica e aplicada nas mais diferentes áreas. A utilização de animais ainda se faz necessária para obtenção de respostas biológicas desconhecidas, principalmente utilizando animais modelo como o camundongo. No entanto, para que a pesquisa seja realizada de forma satisfatória, faz-se necessário seguir procedimento éticos, utilizando o princípio humanitário dos 3 Rs: Reduce (Redução), Refine (Refinamento) e Replace (Substituição) e buscando o bem-estar animal. Nessa perspectiva, o presente trabalho investigou, por meio de teste de preferência, o enriquecimento ambiental selecionado por fêmeas de camundongos da linhagem LG/J. Assim, fêmeas (n=18) em diferentes fases de desenvolvimento (Púberes, Adultas Virgens e Adultas) foram submetidas a dois diferentes tipos de enriquecimentos ambientais: Bateria 1. nidificação (papel toalha e algodão); Bateria 2. abrigo/proteção (rolo de papelão e rolo de PVC). No primeiro contraste (Bateria 1), a preferência das fêmeas, de todas as fases, foi pelo material algodão. No segundo contraste (Bateria 2), houve consenso entre as fêmeas de todas as fases pela escolha do rolo de papelão. No contraste final (Bateria 3), foi testada a preferência das fêmeas pelos enriquecimento escolhidos nas baterias anteriores, o algodão e o rolo de papelão. O enriquecimento ambiental escolhido, pela maioria das fêmeas Púberes, Adultas Virgens e Adultas, foi o rolo de papelão. Esse enriquecimento parece oportuno, pois pode permitir o abrigo/proteção, principalmente para as fêmeas Púberes, mas também pode ser utilizado, quando rasgado, como material de nidificação, importante para termorregulação e na maternidade. Além de ser versátil e de fácil adequação para utilização em biotérios, o rolo de papelão é de baixo-custo e pode proporcionar o bem-estar dos animais de laboratório.

Animal experimentation allows advanced studies in basic and applied research in the most different areas. The use of animals is still necessary to obtain unknown biological responses, especially using animal model such as mice. However, for the research to be carried out satisfactorily, it is necessary to follow ethical procedures, principally by using the humanitarian principle (Reduce, Refine and Replace) and looking for animal welfare. This paper investigated, by using a preference test, the environmental enrichment selected by female LG/J mice. Thus, females (n=18) in different stages of development (Pubescents, Virgins Adults, and Adults) were exposed to two different types of environmental enrichment: 1. Nesting-kind (paper towel and cotton); 2. Shelter/ Protection-kind (cardboard roll and PVC roll). In the first contrast, the preference for females, of all phases, was for cotton. In the second contrast, there was a consensus among the females of all stages for choosing the cardboard roll. In the final contrast, the preference of females for the enrichment chosen in the previous batteries, cotton, and cardboard rolls, was tested. The environmental enrichment chosen by most Pubescent, Virgin Adults, and Adults females, was the cardboard roll. This enrichment seems appropriate, as it may allow shelter/protection, especially for pubescent females, but it may also be used, when ripped, as nesting material, an important source for thermoregulation and maternity. In addition, it is not only versatile, and easy to adapt for use in animal facilities, but also is low-cost and can provide the well-being of laboratory animals.

A link between thrifty phenotype and maternal care across two generations of intercrossed mice.

Maternal effects are causal influences from mother to offspring beyond genetic information, and have lifelong consequences for multiple traits. Previously, we reported that mice whose mothers did not nurse properly had low birth weight followed by rapid fat accumulation and disturbed development of some organs. That pattern resembles metabolic syndromes known collectively as the thrifty phenotype, which is believed to be an adaptation to a stressful environment which prepares offspring for reduced nutrient supply. The potential link between maternal care, stress reactivity, and the thrifty phenotype, however, has been poorly explored in the human and animal literature: only a couple of studies even mention (much less, test) these concepts under a cohesive framework. Here, we explored this link using mice of the parental inbred strains SM/J and LG/J-who differ dramatically in their maternal care-and the intercrossed generations F1 and F2. We measured individual differences in 15 phenotypes and used structural equation modeling to test our hypotheses. We found a remarkable relationship between thrifty phenotype and lower quality of maternal behaviors, including nest building, pup retrieval, grooming/licking, and nursing. To our knowledge, this is the first study to show, in any mammal, a clear connection between the natural variation in thrifty phenotype and maternal care. Both traits in the mother also had a substantial effect on survival rate in the F3 offspring. To our surprise, however, stress reactivity seemed to play no role in our models. Furthermore, the strain of maternal grandmother, but not of paternal grandmother, affected the variation of maternal care in F2 mice, and this effect was mediated by thrifty phenotype in F2. Since F1 animals were all genetically identical, this finding suggests that maternal effects pass down both maternal care and thrifty phenotype in these mice across generations via epigenetic transmission.

Quantitative trait loci for maternal performance for offspring survival in mice.

Maternal performance refers to the effect that the environment provided by mothers has on their offspring's phenotypes, such as offspring survival and growth. Variations in maternal behavior and physiology are responsible for variations in maternal performance, which in turn affects offspring survival. In our study we found females that failed to nurture their offspring and showed abnormal maternal behaviors. The genetic architecture of maternal performance for offspring survival was investigated in 241 females of an F(2) intercross of the SM/J and LG/J inbred mouse strains. Using interval-mapping methods we found two quantitative trait loci (QTL) affecting maternal performance at D2Mit17 + 6 cM and D7Mit21 + 2 cM on chromosomes 2 and 7, respectively. In a two-way genome-wide epistasis scan we found 15 epistatic interactions involving 23 QTL distributed across all chromosomes except 12, 16, and 17. These loci form several small sets of interacting QTL, suggesting a complex set of mechanisms operating to determine maternal performance for offspring survival. Taken all together and correcting for the large number of significant factors, QTL and their interactions explain almost 35% of the phenotypic variation for maternal performance for offspring survival in this cross. This study allowed the identification of many possible candidate genes, as well as the relative size of gene effects and patterns of gene action affecting maternal performance in mice. Detailed behavior observation of mothers from later generations suggests that offspring survival in the first week is related to maternal success in building nests, grooming their pups, providing milk, and/or manifesting aggressive behavior against intruders.

Genetic influences on maternal care.

The basis of social evolution in mammals is the mother-offspring relationship. It is also the primary and most important instance of indirect genetic effects, where genetic variation in one individual affects phenotypic variation among others. This relationship is so important in mammals that often the major factor determining the life or death of newborns is the environment provided by their mother. Variations in these environments can be due to variations in maternal genotypes. In our work with the intercross of two mouse inbred strains, LG/J and SM/J, we uncovered a very severe variation in maternal performance. These females failed to nurture their offspring and showed abnormal maternal behaviors leading to loss of their litter. Rather than this being due to a single gene variant as in knockout mice, we uncovered a complex genetic basis for this trait. The effects of genes on maternal performance are entirely context dependent in our cross. They depend on the alleles present at the same or other epistatically interacting loci. Genomic locations identified in this study include locations of candidate genes whose knockouts displayed similar aberrant maternal behavior. Behaviors significantly associated with maternal performance in this study include suckling, nest building, placentophagia, pup grooming, and retrieval of pups after disturbance.

Genetic architecture of nest building in mice LG/J × SM/J.

Maternal care is critical to offspring growth and survival, which is greatly improved by building an effective nest. Some suggest that genetic variation and underlying genetic effects differ between fitness-related traits and other phenotypes. We investigated the genetic architecture of a fitness-related trait, nest building, in F(2) female mice intercrossed from inbred strains SM/J and LG/J using a QTL analysis for six related nest phenotypes (Presence and Structure pre- and postpartum, prepartum Material Used and postpartum Temperature). We found 15 direct-effect QTLs explaining from 4 to 13% of the phenotypic variation in nest building, mostly with non-additive effect. Epistatic analyses revealed 71 significant epistatic interactions which together explain from 28.4 to 75.5% of the variation, indicating an important role for epistasis in the adaptive process of nest building behavior in mice. Our results suggest a genetic architecture with small direct effects and a larger number of epistatic interactions as expected for fitness-related phenotypes.

Hypothalamic expression of Peg3 gene is associated with maternal care differences between SM/J and LG/J mouse strains.

Maternal care is essential in mammals, and variations in the environment provided by mothers may directly influence the viability of newborns and emotional behavior later in life. A previous study investigated genetic variations associated with maternal care in an intercross of LG/J and SM/J inbred mouse strains and identified two single-locus QTLs (quantitative trait loci). Here, we selected three candidate genes located within these QTLs intervals; Oxt on chromosome 2, and FosB and Peg3 on chromosome 7 and tested their association with maternal care. LG/J females showed impaired postpartum nest building and pup retrieval, a one-day delay in milk ejection, reduced exploratory activity, and higher anxiety-like behavior when compared to SM/J females. The nucleotide sequences of Oxt and FosB were similar between strains, as were their hypothalamic expression levels. Conversely, Peg3 nucleotide sequences showed four nonsynonymous replacement substitutions on LG/J dams, T11062G, G13744A, A13808G, and G13813A, and a 30 base pair (10 aa) in tandem repeat in the coding region with three copies in SM/J and five copies in LG/J. Maternal care impaired LG/J mothers express 37% lower Peg3 mRNA levels in the hypothalamus on the second postpartum day. We also found an association of the Peg3 repeat-variant and poor maternal care in F(2) heterozygote females derived from a LG/J × SM/J intercross. These results may suggest that the maternally imprinted Peg3 gene is responsible for the single-locus QTL on chromosome 7 that has been shown to influence maternal care in these strains. Furthermore, these data provide additional support for an epigenetic regulation of maternal behavior.

Quantitative trait loci for body size components in mice.

Do body size components, such as weights of internal organs and long bone lengths, with different functions and different developmental histories also have different genetic architectures and pleiotropic patterns? We examine murine quantitative trait loci (QTL) for necropsy weight, four long bone lengths, and four organ weights in the LG/J x SM/J intercross. Differences between trait categories were found in number of QTL, dominance, and pleiotropic patterns. Ninety-seven QTLs for individual traits were identified: 52 for long bone lengths, 30 for organ weights, and 15 for necropsy weight. Results for long bones are typically more highly significant than for organs. Organ weights were more frequently over- or underdominant than long bone lengths or necropsy weight. The single-trait QTLs map to 35 pleiotropic loci. Long bones are much more frequently affected in groups while organs tend to be affected singly or in pairs. Organs and long bones are found at the same locus in only 11 cases, 8 of which also include necropsy weight. Our results suggest mainly separate genetic modules for organ weights and long bone lengths, with a few loci that affect overall body size. Antagonistic pleiotropy, in which a locus has opposite effects on different characteristics, is uncommon.