Browsing by study line "ei opintosuuntaa"

Biodiversity is decreasing globally due to human activity. At times, on-time monitoring of the state of habitats and biodiversity is challenging. One useful way to study these is to use certain species as indicators for the state of habitats and biodiversity. One group that are often used, are birds. They are easy to detect and they have specialized to most terrestrial ecosystems. Changes in the population sizes of different bird species can reflect changes in different habitats and their biodiversity. Therefore, it is essential that different bird monitoring practices produce reliable and comparable results. I compared datasets produced by two different Finnish bird census programs in my thesis. The census programs were national breeding bird survey program and Hanko Bird Observatory´s migratory bird census program. Both programs produce data on population abundances and changes in population sizes. Because methods between these programs differ greatly, their data may differ for some species. I compared the datasets by comparing population change indexes of the same species. I also tested whether species traits would be associated with the comparability of the datasets. These traits were mean body size, migratory behavior, favored habitats, and number of sightings for each species. I made models for all combinations of traits. I used a generalized linear model in my analysis. I compared the models by using Akaike´s information criterion (AIC) with correction for small sample sizes (AICc). My results showed that both national breeding bird survey program and Hanko Bird Observatory´s migratory bird census program produce parallel population trends for species. From the tested species traits, only migratory behavior was associated with comparability of the datasets. The datasets were highly comparable for long- and short-distance migratory birds but only moderately comparable for resident birds. This is likely due to migratory bird census program recording the local population dynamics of resident birds of Hanko peninsula. These local population trends may differ from the national trends of the same species. The breeding bird survey program should better reflect the national population trends. My results also showed that more numerous common bird species are declining faster than uncommon species. This is an alarming scenario because it points at extensive habitat degradation and biodiversity loss. Also, population trends of species favoring mires and mountains were clearly declining compared to species favoring other environments. This may be due to endangering of mire and mountain habitats due to climate change and human land use such as peatland drainage. Strong declines of species may also be explained by changes in the wintering areas of these species. Finnish breeding bird survey program and Hanko Bird Observatory´s migratory bird census program both produce overall comparable data on population trends of birds. They could be used to complement one another and to provide supporting evidence on the validity of bird population trends. Producing quality bird census data is highly important in tracking the state of biodiversity and when deciding on conservation acts. The data on the census programs provide support for the research on state of Finnish and European habitats and biodiversity.

Three-dimensional (3D) cellular cultures have been shown to represent tissue formations and functions more accurately than two-dimensional (2D) cultures and have been successfully utilized more accurately in model organisms, e.g., to understand cellular modular functions. However, the applications in non-model organisms are limited, and to our knowledge have not been implemented in ectotherms. At an ecological scale, the technique can enhance our understanding by providing insights on cellular and tissue level molecular mechanisms. A potential implementation of this method in Atlantic salmon is to elucidate the molecular function of the vestigial-like 3 (vgll3) gene, which plays a central role in salmonid maturity, potentially by regulating energy allocation via regulating adipogenesis. In this thesis, a proof of principle study was implemented, where the feasibility of obtaining and maintaining a suspension 3D adipose tissue culture in Atlantic salmon (Salmo salar) was assessed. The harvested visceral white adipose tissue from around the intestinal tissue of mature Atlantic salmon (salmon past smolt stage) was first separated into stromal vascular fraction (SVFs) and mature adipose fraction (MAFs). SVFs contain preadipocytes (precursors of adipocyte cells) in addition to a variety of other cell types. MAFs are mature adipocytes. Both MAFs and SVFs were successfully maintained in-vitro for over four weeks. SVFs were then successfully differentiated into mature adipocytes, demonstrating the feasibility of studying adipogenesis in Atlantic salmon. Proof of this methodology and its further implications may help us to understand the cellular functions of vgll3 and may subsequently help to better understand its causal relation to the maturation process in Atlantic salmon.

As biodiversity is being lost worldwide at an accelerating rate due to anthropogenic activities, the frequency and severity of many infectious diseases has been observed to increase. Together these patterns have brought forth an urgent need to understand the possible linkages between biodiversity and disease risk. Two contradicting hypotheses have been proposed to explain the diversity-disease relationship. The dilution effect hypothesis suggests that increasing host community species diversity ‘dilutes’ disease risk, whereas the amplification effect hypothesis predicts disease risk to increase with increasing diversity. Even though most of the studies support the dilution effect, there remains an intensive debate regarding the generality of this effect. As most of the research efforts to understand the relationship between diversity and disease have focused on animals and crop plants or have been carried out experimentally, one of the research gaps is how relevant the dilution effect is in wild plant communities. In nature, plants and their diseases are affected simultaneously by multiple abiotic and biotic environmental factors that might confound or supersede the effects of diversity. It is also poorly understood, whether we might expect dilution effects to occur not only on diversity gradients driven by anthropogenic diversity loss, but also on natural diversity gradients. To study the possible association between host community species diversity and disease risk in the wild and to test whether this association could be detected after accounting for the effects of abiotic factors, I surveyed grassland vascular plant communities for their species diversity and foliar disease symptoms along a natural diversity gradient driven by elevation. I also recorded data on the mean soil surface temperature in the surveyed plant communities and used structural equation modelling to differentiate and compare the effects of biotic and abiotic variables on disease risk. The data were collected on Mount Calanda in the Swiss Alps during summer 2019. In this thesis I show that host community species diversity and disease risk are negatively associated with each other along a natural diversity gradient driven by elevation. Furthermore, this negative effect can be detected even after accounting for the effects of elevation and mean soil surface temperature on disease. Together the results support the occurrence and the ecological relevance of the dilution effect in wild plant communities along natural diversity gradients and suggest that diversity might protect wild plant communities from increased disease risk. Future studies should aim to identify the exact mechanisms of the association to help us better understand when and where we might expect dilution effects to occur in the wild. This knowledge can be used to predict how epidemics, that affect the well-being of ecosystems, humans and wildlife, are born in the changing world.

Post-transcriptional modifications (PTMs) in RNA are present in all known RNA species and conserved in all kingdoms of life. Transfer RNA (tRNA) has been shown to have numerous conserved modifications, which exemplifies the importance of modifications having impact on the structure of the tRNA and its function as carrier of the amino acids. Ribosomal RNAs (rRNA) are universally modified as well, and modifications are situated at functionally important spots of the ribosome. Given the fact that types and sites of modifications are conserved, it is likely that these modifications have been selected for and that they optimize the ribosomal structure and functions. Stress, such as temperature or infection by a pathogen, is known to change the presence or abundance of modifications in RNA molecules and thereby affect translation efficacy. In line with that, this master’s thesis project sought to gain insight into the dynamics of PTMs in tRNA and rRNA upon oxidative stress, with the goal of utilizing recently optimized UPLC/MS method for identifying modified ribonucleosides. As the specific aim of the thesis was to estimate the change in PTMs in tRNA and rRNA in response to oxidative stress with 0.5 mM and 2 mM hydrogen peroxide H2O2, 3 immediate goals were: (i) to isolate total tRNA from yeast grown in stress conditions, (ii) to isolate rRNA from yeast 80S ribosomes, and (iii) to identify present modifications using mass spectrometry. Yeast was cultured in presence of H2O2 as a stressor in mentioned concentrations, and both treatments considered showed a difference in survival when compared to the control. Rough cell concentration estimates (OD600) did not show the effect of the stressor on cell survival clearly, but when number of viable cells per mL was estimated, it was clear that growth of the stressed yeast cultures was hindered 2 hours after exposure to H2O2 but recovered during the 24 hours. Firstly, using UPLC/MS analysis, 29 modifications were identified in tRNA from control and H2O2 treated yeast. Most identified modifications showed no change in abundance in treatments, which is to be verified with additional replicates. However, distinct dynamics of stress-related change was found for several modifications, revealing additional modifications that may play a role in stress related modificome reprogramming to the previously known signature modifications of oxidative stress. It was expected that recovery of culture growth after 24 hours may be accompanied with modification level recovery. However, that was not demonstrated here as downregulation at 2 hours followed by upregulation at 24 hours was seen for 2-methylthio-N6-methyladenosine, N4-acetylcytidine and 5-methoxycarbonylmethyl-2-thiouridine, and the reverse was shown for N4-methylcytidine. Upregulation in both time points was also shown here for some modifications. Taken together, these results confirm a complex and dynamic control of tRNA modifications in cellular survival responses. Modifications found to be affected by oxidative stress are most frequently located on the wobble position 34 and anticodon loop position 37, so it is expected that changes in their modification levels could directly affect the tRNA function in translation, making them a specific target for future research. Secondly, modifications in rRNA from control yeast cultures were identified, such as expected methylations of all 4 canonical nucleosides. However, further analysis will be needed to confirm the other identified modifications, due to the potential mRNA and tRNA contamination. Optimizing the method for rRNA modifications identifications by acquiring more modified nucleosides specific for the rRNA to use as standards in the analysis, analyzing rRNA types separately and using tandem mass spectrometry would enable getting a deeper understanding of which modifications are present and where they are positioned. Finally, it would enable reliable identification of the signals of novel modifications present in rRNA, such as the tRNA modification 5-carbamoylmethyluridine signal found here. In conclusion, this thesis work lays the foundation to study the evolutionary conserved function of PTM changes during stress as modulators of translation, using the methodological approaches discussed in-depth within the thesis, primarily to confirm the intriguing results found here.

Woodland strawberry (Fragaria vesca) is a perennial herb in the Rosaceae family with dimorphic leaves, summer and winter leaves, adapted to seasonal climate. Woodland strawberry produces a new set of leaves in spring that are photosynthetically active throughout the summer season (summer leaves), and the leaves senescence in autumn when they are replaced by a new set of leaves (winter leaves). The winter leaves retain photosynthetic capacity under the snow cover throughout the winter season, which prolong the photosynthetic period of the species. With the world-wide climate warming, the thickness of winter snow is decreasing, which can affect overwintering and spring phenology of plants. This thesis focuses on springtime ecophysiology and phenology of the senescing winter leaves and the formation of new summer leaves of woodland strawberry genotypes of different European origin. The 15 different genotypes of woodland strawberry are from Iceland, Italy and Norway, and they originate from different environments that are geographically separated from each other, so the populations are genetically distinct. In this study, these genotypes were kept at two different overwintering sites, coastal site at the Åland islands with mild temperatures, and continental site in Lammi with a persistent snow cover. According to the results all 15 genotypes showed earlier development of the summer leaves and earlier senescence of winter leaves in the group with Åland as overwintering site than in the group with Lammi. Another important finding is that the first summer leaves produced in spring begun to senesce shortly after they are fully developed and were replaced by later formed summer leaves. Specifically, the dates of summer leaf formation, flowering and stolon production were advanced, and the dates of winter leaf senescence were also advanced. The value of different leaf types to chlorophyll fluorescence was also lower at the Åland site. Therefore, it can be concluded that overwintering conditions have an effect on the subsequent phenological development in spring. In the context of global climate change, the spring development of woodland strawberry will be earlier, and the senescence of winter leaves will also be earlier.

As a genome editing tool, CRISPR-Cas9 has provided a robust way to generate mutations in the gene of interest, at a certain time point, and in selected cell populations. The impairment of dopaminergic neurons in the substantia nigra is addressed to be one of the main pathologies of Parkinson’s disease. The histopathology of Lewy Bodies, with an undetermined role, accompanies the demise of DA neurons. Development of strategies for the prevention the neurodegeneration has a potential to slow down the progression of Parkinson’s disease. In this study, a novel, neuron-specific CRISPR-Cas9 system was developed for the purpose of dissecting neuroprotective pathways in primary dopaminergic neurons. The optimization of the tool was done by targeting EGFP at TH-positive neurons obtained from transgenic animals expressing EGFP in dopaminergic neurons. Complete loss of EGFP was achieved at day 6 after the introduction of the CRISPR-Cas9 via lentiviral vectors. There were no survival or transduction efficiency differences. Two significant pathways for the survival of dopaminergic neurons, the microRNA biogenesis and GDNF/RET signaling were selected to collect the preliminary data. Dicer, Trbp, Translin, Ago-2 and Ret were targeted with single sgRNAs, which were specifically designed to create indel mutations in these genes, and specific lentivirus vectors were produced with each guide. After transduction with the lentivirus vectors, survival of the TH-positive neurons was unaffected. Data obtained from the quantitative PCR suggested that there was 50-70% decline in transcript levels of Trbp. However, the unchanged transcript levels of the other miRNA-related targets suggest the need for further optimization of the specific guides. Knockdown of Ret was validated by inhibition of pharmacological benefits of GDNF. Overall, this research has shown the further development of this CRISPR-Cas9 tool would be useful to dissect neuroprotective signaling pathways in dopaminergic neurons.

In the process of decomposition soil carbon is transformed into CO2 by microbial respiration, which makes decomposition a key process for understanding carbon cycling an releases of CO2. Since the northern permafrost regions contain half of all belowground carbon and the tundra regions are expected to be markedly affected by climate warming, it is of particular interest to understand how warming will affect decomposition in the tundra. Decomposition is however influenced by many factors, from climatic factors such as temperature and precipitation to the belowground organisms inhabiting the soils and the aboveground system dictating the litter that falls to the ground and is decomposed. Further, grazing has been shown to oppose some of the effects of warming on tundra. In this thesis I analyzed data collected from two long-term field experiments, one in Kilpisjärvi (NW Finland) and the other close to Kangerlussuaq Fjord (SW Greenland), both using fencing for manipulation of grazing regime and open-top chambers for artificial warming. My aim was to not only investigate how warming and grazing affect decomposition, but also to understand whether the magnitude of changes in decomposition can be explained by changes in plant community traits and soil characteristics. I found that in contrast to my hypothesis, warming decreased decomposition in Kangerlussuaq, where the soil was drier and contained less carbon than in Kilpisjärvi. I found no effects of grazing on decomposition, plant community traits nor soil characteristics in neither of the study locations. Neither did I find any consistent associations between changes in decomposition and changes in plant community traits, indicating that the effect of litter quality on decomposition is minor in these areas likely rather limited by climate. I found an association for increased decomposition when plant community C:N ratio and C:P ratio increased as a response to warming, but only in Kilpisjärvi, and since increased plant community C:N and C:P ratios are linked to resistant litter this positive effect is unlikely driven by enhanced litter quality. However, I did find a positive relationship between increased root biomass and increased decomposition as a response to warming that was consistent across areas and grazing regimes, indicating that warming can boost decomposition in different tundra habitats by promoting root growth.

Panssarisiimalevät ovat suuri, morfologisesti ja ekologisesti monimuotoinen kasviplanktonryhmä.Panssarisiimalevien tuntemus on painottunut suurikokoisiin, yhteyttäviin ja laboratoriossa kasvatettaviin lajeihin, jaerityisesti pienten hetero- ja miksotrofisten panssarisiimalevien monimuotoisuus tunnetaan puutteellisesti. Itämerion maantieteellisesti ja ekologisesti eristynyt merialue. Panssarisiimalevien suhteellinen osuus Itämerenkasviplanktonista on kasvanut viime vuosikymmeninä. Tunnistamisen vaikeuden vuoksi Itämerenkasviplanktonseurannoissa pienikokoiset panssarisiimalevät kuitenkin usein sivuutetaan tai niitä käsitellään yhtenä,vain lahkotasolle määriteltynä ryhmänä. Pfiesteriaceae-heimon lajit ja Karlodinium veneficum ovat pienikokoisiahetero- ja miksotrofisia, mahdollisesti toksisia, haitallisia leväesiintymiä muodostavia panssarisiimaleviä. Näistäpanssarisiimalevistä ei ole vahvistettuja havaintoja Itämerestä. Lajeja pidetään kosmopoliittisina ja niidenasettuminen Itämeren murtoveteen on mahdollista. Tutkimukseni tavoite on selvittää pienikokoisten (alle 20 μm)panssarisiimalevien monimuotoisuutta Itämeressä ja arvioida näiden panssarisiimaleväkantojen suhdetta kantojenmuilta merialueilta eristettyihin lajitovereihin. Aineisto kerättiin Hankoniemen itäpuolelta Tvärminnen eläintieteellisen aseman läheisyydestä ja AhvenanmaanFöglöstä matalista ja suojaisista lahdelmista. Vesinäytteistä eristettiin kuoppalevylle valomikroskoopin avullapieniä (alle 20 μm) panssarisiimaleviä puhdasviljelmiä varten. Menestyksellisesti eristettyjäpanssarisiimaleväkantoja kasvatettiin Itämeren murtoveteen valmistetussa f/2-Si-kasvatusliuoksessa. Tiheiksikasvaneista soluviljelmistä eristin DNAn ja selvitin kantojen suku- ja lajitason identiteetin sekvensoimallaribosomaalisen DNAn SSU-, LSU- ja ITS-alueet. Tutkimuksessa luotujen sekvenssien ja GenBank-tietokannastaladattujen sekvenssien avulla laskin aineistosta neljä fylogeneettistä puuta. Kuvasin havaitut lajit morfologisestipyyhkäisyelektronimikroskoopilla. Toksisuusanalyysejä varten suodatin panssarisiimaleväkannoista näytteetlasikuitusuodattimille, jotka lähetettiin tutkittaviksi Yhdysvaltoihin. Tutkimuksessa eristetyistä 512 panssarisiimalevästä 100 kantaa jakautui kuoppalevyillä. Lopullisiin analyyseihinvalikoitui 12 kantaa. Fylogeneettisten ja morfologisten analyysien perusteella Itämerestä eristetyt kannat kuuluvatCryptoperidiniopsis brodyi-, K. veneficum- ja Pfiesteria piscicida-lajeihin. Itämeren K. veneficum -kantojen eitässä tutkimuksessa havaittu tuottavan karlotoksiineja. Itämerestä eristetyt C.brodyi-, K.veneficum- ja P.piscicida-kannat eivät eroa geneettisesti tai morfologisestimuilta merialueilta eristetyistä lajitovereistaan. Taustalla voi olla jatkuva geenivirta lajien eri populaatioidenvälillä. On myös mahdollista, että ribosomaalinen DNA on liian konservatiivinen alue kuvastamaan lajinsisäistävaihtelua. K. veneficum ja P. piscicida voivat tuottaa toksiineja ja muodostaa haitallisia leväesiintymiä. Vaikkatässä tutkimuksessa toksiinituotantoa ei havaittu, luonnonpopulaatiot voivat sisältää toksisuusfenotyypiltäänerilaisia kantoja, ja haitallisten leväesiintymien seurannan ja ennakoinnin kannalta tulisikin selvittää mahdollisestitoksiineja tuottavien kantojen esiintymistä Itämeressä. Suurin osa tutkimuksessa eristetyistä panssarisiimalevistä eimenestynyt laboratorio-olosuhteissa. Tutkimuksessa havaitut lajit eivät siis todennäköisesti edusta Itämerenpienikokoisten panssarisiimalevien koko monimuotoisuutta. Panssarisiimaleviin kohdistettu ympäristönäytteidensekvensointi eristyneessä ja vesimassoiltaan kerrostuneessa Itämeressä voisikin tuottaa lisätietoa niinpanssarisiimalevien monimuotoisuudesta yleensä kuin niiden erityispiirteistä Itämeressä.

The aim of this study was to examine the leaf endophytic bacteria in Plantago lanceolata. The first aim was to get a comprehensive picture of the bacterial diversity, by screening for the different bacterial genera inside the leaves. Furthermore, I aimed to examine the effect of soil and maternal genotype on the endophytic community within P. lanceolata leaves and search for clues of vertical inheritance of endophytes from parent to offspring via seeds. I studied the endophytic bacteria by extracting DNA from the plant leaves and by trying to amplify any bacterial DNA present to get a view of the bacterial diversity in the leaves. My aim was to compare the bacterial community of the mother plants to that of their offspring and also to compare the bacterial communities of plants grown in different soil conditions. Furthermore, I tried to study how the soil conditions affect the growth of P. lanceolata seedlings. I collected seeds and leaf samples of P. lanceolata from Åland, Southwestern Finland, from a population that is part of the ongoing long-term metapopulation research started in Åland in the early 90’s. I marked 21 plant individuals (hereafter referred to as the “mother plants”) in the field in June, when collecting the first leaf samples. In August I collected all seeds from the same plant individuals and a second set of leaf samples. I also collected soil samples from the same location. With the seeds collected from the wild population I executed a growth experiment in Viikki, Helsinki. I grew one set of seeds in twice autoclaved sand (hereafter referred to as the “sterile soil”) and another set in twice autoclaved sand mixed with soil collected from the Åland population (hereafter referred to as the “environmental soil”). I surface sterilized all seeds and then sowed each in their own growth pot and placed them in a growth chamber. During the experiment I took measurements of the leaves. At end of the growth experiment, I took samples of the leaves and surface sterilized them to exclude any epiphytic microorganisms from the analysis. I also surface sterilized the leaf samples taken from the mother plants. I then extracted DNA from the leaf samples and run PCR to amplify certain regions of the bacterial 16S rDNA gene, that is widely used in bacterial taxonomy. The obtained DNA reads where then clustered into Operational Taxonomic Units (OTUs) and assigned taxonomy using SILVA reference database. Mitochondria and chloroplasts of eukaryotic organisms also harbour 16S rDNA regions, so the challenge of studies looking at endophytic bacteria is to exclude the 16S regions of mitochondria and chloroplasts. This proved to be a problem in my study also. More than 86% of all DNA reads obtained turned out to be from P. lanceolata mitochondria and more than 12% from P. lanceolata chloroplasts. Only a bit more than 1% of the reads were eubacterial. This effectively hindered reliable analysis of the endophyte community. I nevertheless analysed the observed eubacterial diversity although the results must be taken as only preliminary and with utmost caution. The eubacterial reads clustered into 218 OTUs, representing 71 different bacterial genera. Six most common genera constituted over 83% of eubacterial reads. Most of these bacteria, most notably Shewanella, Ralstonia and Halomonas, could be identified as being clearly contaminants and not real endophytes. For the 65 less common bacterial genera I performed community analysis using Bray-Curtis Dissimilarity index and Analysis of Similarities (ANOSIM). The results showed that there was a significant difference between the different soil treatments (P = 0.014, R = 0.3787) and also between the two growth chambers (P = 0.011, R = 0.5493). I found no effect of maternal genotype on the bacterial community. Therefore, I observed no sign of vertical inheritance of endophytes. The growth experiment results showed that germination percentage was significantly lower in the environmental soil than in the sterile soil for all genotypes (F = 10.78, P = 0.0012). However, seedling in the environmental soil grew bigger than the seedlings in the sterile soil (F = 10.91, P < 0.0001). For future studies on similar topics, validating molecular methods before large scale sequencing could yield more reliable results. Size fractionating the DNA products of the first PCR round could exclude most mitochondrial sequences and hence allow better analysis of endophytes. This would enable studying interesting questions on coevolution and ecology of host-endophyte interactions. Although I did find some differences in the bacterial communities of different treatments, these results must be considered with caution and as only preliminary.

Individuals of long-lived animal species can improve their reproductive success through experience. While individual’s resources available for survival and reproduction decrease toward the end of its lifespan through senescence, terminal investment hypothesis predicts the less likely old individuals reproduce again the more they invest in their current offspring. Experience gained through a long lifespan might have an important role in changing behavior to optimize the use of resources and compensate the effects of senescence. In addition, behavioral plasticity allows animals to respond changes in their habitat within much shorter timespan than on an evolutionary timescale. Svalbard reindeer (Rangifer tarandus platyrhynchus) is a wild subspecies of reindeer. It is only found in Svalbard, a remote archipelago in the Arctic with extreme weather conditions rapidly changing due to climate change. It has been isolated at least 5000 years and adapted to a barren habitat with nearly no hunting, predation or harassment of flying insects. The objective of this study is to investigate the effect of age and a calf at heel in Svalbard reindeer females’ maternal, vigilant, and social behavior and time budget in the light of life history theory and its senescence and terminal investment hypotheses. I carried out the field work for the study in two periods in summer in Semmeldalen valley and the south-western part of Reindalen valley on the island of Spitsbergen, Svalbard. I collected behavioral data on marked individuals by instant scan sampling and focal watch methods, wrote observations down manually and later fed them into computer. In addition, I have used birth year data collected by the long-term monitoring program by the Norwegian Polar Institute. I used generalized linear mixed models to analyze the effects of age and calf at heel to the behavior of females. The main results include that young dams maintained shorter distance to their calf in July than in August, and old females were less vigilant. Younger dams and older females without calves were in smaller groups than older dams and younger females without calves. In addition, females with calves spend proportionately less time lying down than females without calves. Dams maintained a longer distance to the nearest neighbor than females without calves. Older dams spend proportionately more time feeding and in groups in August than younger dams. These results show that the age and calf at heel do play a role in the behavior of Svalbard reindeer females and the effect varies over the course of the short Arctic summer. Experience may make older females more effective mothers by optimize the use of resources for example from vigilance to feeding in a predator-free environment. On the other hand, senescence may affect the amount of energy females can spend on their calves, potentially influencing their survival.