J. Dairy Sci. TBC https://doi.org/10.3168/jds.2024-24947 © TBC, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Potential use of HSP70 as an indicator of heat stress in dairy cows–A review M. R. H. Rakib,1,2,* 1 V. Messina,1 J. I. Gargiulo,1,3 N. A. Lyons,4 and S. C. Garcia1
Dairy Science Group, School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, Camden, NSW 2570, Australia Bangladesh Livestock Research Institute, Savar, Dhaka 1341, Bangladesh NSW Department of Primary Industries and Regional Development, Menangle, NSW 2568, Australia 4 DairyNZ, Hamilton 3240, New Zealand 2 3
ABSTRACT
Heat stress (HS) poses significant challenges to the dairy industry, resulting in reduced milk production, impaired reproductive performance, and compromised animal welfare. Therefore, understanding the molecular mechanisms underlying cellular responses to HS is crucial for developing effective strategies to mitigate its adverse effects. Heat shock protein 70 (HSP70) has emerged as a potential player involved in cellular thermotolerance in dairy cows. This review provides a comprehensive overview of the role of HSP70 as a molecular chaperone in cellular thermotolerance in dairy cows under HS. HSP70 facilitates proper protein folding and prevents the aggregation of denatured proteins. By binding to misfolded proteins, it helps maintain protein homeostasis and prevents the accumulation of damaged proteins during HS. Additionally, HSP70 interacts with various regulatory proteins and signaling pathways, contributing to the cellular adaptive response to HS. The upregulation of HSP70 expression in response to HS is regulated by a complex network involving heat-shock factors (HSFs), heat-shock element-binding proteins, and HSF co-chaperones. Therefore, HSP70 holds the potential to be a useful indicator of tissue stress due to its role in maintaining cellular balance, and as it is released both inside and outside cells in response to stress. Traditional methods of measuring HSP70 in blood samples are labor-intensive, and with the process being potentially stressful for the animals and may subsequently affect the results. Therefore, measuring HSP expression in cow's milk has shown promise as an easy, non-invasive, and accurate way to detect HS in dairy cows. Monitoring HSP70 levels in milk can be applied as a supplementary approach to identify HS or HS resistance of individual Received March 24, 2024. Accepted July 16, 2024. *Corresponding author: Md Rezaul Hai Rakib; Dairy Science Group, School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, Camden, NSW 2567, Australia; Phone number: +61480477401; Email: rezaul.rakib@sydney.edu.au
cows, selection of suitable animals and to guide targeted management strategies. However, despite the potential advantages of using HSP70 as a biomarker for monitoring HS on dairy cows, challenges remain in standardizing measurement protocols, establishing species-specific reference ranges, addressing inter-individual variations, and determining the specificity of changes in HSP70 due to HS. Future research should focus on developing non-invasive techniques for HSP70 detection, with consideration of climatic conditions, and unravelling the molecular interactions and regulatory networks involving HSP70. Keywords: Animal welfare, Biomarker, ELISA, Heat shock protein, Milk INTRODUCTION
Heat stress (HS) in dairy cows refers to an environment that raises the body temperature of cows due to exposure to high temperatures and humidity levels beyond their ability to dissipate heat effectively (Dunshea et al., 2013; Hyder et al., 2017). This can lead to reduced feed intake, lower milk production, and compromised reproductive performance in cows (Bernabucci et al., 2014; Polsky and Von Keyserlingk, 2017; Becker et al., 2020; Rakib et al., 2020). To cope with HS, cows attempt to regulate their body temperature through panting and sweating, leading to increased water consumption and dehydration (Islam et al., 2021). A combination of observed behavioral changes and physiological indicators are used to diagnose HS in cows, including monitoring increased respiration rates, rectal temperature, panting, drooling, and reduced activity (Tresoldi et al., 2018). The ability of an animal to maintain homeostasis in response to thermal stress is known as thermotolerance, and it is essential for survival under these conditions. Heat shock proteins (HSPs) are a class of molecular ‘chaperones’ (i.e., assistants or helpers) that play a crucial role in maintaining cellular homeostasis and promoting thermotolerance in cells exposed to high temperatures (Mayer and Bukau, 2005). Among the HSP
The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. Rakib et al.: HSP70 as an indicator of heat stress
family, HSP70 is the most extensively studied as it is a widespread protein that acts as a critical regulator of protein folding, stabilization, and degradation under various environmental stresses (Mosser and Morimoto, 2004; Mayer and Bukau, 2005). The HSP70 is known to be induced in response to HS, and its expression levels have been shown to increase in various tissues of dairy cows under HS conditions (Aggarwal et al., 2012). The increased expression of HSP70 has been suggested to play a role in the cellular response to thermal stress in dairy cows, protecting proteins against damage and reducing the risk of cellular dysfunction (Gaughan et al., 2013; Hassan et al., 2019). While the functions of HSP70 have been thoroughly examined by Hyder et al. (2017) and Archana et al. (2017), recent efforts to understand the role of HSP70 in the cellular response of cows to HS are limited. Additionally, there is a significant gap in the literature regarding methods for detecting HSP70. Several studies have investigated HSP70 detection using different sample types. While some focused primarily on blood plasma (Aggarwal et al., 2012; Haque et al., 2012; Gaughan et al., 2013; Kumar et al., 2018; Kumar et al., 2020) others reported HSP70 detection through salivary concentration (Lamy et al., 2017). Furthermore, Pathirana and Garcia (2022) developed a competitive ELISA test for detecting HSP70 in milk samples, that presents promising avenues for further exploration. This suggests the potential for non-invasive sampling methods to monitor the presence of HS response biomarkers in cattle, which could offer practical advantages in field settings. This review aims to present a comprehensive overview of the existing literature concerning the role of HSP70 as a molecular chaperone for cellular thermotolerance in dairy cows during HS. Heat stress and its adverse impacts on the health and productivity of dairy cattle is explored, along with the currently available methods for detecting HS. It then focuses on heat shock proteins (HSPs), particularly HSP70, providing insights into the protein's structure, function, and regulatory mechanisms. The current understanding of HSP70s role in the cellular response to thermal stress in dairy cows is investigated, encompassing its impact on cellular protein folding, degradation, and apoptosis. Finally, the potential mechanisms through which HSP70 may enhance thermotolerance in dairy cows are reviewed. This includes its possible role in regulating cellular signaling pathways, metabolism, and immune function, along with discussing available HSP70 detection methods to highlight the possibilities for using non-invasive approaches to detect HS in dairy cattle.
HEAT STRESS IMPACT ON THE PERFORMANCE OF DAIRY COWS
Heat stress in lactating dairy cattle trigger physiological responses that leads to reduced feed intake and decreased milk production, lower reproductive efficiency, and increased susceptibility to disease, which can have significant economic consequences for the dairy industry (Becker et al., 2020; Rakib et al., 2020) (Figure 1). Homeothermic animals, such as cattle, have a thermoneutral zone (TNZ), defined as a temperature range where they do not spend extra energy to maintain their core body temperature, allowing more energy to be diverted toward production (Hyder et al., 2017). For most dairy cattle, the TNZ is between 4°C and 25°C, although there is some variation based on age, species, breed, lactation stage, dietary intake and composition, housing facilities and management, temperature and humidity of the barns, preceding temperature, acclimation, productivity, and behavior of the animal (West, 2003). To regulate internal temperature, animals must balance the heat they acquire from the environment, and that they generate through metabolism, and release excess heat to the environment (Dunshea et al., 2013). The challenge of managing HS has become more complex due to the increasing number of animals with higher genetic merit for production, greater metabolic activity, and the impact of climate change including an increased frequency of extreme heatwaves (Polsky and Von Keyserlingk, 2017). Since the 1800s, average global temperatures have increased by 1.0°C, and are expected to exceed pre-industrial levels by 1.5°C as early as 2030 (IPCC, 2021). Nidumolu et al. (2014) reported that, in Southern Australia, the average duration of HS events doubled from 2 to 4 consecutive days between 1960 and 2008, where a HS event refers to a period of sustained high temperatures that exceed the threshold for what is considered normal or comfortable for this region. Dairy cows are highly sensitive to temperature and humidity changes, making them susceptible to HS (Polsky and Von Keyserlingk, 2017). The diurnal temperature range is crucial for preventing HS by enabling animals to cool down during the lower nighttime temperatures. This natural cooling cycle effectively regulates body temperature, reducing the risk of HS (Veissier et al., 2017). In intensive, indoor or contained housing systems, optimal indoor climate conditions, such as temperature, relative humidity, light and ventilation can be controlled more easily than in pasture-based systems, to ensure the wellbeing of the dairy cattle (Veissier et al., 2017). Farmers can implement various strategies to increase cow comfort, including decreasing animal density in the barn, active cooling with sprinklers, air movement via fan and proper ventilation, dietary changes, feed supple- Rakib et al.: HSP70 as an indicator of heat stress
Figure 1. Impact of HS on the health and productivity of dairy cows [Expanded from (Rakib et al., 2020)]
mentation and adjusting feeding schedules. These strategies can help mitigate the impact of HS and ensure the wellbeing and productivity of their cows during periods of hot weather (Rakib et al., 2020). In Australia, most dairy cows graze pasture throughout the year and receive low to moderate levels of concentrate and supplements (Garcia et al., 2013). Under typical seasonal conditions, Dairy Australia (2022) reports that approximately 60–65% of cattle feed requirements are fulfilled through grazing. Dairy cows fed with pasture are also influenced by the effect of the temperaturehumidity index (THI) which is considered the most appropriate and straightforward parameter for assessing environmental HS in dairy cattle (Polsky and Von Keyserlingk, 2017). While THI serves as a reliable indicator in indoor or barn settings, its effectiveness diminishes when applied to cows grazing in pastures. A study conducted by Bryant et al. (2023) in the Waikato region of New Zealand developed a grazing heat load index (HLI) incorporating ambient temperature, solar radiation, and wind speed to predict respiration rates in extensively grazed dairy cattle. The study demonstrated increased accuracy compared with existing indices such as THI, with observations indicating that a grazing HLI exceeding 70 may indicate compromised welfare due to HS, although the respiration rate begins to steeply increase before this threshold. However, Wildridge et al. (2018) reported a significant correlation between THI and both milk yield and milking frequency in dairy cows within pasturebased automatic milking systems. Dairy cows with high milk yields experience a reduction in milk production Journal of Dairy Science Vol. TBC No. TBC, TBC
when the THI reaches approximately 68 (Collier et al., 2012). A significant correlation was reported between the THI and the physiological responses of Australian dairy cows during summer, with notable increases observed in respiratory rate (66.7, 84.7, and 109.1 breaths per minute), panting scores (1.4, 1.9, and 2.3), and average body temperatures (38.4, 39.4, and 41.5 ◦C) as THI levels increased from low (≤72) to moderate (73–82) to high (≥83) levels (Osei-Amponsah et al., 2020). Moreover, during periods of moderate and high THI, the cows tended to seek shade, spend more time around watering points, and exhibited signs of distress, such as excessive salivation and open-mouth panting (Wildridge et al., 2018; Osei-Amponsah et al., 2020). Although THI being widely used, it has limitations because it only considers air temperature and humidity, ignoring other crucial factors such as wind speed and solar radiation that are important for assessing environmental conditions (Dunshea et al., 2013). Additionally, THI lacks animal-specific parameters, and the threshold for cow HS varies depending on the specific THI calculation used. Heat stress also affects the cow’s appetite, increasing metabolic maintenance requirements by 7 to 25%, and resulting in prolonged negative effects on milk yield, composition, and quality (Bernabucci et al., 2014). Decreases in daily milk production (14%) and increases in milk temperature (3%), fat percentage (3%) and protein content (2%) were correlated with increases in THI observed by Osei-Amponsah et al. (2020). Dairy farms equipped with efficient cooling systems and located in temperate regions may encounter about 10–15% decline
Rakib et al.: HSP70 as an indicator of heat stress in milk production during HS, characterized by prolonged periods of elevated temperatures surpassing the region's typical comfort threshold. Conversely, dairy operations lacking cooling infrastructure or situated in regions prone to severe heatwaves may face a more substantial reduction of 40–50% in milk yield (Dunshea et al., 2013). Acclimation of the animal plays an important role in alleviating the effects of HS (Becker et al., 2020). In temperate climates, dairy cattle may exhibit lower levels of heat acclimation compared with cows in tropical, subtropical, and Mediterranean climates as these later regions often experience prolonged periods of HS, which can hinder the recovery of cattle from its detrimental effects (Becker et al., 2020). Additionally, when animals experience short bursts of HS, production is negatively affected for about a 5-d recovery period (Ominski et al., 2002). Although HS-related performance decline is typically associated with summer, adverse effects can persist into autumn months, even if cows are no longer exposed to HS (De Rensis and Scaramuzzi, 2003). In addition to reduced productivity, HS in livestock has negative effects on reproductive efficiency and disease susceptibility. According to Becker et al. (2020), it can have a detrimental impact on multiple aspects of livestock reproductive physiology, such as changes in estrus duration, uterine function, endocrine status, follicular growth and development, luteolytic mechanisms, early embryonic development and survival, fetal growth, and colostrum quality. Additionally, it reduces conception rates, dropping below 35% during periods of HS (De Rensis and Scaramuzzi, 2003). Heat stress especially in summer, also has adverse effects on the bulk tank somatic cell count (BTSCC) and the incidence of clinical mastitis in dairy herds (Rakib et al., 2020). According to Nasr and El-Tarabany (2017), there is a positive linear relationship between HS and BTSCC, with BTSCC increasing up to 36% as the THI increases from low (≤70) to moderate (70–80) to high (80–85)levels and with the advancement of the cow's parity. Dry cows have lower feed requirements (Do Amaral et al., 2011) and generate less metabolic heat than lactating cows, HS can still lead to adverse effects, including increased rectal temperature and respiration rate (West, 2003). In addition to these effects, HS can affect dry cows' immune function, particularly when cooling measures are absent, leading to a reduction in lymphocyte proliferation (Do Amaral et al., 2011). According to Ferreira et al. (2016), if dry cows are not provided with cooling measures HS results in annual economic losses of over $800 million in the US dairy industry. The losses are expected to rise in the coming years due to ongoing global climate change. Journal of Dairy Science Vol. TBC No. TBC, TBC
Detection and prevention of HS is critical for the overall welfare of dairy cows and the economic viability of dairy farming. Several methods have been developed to detect and quantify HS in dairy cows in recent years, ranging from physiological and behavioral measurements to advanced technologies such as remote sensing and machine learning (Becker et al., 2021). In this context, understanding the principles and applications of HS detection methods is essential for dairy farmers and industry professionals to effectively detect, mitigate and manage the adverse effects of HS and ensure sustainable dairy production. Several parameters are currently being used to determine HS in dairy cattle. Among these, THI is widely considered as the most suitable and straightforward indicator for evaluating environmental HS in this context (Polsky and Von Keyserlingk, 2017). Researchers have utilized diverse THI formulas, depending on their unique assessments of humidity and temperature (Table 1). However, THI has limitations as it only considers air temperature and relative humidity, neglecting factors like wind speed or solar radiation, which are useful indicators to understand environmental conditions (Dunshea et al., 2013). Additionally, THI does not include any animalspecific parameter. Therefore, dairy cows with high milk yields experience a reduction in milk production when the THI reaches approximately 68 (Collier et al., 2012). However, it is important to note that the value at which cows experience HS depends on the specific THI calculation method used, as there are different formulas and approaches to calculating THI (Table 1). When exposed to HS, animals exhibit various physiological and behavioral changes include increased respiration rate (RR), panting, open-mouth breathing, standing time and elevated body temperature that can serve as indicators (Tresoldi et al., 2018). Alterations in their behavior are also evident, such as reduced eating, rumination, and lying down, as well as an increase in drinking, and seeking shade (Islam et al., 2021). Measuring rectal temperature (RT) has been a widely accepted method for monitoring core body temperature (CBT) in animals. However, one of the challenges of using RT is the interference of defecation, which can impact the accuracy and reliability of RT measurements (Islam et al., 2020; Islam et al., 2021). As a result, alternative methods for measuring CBT in animals have been explored, including non-invasive techniques such as thermal imaging and implantable devices that can provide continuous temperature monitoring without the need for manual intervention. Wearable sensor technology for individual-level HS monitoring has recently gained substantial popularity in
Rakib et al.: HSP70 as an indicator of heat stress the dairy industry, presenting a methodological advancement. This technology offers a promising approach to address HS management at the individual animal level, leading to improved animal welfare, increased productivity, and reduced heat management costs (Islam et al., 2020; Becker et al., 2021). Ongoing research focuses on evaluating various remote and automated monitoring techniques, some of which have already been validated for monitoring cattle behavior and health concerning HS. A study conducted by Islam et al. (2020) examined Australian feedlot cattle and employed ear tag-based sensors to monitor panting and individual variability of HS-related behaviors. The findings revealed that heatsusceptible cattle exhibited higher levels of panting and eating behavior while experiencing reduced resting time, particularly during hotter periods of the day. These sensors have also proven effective in detecting variations in panting behavior associated with the breed, coat color, and individual animals. However, Stygar et al. (2021) reported that among currently available commercial sensors, only 18 (14%) have been externally validated, with accelerometers demonstrating the highest validation rate (30%), while other sensor types showed lower rates. These authors also highlighted the limited potential of existing sensors to evaluate appropriate behavior in dairy cows, underscoring the importance of future validation research, particularly in commercial herds. On the other hand, there has been relatively less focus on cellular-level indicators of HS, especially the relationship between extracellular expression of HSP70 and HS. Hassan et al. (2019) reported that, HSPs particularly HSP70 in bovine provided a direct and quantitative measurement to assess the cellular stress response with enhanced sensitivity and precision. This underscores its potential as a biomarker, offering a supplementary method for identifying HS or HS resistance in dairy cows. The measurement of HSP70 can also enhance predictive models when combined with other physiological and behavioral variables, leading to more precise predictions
of HS. Additionally, elevated levels of HSP70 have been linked to HS in cattle across various sampling methods, including blood, skin (dermal fibroblasts), mammary epithelial cells, milk, and saliva (Gaughan et al., 2013; Lamy et al., 2017; Pathirana and Garcia, 2022). TYPES OF HSPS ASSOCIATED WITH LIVESTOCK DURING HS
HSPs are classified based on their molecular weight and biological functions, and the different types include HSP110, HSP100, HSP90, HSP70, HSP60, HSP40, HSP27, and HSP10. Among these, HSP110, HSP70, HSP90, HSP60, and HSP27 are significantly linked to thermotolerance in livestock species (Fujimoto and Nakai, 2010; Belhadj Slimen et al., 2016). These proteins are critical for maintaining cellular homeostasis and protecting cells from HS, making them essential for coping with HS in animal agriculture. HSP70 and HSP90 have been identified in several studies as important proteins associated with the development of thermotolerance in various farm animals such as cattle, buffalo, sheep, goats, and broilers (Belhadj Slimen et al., 2016). Cells detect heat and respond by increasing the expression of HSPs through various mechanisms. Heat stress can lead to protein denaturation and misfolding, which triggers the activation of heat-shock factors (HSFs). Activated HSF then promotes the transcription of HSP genes, aiding in the refolding of damaged proteins (Fujimoto and Nakai, 2010). Although not all cells have specialized thermal receptors, these mechanisms enable a wide range of cells to sense and respond to HS effectively, thereby protecting themselves from thermal damage (Doberentz and Madea, 2018). The heat shock response is primarily regulated transcriptionally by 4 HSFs, including HSF1, HSF2, HSF3, and HSF4, which bind to heat shock elements (HSEs) in DNA to increase the expression of heat shock proteins (Fujimoto and Nakai, 2010). The HSF1 is mainly as- Table 1: Summary of frequently used THI formulas for dairy cattle THI equations