The impact of heat stress on performance, fertility, and adipokines involved in regulating systemic immune response during lipolysis of early lactating dairy cows

The aim of this study was to assess the potential impact of heat stress on dairy cow productivity, fertility, and biochemical blood indices during the early lactation stage in a temperate climate. Additionally, the study aimed to determine the role of leptin and adiponectin in regulating the immune response accompanying lipolysis after calving in dairy cows. The study included 100 clinically healthy Polish Holstein-Friesian dairy cows selected based on parity and 305 d of milk yield from 5 commercial farms with similar herd management and housing systems. Prospective cohort data were recorded from calving day until 150 d in milk, and microclimate loggers installed inside the barns were used to record temperature and relative humidity data to calculate daily temperature-humidity index (THI) on the calving day, through +7, +14, and +21 d during early lactation. Additionally, monthly productive performance parameters such as milk yield, chemical composition, fatty acids composition, and fertility indices were analyzed. Results showed that the THI from calving day through +7, +14, and +21 d during early lactation was negatively associated with fertility parameters such as delayed first estrus postpartum and an elongated calving interval, respectively by 29, 27, 25, and 16 d. Furthermore, an increase in THI value during early lactation was associated with an elongated artificially inseminated service period, days open, and inter-calving period. Increasing THI from calving day to +21 d during early lactation was also linked to decreased milk yield by 3.20, 4.10, 5.60, and 5.60 kg, respectively. The study also found that heat stress during early lactation was associated with a lower body condition score in dairy cows and higher concentrations of leptin, nonesterified fatty acids, and β-hydroxybutyrate, accompanied by a drastic reduction in adipose tissue-secreted adiponectin levels after calving. Additionally, heat stress-induced lipolysis in adipose tissue caused an inflammatory response that increased biochemical blood indices associated with immune responses such as cytokines, acute phase proteins, and heat shock


INTRODUCTION
Heat stress (HS) is a combination of internal and external environmental factors that cause behavioral and physiological changes that negatively impact animal welfare (Becker et al., 2020).Genetic selection for milk production traits leads to increased metabolic heat production, as cows consume more feed for processes such as body tissue synthesis and milk secretion (Collier et al., 2012;Gauly et al., 2013).Meanwhile, global temperatures have risen by 1.0°C since the 1800s, and an additional 1.5°C increase is expected between 2020 and 2052 (IPCC, 2018).Dairy cows in temperate regions are not well-adapted to the excessive thermal stress caused by global warming (Becker et al., 2020) and are unable to recover from the negative effects of HS The impact of heat stress on performance, fertility, and adipokines involved in regulating systemic immune response during lipolysis of early lactating dairy cows as quickly as cows in tropical or subtropical climates (Ominski et al., 2002).
Short-term periods of HS negatively affect production for about a 5-d recovery period following the HS condition (Ominski et al., 2002).Long-term exposure to high temperatures and humidity can also have carry-over effects on the behavioral and physiological changes of high-yielding dairy cows.While decreased performance due to HS is typically associated with summer months from June to September, the negative consequences can extend into autumn, even after cows are no longer experiencing HS (De Rensis and Scaramuzzi, 2003).As a result, managing and preventing HS has become a significant challenge due to the increasing number of production animals with high milk yields and ongoing global warming.
High-yielding dairy cows undergo extensive physiological and metabolic adaptations during the transition period due to increased energy requirements for lactation compared with pre-calving requirements (Heirbaut et al., 2023).These adaptations involve increased lipid mobilization and lipolysis of white adipose tissue (AT), which is a major energy reserve in the body.Nonesterified fatty acids (NEFA) are released starting around 3 weeks before calving and up to 4-5 weeks after calving (Contreras et al., 2018), and are used for β-oxidation and energy generation in the TCA cycle, leading to hepatic gluconeogenesis.However, NEFA can also be incompletely oxidized to ketone bodies, such as β-hydroxybutyrate, or re-esterified as triglycerides (TG), leading to negative energy balance (NEB) occurrence (Mann, 2022).In addition to being an energy source, AT is also considered an endocrine organ that produces factors regulating inflammation, lipid homeostasis, insulin sensitivity, glucose metabolism, and fat distribution (Coelho et al., 2013).The lipolytic activity around parturition and the onset of lactation leads to AT remodeling, a process characterized by cellular proliferation, enhanced immune cell production, and inflammatory response.Signal molecules produced and secreted by AT during the transition period are called adipokines, which include inflammatory cytokines [e.g., tumor necrosis factor-α (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), Funcke and Scherer (2019)], acute phase proteins [e.g., haptoglobin (Hpt) and serum amyloid A (SAA), Ceciliani et al. (2012)], hormones, growth factors, chemokines, complement factors, and other proteins (Haussler et al., 2022).Adipokines play a critical role in cell communication within AT and endocrine crosstalk with other tissues, such as the liver, pancreas, skeletal muscle, heart, and brain (Romacho et al., 2014, Kita et al., 2019).Lipolysis from AT depots during the transition period coincides with a period of induced AT remodeling (Contreras et al., 2017(Contreras et al., , 2018) ) and decreased insulin sensitivity in hepatocytes, adipocytes, and monocytes, redirecting energy toward milk synthesis (De Koster and Opsomer, 2013).Prolonged lipolysis leads to changes in the secretion pattern of adipokines, which in turn modulate bioactive compounds (Contreras et al., 2017).Bovine adipocytes become more sensitive to insulin as lactation progresses, leading to decreased rates of lipolysis and increased lipogenesis (Contreras et al., 2017).While moderate insulin resistance in AT may support healthy and productive lactation during the transition period, intense insulin resistance in AT can predispose dairy cows to inflammation and metabolic dysfunction by limiting the AT's ability to expand its energy-buffering capacity (De Koster and Opsomer, 2013).In ruminants, research on adipokines has mainly focused on mRNA, and there is limited information on the role of new regulatory peptides such as leptin (LEP) and adiponectin (ADP) from adipokines in AT in regulating the systemic inflammatory response during the transition period in high-yielding dairy cows (Haussler et al., 2022).LEP is a protein that modulates insulin secretion and regulates lipid metabolism by reducing the synthesis of TG and fatty acids, leading to increased lipolysis of AT and NEFA concentration in the blood (Zhang et al., 2019).ADP is negatively correlated with NEFA concentration in the blood, suggesting that it can reduce lipolysis of AT and increase cell sensitivity to insulin (Zachut et al., 2020).
HS causes similar disruptions in nutrient metabolism in the liver as those observed in NEB during the transition period.HS reduces glucose concentration and increases both insulin and NEFA concentrations in the blood, contributing to the development of insulin resistance (Zachut et al., 2020).Currently, researchers are investigating biomarkers of biological mechanisms connecting HS and NEB occurrence with the immunity of dairy cows in the transitional period after calving, which may affect performance and fertility during lactation (Zachut and Contreras, 2022).However, the biological mechanism of both LEP and ADP as immunomodulators of the inflammatory reaction during lipolysis of AT in dairy cows during the postpartum transition period are currently poorly understood, and the current results are inconclusive (Salcedo-Tacuma et al., 2020).
Our study was designed to test 2 hypotheses.First, we hypothesized that HS during early lactation in a temperate climate would be associated with a decrease in productive performance, fertility, and biochemical blood indices in dairy cows.Second, we hypothesized that adipokines such as LEP and ADP might play a regulatory role in the immune response during lipolysis after calving in dairy cows.Therefore, the first aim was to evaluate whether HS during early lactation in a temperate climate affected variation in productive performance, fertility, and biochemical blood indices in dairy cows.The second aim was to investigate the role of LEP and ADP in the regulation of the immune response accompanying lipolysis after calving in dairy cows.

Ethics approval
All procedures for the study were conducted in compliance with the guidelines of the Polish Council for Animal Care (Act on the Protection of Animals Used for Scientific Purpose in Poland) and the EU directive (no.2010/63/EU) for the protection of animals used for scientific purposes (European Commission, 2010).These practices are standard for animal health assessment and monitoring; in particular, blood samples were collected during standard veterinary activities, and all procedures were approved by the appropriate authorities.

Farms and animal management
For this prospective cohort study, data were collected from 5 commercial farms between June to September 2018.The selected farms had more than 80 lactating cows (85-110, with an average of 93) and a milk yield of the previous lactation greater than 9,500 kg (9,987-10,640 kg, with an average of 10,173 kg).The study included 100 clinically healthy Polish Holstein-Friesian breed (20 cows from each farm) dairy cows selected according to parity (n = 41 primiparous and n = 59 multiparous) and their 305 d milk yield was more than 9,000 kg (9,252-10,800 kg, with an average of 10,055 kg).From a total of 100 Polish Holstein-Friesian breeds, 6 cows were excluded after calving (up to 60 d) due to health complications/disease (n = 4) and mastitis (n = 2), but data obtained for these cows that were dropped during the lactation period were included in the analysis until the time they were eliminated from the study.The cows were kept under normal dairy farm management in free-stall housing systems with naturally ventilated barns bedded with chopped straw during lactation.The cows were dried off once weekly approximately 56 (±3) d before the expected calving date.The prospective cohort recorded data including the early lactation period from the calving day until 150 d in milk (DIM).The cows were kept in a similar ambient photoperiod, including approximately 14 h of natural light and 10 h of dark, and provided with a light intensity of approximately 230 lx during the night while being kept on the farm from 2000 to 0600 h.
Representative samples of silages, such as corn, grass, and alfalfa were collected monthly and analyzed using the near-infrared reflectance spectroscopy method according to PN-EN 12099:2017-10 with a FOSS 6500 (Foss Electric, Hillerod, Denmark) for dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber, ether extract, and starch.The nutritional value of the feed components was calculated based on the analyzed content of nutrients using the AMTS.Cattle.Pro version 4.7 (2017, AMTS LLC, Groton, NY).The cows were fed a total mixed ration (TMR) diet twice daily at 0600 and 1400 and delivered ad libitum.Orts were removed daily before the morning feeding.The diets were formulated monthly to meet the nutritional requirements according to National Research Council guidelines (NRC, 2001) and were based on ensiled forages such as corn, grass, alfalfa, ensiled high-moisture corn grain, sugar beet pulps, grains, rapeseed, soybean meals, and mineral-vitamin premix.The cows were fed an early FRESH lactational diet from the calving day up to 21 DIM, with an average feed intake of 24 kg dry matter intake (DMI) and 35 kg milk yield.Next, the cows received TMR I from 22 to 150 DIM, formulated to meet the nutritional requirements of dairy cows consuming 28 kg DMI and producing 42 kg of milk.The early lactation FRESH diet contained a range of  mEq/kg dietary cation-anion balance (DCAB) on a DM basis.The TMR I diet contained a range of 41.7-55.8%forage, 15.9-17.5% CP, 28.1-31.5% NDF, 25.5-28.4% starch, and 286-320 mEq/kg DCAB on a DM basis.The cows had ad libitum access to fresh water throughout the study, which was available in self-filling troughs.Body condition score (1 = thin to 5 = fat; Edmonson et al. ., 1989) was recorded at 3 weeks after calving (on d 21 ± 3).

Environmental data collection
Microclimate barn indices were collected during the study, which included the temperature (T, °C) and relative humidity (RH, %).These were recorded at 15min intervals using data loggers (WatchDog A-Series, SpecWare 9 Basic, Spectrim Technologies Inc., Aurora, USA; temperature range −45-85°C and accuracy ± 0.6°C; humidity range 0-100% and accuracy ± 3%), placed inside the building at a height of 3 m above the cow area, as described by Lambertz et al. (2014) and Hut et al. (2022).The temperature-humidity index (THI) was calculated according to the National Research Council recommendation (NRC, 1971), using The cows were classified based on their THI exposure during the early lactation period, and daily THI values were calculated on the calving day (0 d), +7, +14, and +21 d, and divided into 3 categories: THI <68 for the thermal comfort zone, 68-72 for mild HS, and THI >72 for HS in dairy cows.This formula and THI categories were selected because they have been used previously in dairy cow trials conducted in a temperate climate (Hammami et al., 2013).

Fertility performance
Cows were observed weekly from 28 d (±3) after calving using a color Doppler ultrasound scanner (SSD-5500, Aloka Co., Japan) equipped with a 7.5 MHz convex transducer (UST-995-7.5, Aloka Co., Japan) by a veterinarian.Reproductive performance was evaluated based on the first postpartum estrus (number of days from calving to the first estrus), calving interval (number of days from calving to the first service), the period of insemination service (number of days from the first artificially inseminated (AI) service to conception), services per conception (number of AI services required for conception), days open (number of days from calving to the next fertilization), and inter-calving period (number of days from one calving to the next calving).

Blood sampling and analyses
Blood samples were collected from each cow at 3 weeks after calving (on d 21 ± 3) during standard veterinary activities, 4 ± 0.5 h after morning feeding, from the tail vein into a blank 10 mL vacutainer for serum (KABE, Poznan, Poland).The vacutainers with samples were then transported to the laboratory in a refrigerated vehicle.Subsequently, the blood samples were centrifuged at 3,000 × g for 15 min at 4°C, and the serum was separated and stored at -20°C until analyzed.The serum was analyzed in duplicate using a microplate spectrophotometer (Synergy 2, BioTek Biokom) for the determination of the concentration of biochemical blood indices (Table 1).The interassay and intra-assay variation was controlled by limiting the coefficient of variation to ≤ 5% for all blood variables.

Statistical analysis
The data were checked for normality using PROC UNIVARIATE in SAS (version 9.4, SAS Institute, 2014) before analysis.To analyze fertility parameters such as service per conception and AI period service, a logistic transformation function was applied.The explanatory variables, THI during early lactation (0, +7, +14, and +21 d), were divided into 3 categories, and this classification was consistent across the analysis of productive performance, fertility, and biochemical blood indices.The effect of HS defined by THI during early lactation on productive performance, fertility, and biochemical blood indices was determined using the MIXED procedure of SAS under the models described in Equations.The best covariance structure was determined using the lowest Bayesian information criterion fit statistic level, and the autoregressive variance-covariance structure was selected accordingly.The statistical model used to analyze productive performance was Y ijklmn = μ + L i + H j + B k + β 1 afc l + β 2 dl m + e ijklmn , where Y ijklmn is the value of the dependent variable; μ is the overall mean; L i is the fixed effect of parity of dam (i = 1, 2); H j is the fixed effect of the farm (j = 1, 2, 3, 4, 5); B k is the explanatory variables (k = effect of the category of THI on the 0, +7, +14, and +21 d during early lactation; listed in Table 2); β 1 and β 2 are the The same statistical model was used to analyze the fertility indices but without the second partial regression coefficient (β 2 d lm ).The biochemical blood indices were analyzed using the following model: Y ijklmn = μ + L i + H j + B k + β 1 afc l + β 2 d sm + e ijklmn , where Y ijklmn is the value of the dependent variable; μ is the overall mean; L i is the fixed effect of parity of dam (i = 1, 2); H j is the fixed effect of the farm (j = 1, 2, 3, 4, 5); B k is the explanatory variables (k = effect of the category of THI on the 0, +7, +14, and +21 d during early lactation); β 1 and β 2 are the partial linear regression coefficients; afc l is age at first calving; d sm is day of blood sampling; and e ijklmn is the random error.Individual comparisons between the explanatory variables (category of THI on the 0, +7, +14, and +21 d during early lactation) were conducted using Duncan's adjustment in cases of significance.Statistical significance was determined at P ≤ 0.05, while trends were denoted by 0.05 < P ≤ 0.1.
The Pearson correlation coefficients were calculated using the PROC CORR procedure in SAS.

RESULTS
In the present study, a negative correlation was found between THI on the calving day (0 d), +7 d (P ≤ 0.05), +14 and +21 d (P ≤ 0.01) during early lactation and reproductive performance, including first estrus, calving interval, days open, and inter-calving period (Table 3).We also observed a negative correlation between THI and lactation performance, especially milk yield (P ≤ 0.01) during early lactation up to +21 d.Also, was found that THI was positively correlated with the percentages of milk fat (P ≤ 0.01), SCC, and MU (P ≤ 0.05), and negative with the percentages of protein (P ≤ 0.05) from the calving day up to +14 d.Furthermore, THI from the calving day up to +14 d showed a negative correlation with saturated fatty acids (SFA) (P ≤ 0.01), short-chain fatty acids (SCFA) (P ≤ 0.05), The fertility and lactation performance of dairy cows were found to be associated with heat stress experienced during early lactation, as shown in Table 4.An increasing THI value from the calving day through +7, +14, and +21 d during early lactation was associated with the later manifestation of first postpartum estrus and an extended calving interval, by approximately 29 d (P ≤ 0.01), 27 d (P ≤ 0.01), 25 d (P ≤ 0.05), and 16 d (P ≤ 0.05), respectively.Additionally, an increasing THI value on the calving day, +7 d (P ≤ 0.01), +14, and +21 d (P ≤ 0.05) was associated with deteriorated fertility indices through an elongated AI service period, days open, and inter-calving period.The increasing value of THI on the calving day was associated with a higher number of services per conception (P ≤ 0.01) by about 1.30.
In this study, we also observed that the THI value from the calving day up to +14 d postpartum was linearly associated with a decrease in body condition score (BCS) on the 21 d after calving (P ≤ 0.05).Lactation performances of dairy cows were associated with increasing THI values during early lactation, resulting in a decrease in milk yield (P ≤ 0.01).With an increase in the THI from the calving day up to +21 d during early lactation, we recorded decreased milk yield by approximately 3.20, 4.10, 5.60, and 5.60 kg, respectively.We also noted that the decrease of 3.5% FCM, 3.5% FPCM, and ECM was associated with an increase of THI on +14 d (P ≤ 0.05) and +21 d (P ≤ 0.01).Conversely, decreasing THI from the calving day up to +14 d during early lactation was associated with lower milk fat and higher protein contents (P ≤ 0.01 and P ≤ 0.05, respectively).Furthermore, an increasing value of THI on the calving day, +7, and +14 d were associated with an increase in SCC and MU (P ≤ 0.05).
The milk fatty acid groups were also found to be associated with the THI during early lactation of dairy cows, as shown in Table 5.The higher levels of SFA (P ≤ 0.01) and LCFA (P ≤ 0.05) and lower levels of UFA (P ≤ 0.01), SCFA (P ≤ 0.05), and MCFA (P ≤ 0.05) contents were associated with increasing THI values on the calving day, +7, and +14 d during early lactation.
The current study found that the biochemical blood indices of dairy cows were associated with various parameters including THI during early lactation (Table 6).Increasing THI values from the calving day through +7 and +14 d during early lactation were associated with higher concentrations of TG (P ≤ 0.01), NEFA (P ≤ 0.05), BHBA (P ≤ 0.05), LEP (P ≤ 0.05), BUN (P ≤ 0.05), and TNF-α (P ≤ 0.05), as well as lower concentrations of IGF-I (P ≤ 0.01), ADP (P ≤ 0.05), FSH (P ≤ 0.05), and LH (P ≤ 0.05) in the blood.Additionally, increasing THI values on +7 and +14 d were associated with lower concentrations of glucose (P ≤ 0.01) and higher concentrations of insulin (P ≤ 0.05), cytokines (IL-1 and IL-6; P ≤ 0.05), acute phase proteins (SAA and Hpt; P ≤ 0.05), and heat shock protein (HSP70; P ≤ 0.05).No association was found between THI values on the calving day, through +7, and +14 d during early lactation and concentrations of hepatic enzymes such as AST and ALT, cholesterol and its fractions (LDL and HDL), and total serum protein and albumin (P > 0.05).

DISCUSSION
Considerable evidence exists in the literature regarding the potential effects of heat stress during early lactation on postpartum disease incidence (Menta et al., 2022), fertility (Gernand et al., 2019), and productive performance (Gunn et al., 2019), all of which have economic consequences for the dairy industry and can cause harm to animal welfare (Polsky et al., 2017).Furthermore, most of the studies inducing heat stress were conducted in climate chambers (Hou et al., 2021) or using electric heat blankets (Al-Qaisi et al., 2019), and were associated with different cooling and fan systems (Perano et al., 2015;Kim et al., 2022), as well as the effectiveness of the feed additives and feeding solutions (Coleman et al., 2022;Danesh Mesgaran et al., 2022;Ruiz González et al., 2023) used to prevent negative consequences of heat stress occurrence.However, currently, there is a need to explain the adapta-    Means within the row with different letters differed significantly (P ≤ 0.05).

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tion mechanisms during the still little-known transition period, particularly during heat stress occurrence.To our best knowledge, limited results are associated with determining the role of LEP and ADP in regulating the immune response during lipolysis after calving in dairy cows.
The current study included prospective cohort data and the HS occurrence model was similar to previous studies (Lambertz et al., 2014;Hut et al., 2022).The cows were classified based on their THI exposure during the early lactation period and divided into 3 categories because they were used previously in dairy cow trials conducted in a temperate climate (Hammami et al., 2013).The 3 categories from thermoneutral comfort zone (THI < 68), through mid-HS (THI 68-72) up to heat stress (THI > 72) allowed observed productive and physiological changes in early lactation dairy cows.Also, our HS model included the data collected only during one summer season and cows were kept in a similar ambient photoperiod.Consequently, the observed association in performance and other variables is expected to be influenced by the heat challenge alone.On the other hand, we performed a prospective cohort study on 5 commercial dairy cow farms, therefore due to the character of a field study, individual DMI, body weight, and its changes were not possible to measure, which is a limitation and may affect the results and might possibly have a masking effect.Also, the blood samples were collected one time at 3 weeks after calving during standard veterinary activities, and was not possible a collect samples at repeated times.However, the observed association of physiological changes is for us as an observational study, and reason for further precisely research in controlled conditions, especially in the context of the need to explain the adaptation mechanisms during the still little-known transition period and limited results associated with determining the role of LEP and ADP in regulating the immune response during lipolysis after calving.
In the current study, it was observed that an increase in THI values from the calving day (0 d) through +7, +14, and +21 d during early lactation was associated with a later manifested first estrus postpartum and elongated calving interval, by approximately 29, 27, 25, and 16 d, respectively.Furthermore, the fertility indices were negatively affected by the increase in THI, which led to an elongated AI service period, days open, and inter-calving period.Additionally, an increase in THI on the calving day was associated with a higher number of services per conception, approximately 1.30.The decrease in fertility parameters during heat stress occurrence might be associated with a reduction in blood concentrations of key metabolic hormones and growth factors required for normal follicular development to suboptimal levels (De Rensis et al., 2017).Studies have shown that the concentrations of IGF-I and glucose were lower in the summer compared with winter months, during the postpartum period (De Rensis et al., 2002).Similarly, Roth (2020) found that HS leads to a lower concentration of LH and FSH, which are associated with oocyte development.In the current study, increasing THI values during early lactation were associated with lower concentrations of IGF-I, glucose, LH, and FSH, which were below the reference values.The components of IGF-I are predominantly found in the liver under the influence of growth hormone but are also found in reproductive tissues, where they serve several roles, including positive effects on embryo development (Thatcher et al., 2003).The serum IGF-I concentration in the periparturient period and at the time of insemination has been described as a useful predictor of reproductive performance in dairy cattle (Wiltbank et al., 2006).Similarly to our results, Aungier et al. (2014) found that cows with a serum IGF-I of < 40 ng/mL during the first week after calving were less likely to conceive after the first service.A lesser concentration of IGF-I in the postpartum period was also associated with an elongated calving interval.Both IGF-I and glucose play a stimulatory role in follicular growth and implantation, with glucose serving as the primary metabolic fuel for the ovary.Glucose is directly involved in modulating pulsatile LH secretion at a hypothalamic level, and insulin is required for the normal development of follicles and has beneficial effects on oocyte quality (Bucholtz et al., 1996).Cows exposed to HS have impaired endocrine pathways that alter   Means within the row with different letters differed significantly (P ≤ 0.05).

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the production and secretion of ovarian hormones such as estradiol and inhibin (Wolfenson and Roth, 2019), which affects follicular dominance.HS alters the secretion of LH and progesterone (Wolfenson et al., 2002), which can impair the final maturation and ovulation of the preovulatory follicle.As a consequence, hypothermia and heat stress negatively affect follicle growth, oocyte quality, early embryo development (Schuller et al., 2017), uterine function (Kawano et al., 2022), the duration of estrus (De Rensis et al., 2017), and estrus behavior in dairy cows (Wolfenson and Roth, 2019).Cows exposed to HS 3-5 weeks before and 1 week after the day of service had decreased conception rates (Morton et al., 2007).Schuller et al. (2017) found that exposure to HS 3 weeks before the day of service can negatively affect conception rates.The development of the preovulatory follicle begins months before ovulation, and insults such as HS (Torres-Junior et al., 2008) or diseases (Ribeiro et al., 2016) can have long-lasting effects on reproduction.Torres-Junior et al. (2008) reported that oocytes can be damaged by HS as early as 105 d before ovulation, reducing the quality of the morula and conceptus and inducing damage that carries on during later stages of pregnancy.Therefore, according to the results of the current study, it is fully justified to change herd management and implement cooling measures for cows during early lactation to reduce the negative effects of heat stress on the antral follicle containing the oocyte and improve fertility performance in dairy cattle.
In the current study, an increase from < 68 to > 72 in THI from the calving day (0 d) through +7, +14, and up to +21 d during early lactation was associated with a decreasing daily milk yield by approximately 3.20, 4.10, 5.60, and 5.60 kg, respectively, and a deterioration of 3.5% FCM, 3.5% FPCM, and ECM when THI was > 72 on +14 and +21 d.Similarly, other studies have reported a reduction in milk production related to an increase in THI.A THI of 68 is often used as an indicator of heat stress in both lactating (Zimbelman et al., 2009;Collier et al., 2012) and dry cows (Ferreira et al., 2016;Fabris et al., 2019).This threshold was retrieved from a series of 8 studies where the milk yield of 100 multiparous high-producing Holstein cows decreased by 2.2 kg/d for each 24 h at a daily THI of 68.Also, Ranjitkar et al. (2020) found that exposing cows to a THI higher than 68 affected losses of milk yield from 0.7 to 4.0 kg/cow/d, and these losses were estimated to further increase from 1.50 to 6.50 kg in 2050 and from 2.0 to 7.50 kg in 2070.West (2003) reported that mean THI had a greater negative correlation with milk production, and for each unit increase of THI higher than 68, cows decreased milk production by 0.88 kg.Gao et al. (2017) found that cows under heat stress had reduced milk yield by 17%, milk protein by 4.1%, and 3.5% FCM by 23% compared with pair-fed thermoneutral cows.Baumgard and Rhoads (2013) explained that the decrease in milk yield is associated with decreased dry matter intake and reduced nutrient supply, accounting for only 50% of the decreased milk yield, with the remaining portion due to changes in nutrient partitioning, metabolism, and immune activation.Also, Ruiz-González et al., (2023) observed that 67% of the decrease in both actual milk yield and ECM yield, and 57, 61, and 65% of the observed reduction in fat, protein, and lactose yields respectively was explained by reduced DMI, which suggests nutrient use in tissues other than the mammary gland and decreased production was likely associated with HS effects on nutrient partitioning.In the current study, HS occurrence affected the decreasing milk production and also was associated with decreasing serum glucose concentration, promotion of lipolysis, and inflammation response.Inflammation is a multifaced biological process encompassing various physiological and pathological responses aimed at eliminating harmful stimuli and facilitating tissue heading (Medzhitov, 2010).Around calving, inflammation is commonly observed in nearly all cows, triggered by nonpathogenic factors such as tissue remodeling, as well as pathogenic factors like bacterial infections (Sheldon et al., 2019).Results showed by Kvidera et al., (2017a) hyperactivated immune system of lactating dairy cows can use the increased amount of glucose (>1 kg over a 12-h period), resulting in its availability for milk production.On the other hand, inflammatory dysfunction has the potential to impact the metabolic status of dairy cows through the promotion of lipolysis (Abuelo et al., 2019).In the current study, individual DMI was not measured due to the character of a field study, which is a limitation and may affect the results and might possibly have a masking effect.However, in line with earlier results presented by Baumgard and Rhoads (2013) and Ruiz-González et al., (2023), where in response to heat challenge, the observed decreased milk production and its chemical composition might be associated with induction of increased nutrient use by the immune system.Furthermore, the stage of lactation also plays an important role in the severity of heat stress and the amount of milk lost (Tao et al., 2018).Up to 60 d postpartum, cows are in negative energy balance, and to make up for the excess energy loss, body stores are mobilized.Due to increased metabolic heat, the first 60 DIM and the peak of lactation are critical for managing heat stress to minimize the effect on milk production (Becker et al., 2020).Additionally, postpartum cows exposed to HS have a reduced number of mammary epithelial cells and display a greater risk Stefanska et al.: HEAT STRESS DURING EARLY LACTATION AND COW PERFORMANCE of programmed cell death, resulting in reduced milk yield (Collier et al., 2006).
In the current study, it was found that HS occurrence during early lactation not only was associated with a decrease in milk yield but also had an effect on the chemical composition of milk.Increasing THI values from the calving day through +7 and +14 d during early lactation were associated with lower milk protein content and higher milk fat percentage, SCC, and MU.We noted also that higher SFA and LCFA, and lower UFA, SCFA, and MCFA contents were associated with increasing THI values on the calving day, +7, and +14 d during early lactation.The exact reason why HS decreases milk protein content is not clear, but it negatively affects the transcriptome of milk protein genes in mammary tissue (Gao et al., 2019), and an increase in extramammary amino acid (AA) utilization, especially 3 essential AAs, such as Ile, Val, and Met used for the synthesis of milk protein (Gao et al., 2017), and downregulation of mammary protein synthesizing are probable explanations (Cowley et al., 2015).The occurrence of HS causes an increase in BUN and MU, which has been previously reported (Wheelock et al., 2010;Cowley et al., 2015) and is likely due to skeletal muscle mobilization to support processes like gluconeogenesis and acute phase protein synthesis.This study also found an increase in milk fat content and changing its chemical composition through higher SFA and LCFA, and lower UFA, SCFA, and MCFA contents, which can be attributed to a decrease in BCS, excessive adipose mobilization, and increasing circulating NEFA associated with NEB occurrence during HS in early lactation dairy cows.Milk fat can either originate from blood circulation or from de novo synthesis in the mammary gland (Rico and Razzaghi, 2023).During early lactation, approximately 40% of NEFA is used as a source of fatty acids in milk triacylglycerols (Bell, 1995), therefore, changes in AT mobilization may substantially impact milk fat synthesis.According to Miller et al. (1991) about 56% of the variation in mammary uptake of NEFA is explained by their concentrations in arterial blood.These observations explain why performed fatty acids (i.e., fatty acids >16 carbons) typically found in AT (Rukkwamsuk et al., 2000) are increased when NEFA is increased in early lactation (Mann et al., 2015;Henno et al., 2021).In support of this, NEFA is positively correlated with milk fat concentration (r = 0.76; Pullen et al., 1989).Mann et al., (2015 and2016a) reported that cows with higher concentrations of NEFA exhibit also higher concentrations of LCFA in milk, suggesting an important role of AT mobilization in the availability of substrates for milk fat synthesis, similar to previous reports (Miller et al., 1991;Nielsen and Jakobsen, 1994).Similar to our and Mann et al., (2015and 2016a) results, Hammami et al. (2015) reported that milk fat samples collected during HS tended to have higher proportions of LCFA but lower proportions of SCFA and MCFA compared with those collected during temperate conditions.Milk fat content is often unchanged (Shwartz et al., 2009) or slightly increased under experimentally induced HS (Rhoads et al., 2009), whereas, although not always reported, milk fat yield has been consistently shown to be reduced (Knapp and Grummer, 1991;Ruiz González et al., 2023), showing a negative effect on mammary fat synthesis.Knapp and Grummer (1991) observed a 19% reduction in milk fat yield when cows were exposed to HS over a 15-d period.However, this effect was likely a combination of reduced DMI and HS-related alteration in nutrient partitioning, as this experiment did not include dairy cows in thermoneutral conditions.Also, Ruiz González et al., (2023) presented a 10% reduction in milk fat yield in 14-d period HS cows as a response observed concomitantly with a 58% increase in insulin and a 64% reduction in NEFA concentrations, suggesting that reduced availability of LCFA may have limited mammary fat synthesis to some extent.These authors explained that lower NEFA could be a consequence of reduced adipose tissue lipolysis in response to increased insulin concentration in the HS dairy cows group.On the other hand, the above results are in contrast to the current study where HS occurrence was associated with decreasing BCS, higher milk fat and LCFA contents, and also higher NEFA and insulin concentrations, which in our hypothesis might be associated with reduced insulin sensitivity and the development of insulin resistance in AT during early lactation.Similarly, Wu et al., (2020) found that over-conditioned dairy cows during early lactation are prone to greater insulin resistance associated with higher NEFA and insulin concentration to adapt to negative energy balance and greater lipolysis in transition.On the other hand, a recent study by Chirivi et al. (2022) reported the development of insulin resistance in AT during a lipopolysaccharide (LPS) challenge, thus enhancing cow's susceptibility to lipolysis.Therefore, importantly, a better understanding of the potential role of endotoxemia on AT metabolism may be relevant under commercial conditions, where infectious diseases such as mastitis and metritis result in endotoxemia (Suojala et al., 2013;Magata et al., 2015).Also, the duration of the HS challenge can influence the lipolytic response of AT as recently shown by Hou et al. (2021), who reported lower NEFA in cows subjected to a 7-d relative to a 3-d heat stress challenge, and no differences in insulin concentration between both groups.
Previous studies similar to ours have also shown an increase in SCC during HS, although the mechanism  2023) HS concurrence was associated with a higher concentration of markers of endotoxemia and immune system activation, as well as increased milk SCC.Interestingly, SCC is known to increase under scenarios of immune activation, such as LPS challenge and leaky gut (Kvidera et al., 2017 a, b).In cattle, HS has been found to affect the integrity of the intestinal barrier, which allows the passage of LPS to the circulatory system to cause the leaky gut condition, and, thus, stimulates the production of proinflammatory cytokines (Lian et al., 2020;Patra and Kar, 2021;Fontoura et al., 2022).Also, the inflammatory state might be due to the increased release of HSP and the HS-induced leaky gut (Koch et al., 2019), which stimulates to generate a proinflammatory response in cells expressing TLR4 and CD14, and proinflammatory cytokines such as IL-1, IL-6, and TNF-α.Indeed, as already mentioned, HSP70 can activate the TLR4 of dendritic cells (Archana et al., 2017;Bagath et al., 2019) and a variety of HSP, including HSP70, which are upregulated during HS (Mishra, 2021).Our results also showed an increase in HSP70 levels with an increasing THI on d 7 and 14.This finding is consistent with the fact that HSP70 is one of the most recognized cellular responses to hyperthermia and exerts cytoprotective effects on stressed cells, exhibiting antiapoptotic properties (Lanneau et al., 2007).Increasing concentrations of HSP70 protects cells and organisms by preventing protein degradation and repairing unstable proteins against heat stress.
In the current study, we found that as the THI value increased from the calving day through +7 and +14 d during early lactation, there was an increase in NEFA and BHBA concentrations.This increase was also accompanied by an increase in the concentrations of insulin, LEP, acute phase proteins such as SAA and Hpt, cytokines such as TNF-α, IL-1, and IL-6, and a decrease in ADP concentration.One possible explanation for this response is that during NEB occurrence after calving, AT reserves are mobilized to provide NEFA as energy sources.The excess of acetyl CoA that could not enter the TCA cycle caused a shift in the pathway toward ketone body production, such as BHBA (Kuhla, 2020).The occurrence of HS during early lactation might further deepen the NEB and lipolysis of AT.Besides its metabolic function, AT is an active endocrine organ that secretes a range of adipokines, cytokines, and acute-phase proteins that regulate energy metabolism and inflammation.The deterioration of BCS in dairy cows after calving, which is associated with NEB, is accompanied by higher concentrations of LEP and a greater lipolytic capacity, resulting in higher NEFA concentration (Kuhla et al., 2016).Similarly, Sadri et al. (2011) observed an increase in LEP concentrations with the greatest NEFA concentrations in the blood of dairy cows 1 week after parturition, due to either NEB or increased turnover rate during lactation.At the same time, ADP secreted by AT is drastically reduced after calving and then steadily increases to a peak at around 60 DIM (Ohtani et al., 2012).Remarkably, the content of circulating ADP is inversely associated with NEFA, as reported by Kabara et al. (2014).ADP plays a crucial role in regulating energy metabolism by promoting insulin sensitivity and adipogenesis while inhibiting lipogenesis in adipocytes, and NEFA β-oxidation in monocytes and hepatocytes, according to Stern et al. (2016).Increased concentrations of this adipokine during the transition period are speculated to enhance glucose partitioning in the mammary gland, as suggested by Giesy et al. (2012).Lipolysis triggers a remodeling process within the AT that results in an inflammatory response, leading to changes in immune cell trafficking, the proliferation of specific cell types, and rearrangements of the extracellular matrix (Contreras et al., 2017).The intense lipolysis of AT generates local inflammatory responses, participating in the regulation of the innate immune system during the transition period.Trevisi et al. (2012) reported higher serum TNF-α, IL-1, and IL-6 concentrations in cows with greater body fat mobilization, as indicated by higher plasma NEFA concentrations during the transition period after calving.Mann et al. (2016b) also found that the AT's inflammatory process associated with lipolysis during the transition period focused on the upregulated synthesis of proinflammatory cytokines, such as TNF-α, IL-1, and IL-6, reflected in high concentrations of NEFA.On the other hand, markers of later inflammatory stages, primarily positive acutephase proteins such as Hpt and SAA, accumulate in early lactation, with their serum concentration changing by > 25% in response to inflammatory cytokines such as IL-1, IL-6, and TNF-α.They are also potential biomarkers for immune response during heat stress in ruminants (Hamzaoui et al., 2013).Additionally, in line with the above-discussed findings, IGF-I concentration during HS occurrence in the current study shows an antagonistic relationship with proinflammatory cytokines and plasma IGF-I, as found by Kasimanickam et al. (2013).Serum IGF-I may antagonize proinflammatory activity by decreasing expression of the IL receptors and via suppression of cytokine-signaling proteins.Several large epidemiological studies demonstrate that excessive lipolysis is associated with NEB and a higher incidence and prevalence of disease in the transition period of dairy cows (Contreras et al., 2017).Furthermore, the mechanisms linking HS occurrence during the transition period after calving and lipolysis are not entirely understood, but several studies, including ours,

CONCLUSIONS
The results of this study indicate that the THI from the calving day, through +7, +14, and +21 d during early lactation had a negative impact on the fertility parameters of dairy cows.Specifically, increasing THI values were associated with a delay in the first estrus postpartum and an elongated calving interval both by about respectively, 29, 27, 25, and 16 d.Additionally, the increasing value of THI during early lactation was linked to the deterioration of fertility indices through an elongated AI service period, days open, and inter-calving period.Furthermore, increasing THI values during early lactation were also associated with decreased milk yield by approximately 3.20, 4.10, 5.60, and 5.60 kg, respectively, from the calving day up to +21 d.The study also found that HS occurrence during early lactation led to deteriorated BCS of dairy cows after calving, accompanied by a higher concentration of LEP and a greater lipolytic capacity resulting in higher NEFA and BHBA concentrations.Additionally, lipolysis triggered a remodeling process within the AT, which led to drastically reducing adiponectin levels and increased biochemical blood indices associated with immune responses such as cytokines (TNF-α, IL-1, and IL-6), acute phase proteins (SAA and Hpt), and heat shock protein (HSP70) after calving.These results suggest that exposing dairy cows to HS during early lactation had negative consequences on productive performance, fertility, and biochemical blood indices in the subsequent lactation.Therefore, it is necessary to implement management changes on the farm to mitigate the negative impact of HS occurrence during early lactation.However, the observed association of the biological indices changes is for us as an observational study, and reason for further precise research in controlled condition trials.

Figure 1 .
Figure 1.Model of the relationship between nonesterified fatty acids (NEFA) and immunometabolic blood indices during heat stress occurs in the early lactation of dairy cows.* significant correlations P ≤ 0.05, ** significant correlations P ≤ 0.01 = temperature and humidity index calculated on the calving day; THI +7 d = temperature and humidity index calculated on the 7 d after calving; THI +14 d = temperature and humidity index calculated on the 14 d after calving.a,b,c Stefanska et al.: HEAT STRESS DURING EARLY LACTATION AND COW PERFORMANCEsupport the deleterious effect of high rates of lipolysis on both innate and adaptive immune responses.
Stefanska et al.: HEAT STRESS DURING EARLY LACTATION AND COW PERFORMANCE Stefanska et al.: HEAT STRESS DURING EARLY LACTATION AND COW PERFORMANCE partial linear regression coefficients; afc l is the age at first calving; d lm is DIM; and e ijklmn is the random error.

Table 1 .
Stefanska et al.:HEAT STRESS DURING EARLY LACTATION AND COW PERFORMANCE Specification of bovine kits used for determining biochemical blood indices

Table 2 .
Stefanska et al.: HEAT STRESS DURING EARLY LACTATION AND COW PERFORMANCE Descriptive statistics of evaluated parameters (explanatory variables) during early lactation of dairy cows

Table 3 .
Pearson correlation coefficients (r) between heat stress occurs during early lactation and fertility indices 1

Table 4 .
The effect of heat stress occurs during early lactation on fertility indices and lactation performance of dairy cows Item

Table 5 .
The effect of the heat stress occurs during early lactation on milk fatty acids composition of dairy cows Item

Table 6 .
The effect of the heat stress occurs during early lactation on biochemical blood indices of dairy cows Item Stefanska et al.: HEAT STRESS DURING EARLY LACTATION AND COW PERFORMANCE involved is not yet clear.According to Ruiz-González et al. (