Effect of Short Duration Heat Stress on the Physiological and Production Parameters of Holstein-Friesian Crossbred Dairy Cows in Bangladesh

Abstract

Heat stress is a major concern for lactating dairy cows. This study evaluated the effects of heat stress on six Holstein-Friesian crossbred dairy cows exposed to three thermal conditions represented by the Temperature-Humidity Index (THI). These conditions included a baseline pre-treatment phase at THI-72, a heat stress treatment phase at THI-75 and THI-80, and a post-treatment recovery phase at THI-72. The duration of the heat stress treatment phase was 24 h. A total of four trials, each involving three cows, were conducted in an IoT-based climatic chamber to assess various physiological, hematological, biochemical, and production parameters across these phases. Compared to the baseline (THI-72), cows showed significant increases (p < 0.05) in rectal temperature (RT), heart rate (HR), respiration rate (RR), and water intake (WI) at both THI-75 and THI-80, with the highest elevations observed at THI-80 (RT: 5.1%, HR: 8.6%, RR: 23.5%, and WI: 19.1%). Feed intake declined significantly (p < 0.05) by 6.5% and 14.0%, and milk yield dropped by 5.3% and 14.7% at THI-75 and THI-80, respectively; milk fat and protein percentages decreased by 1.1-fold and 1.2-fold. Hemoglobin, platelet, and lymphocyte counts, along with biochemical parameters (excluding serum creatinine) also decreased significantly (p < 0.05). The different levels of THI influenced pairwise correlation patterns, with THI-75 showing intense interactions and THI-80 exhibiting greater variability. The findings highlight that Holstein-Friesian crossbred dairy cows are particularly vulnerable to heat stress, even with short-term exposure. This vulnerability can lead to economic losses for Bangladeshi dairy farmers rearing Holstein-Friesian crossbred cows.

1. Introduction

The livestock sector in Bangladesh is experiencing swift expansion, making it one of the rapidly growing industries. It has contributed over 1.8% to the country’s gross domestic product (GDP) and over 16.3% to the agricultural sector in the fiscal year 2023–2024 [1]. Bangladesh possesses an estimated 25.01 million cattle for the fiscal year 2023–2024 [1]. Among these, nearly 6.0 million are specifically dairy, with crossbred cows accounting for 10–15% and indigenous breeds constituting 85–90% [2]. Moreover, dairying in Bangladesh is vital for small- and medium-scale farmers, predominantly in rural, peri-urban, and even urban areas. A substantial proportion of Bangladesh’s dairy farming population, approximately 70%, consists of small-scale producers who rear 1–3 dairy cows, contributing about 70–80% of the nation’s total milk production [3]. Although indigenous breeds and exotic zebu cattle are frequently reared, there has been a growing trend of rearing Holstein-Friesian and Jersey crossbred dairy cows nowadays. According to a report generated by FAO [4], crossbred cows produce more milk (7.3 kg/cow/day) than that of local cattle (2.5 kg/cow/day). These crossbred cows follow a semi-intensive/stall-feeding-based production system characterized by zero grazing, purchased concentrate and rice straw (sometimes treated with urea and molasses), and green fodder. The country is projected to require around 15.67 million tons of milk yearly, but its production is estimated to be around 15.04 million tons during the fiscal year 2023–2024 [1]. Due to this rationale, the current quantity of milk accessible to each person is 234.5 mL per day, which falls below the required minimum daily intake of 250 mL [1]. Consequently, crossbred dairy farming is becoming more frequent nowadays to satisfy the growing demand for milk. Various climatic variables and management strategies greatly influence the productivity of dairy cows. Lactating cattle experience stress due to fluctuations in environmental factors such as temperature, humidity, wind velocity, and solar radiation. These disparities adversely affect the productivity of dairy cattle [3,5]. Heat stress is one of the major concerns among livestock owners across nearly every region worldwide. For each incremental rise in the Temperature-Humidity Index (THI), the milk output of Holstein cows dropped by 0.69 kg, while that of Jersey cows declined by 0.45 kg [6]. Heat stress occurs when animals generate and absorb more heat through metabolic activity and environmental exposure than they can dissipate through respiration, sweating, and convective cooling, leading to thermal imbalance [7,8]. Recent reviews stated that cows affected by heat stress exhibit several de novo physiological changes, such as elevated body temperature, respiratory rate (RR), heart rate (HR), rectal temperature (RT), sweating rate, panting, and drooling scores [9,10]. Consequently, cows undergo various physiological adaptations, including activation of their thermoregulatory mechanisms to mitigate heat stress [11]. The appetite center of the hypothalamus is directly and negatively affected by increased environmental temperatures, leading to a reduction in feed intake (FI) [6,12,13,14] as well as feed conversion efficiency [15,16]. The water intake (WI) is also reported to be significantly altered under heat stress conditions as reviewed by Giannone et al. [10] and Chen et al. [14]. In lactating cows, FI begins to decline at air temperatures of 25–26 °C, with a more rapid reduction observed at temperatures exceeding 30 °C in temperate climates. At 40 °C, FI may decrease by as much as 40% [17]. Reduced feed consumption and rising ambient temperature gradually lead to declining milk yield (MY) [12,18,19,20,21]. Elevated temperatures not only reduce MY but also impact milk composition and somatic cell count. Reviews of multiple research findings indicate a negative correlation between heat stress and milk fat (MF) and milk protein (MP) content; however, MP content is more significantly affected than MF content. Analysis of data from 34 studies suggests that MP is reduced by 3.9%, while MF decreases by only 0.1% [10,14]. Somatic cell count is considered an early indicator of heat stress. Milk obtained from heat-stressed cows contains a higher somatic cell score [22]. The hematological and biochemical parameters are also important indicators to recognize heat stress in dairy cattle [18,23]. Many studies have reported reductions in both White Blood Cell (WBC) and Red Blood Cell (RBC) counts in heat-stressed cows along with declines in biochemical parameters such as serum glucose (S.Glu), protein (S.Pro), cholesterol (S.Cho), creatinine (S.Cre), and triglycerides (S.Tg) in heat-stressed cows [23,24,25]. Hematological and biochemical parameters reported in these studies were analyzed using both manual techniques and automated blood and biochemistry analyzers. Methods employed for quantifying various blood metabolites included colorimetric assays, enzymatic methods, ion-selective electrode analysis, UV assays, and kinetic colorimetric techniques. Accurate measurement of heat stress indicators in particular physiological traits is essential for precisely assessing animal condition. A comprehensive review [8] on the measurement of physiological parameters reports that core body temperature can be assessed using infrared thermography or thermometers with thermistors, while RT is measured with a digital veterinary thermometer. Milk composition is analyzed using automatic milk analyzers, RR is determined by counting thoracoabdominal movements, HR is assessed through auscultation, and pulse rate is observed by monitoring pulsations in the middle coccygeal artery at the base of the tail. Additionally, several precision livestock farming tools have recently been developed, which can be applied in large-scale field studies and assist farmers in effectively managing heat stress in livestock [26].

Environmental parameters, animal-specific traits, or a combination of both are commonly used to assess the influence of climatic conditions on cattle. However, obtaining validated data on diverse microclimatic factors and accurately measuring physiological traits across large populations of animals is challenging, costly, and complex [8,27]. Additionally, considerable heterogeneity in assessing the effects of heat stress has been reported. These challenges could be even more complex when seasonal studies (heat stress effects in different seasons) are used to assess the impact of heat stress on dairy cattle as compared to chamber studies (climate chambers designed to simulate heat stress conditions). A recent meta-analysis [14] indicates that out of 31 studies conducted on Holstein cows across seven countries, only 6 focused on seasonal studies, while 25 were followed by chamber studies. Several seasonal heat stress studies on crossbred dairy cows in Bangladesh, India, and Sri Lanka demonstrated that increases in the THI negatively affect physiological and production parameters, including RT, RR, MY, and milk composition [3,28,29,30,31,32]. Therefore, the present study employed a climate-controlled chamber system to investigate the impact of heat stress on the physiological, biochemical, and production parameters of Holstein-Friesian crossbred cows in Bangladesh.

2. Materials and Methods

2.1. Ethical Approval

The animal study protocol was approved by the Animal Research Ethics Committee of Bangabandhu Sheikh Mujibur Rahman Agricultural University (Ref. No. FVMAS/AREC/2022/01).

2.2. Construction of Climatic Chamber

A climatic chamber with a dimension of 4.5 L × 4.5 W × 3.0 H (m) was constructed and equipped with an electric resistance air heater (with 10,000 watts of power), a hot/cold split air conditioner of 72,000 BTU h⁻¹, and an air humidifier with a capacity of 30 L and fog flow (mean value) of 10 L h⁻¹. The heater and humidifier were operated by an electronic controller and an SCD-30 (Seeed Technology Co., Ltd., Shenzhen, China) temperature and humidity sensor (temperature range: 10 to 70 °C, with a resolution of 0.01 °C; humidity range: 20 to 95% RH, with a resolution of 0.01% RH). The ventilation was applied using KDK ceiling mount fans, AC 250 V 0.2 A, with automatic activation, to maintain the O₂ and CO₂ levels at 21% and 1000 ppm, respectively. Sensors were used to monitor the O₂ and CO₂ levels in the climatic chamber. The data acquisition system of the climate chamber comprised a data logger and application software through the cloud.

2.3. Experimental Design and Treatments

A completely randomized design was used in this study. Dairy cows were kept under three distinct thermal conditions, expressed as the THI, which was calculated according to the formula proposed by Mader et al. [33]. The THI within the climate chamber was maintained at 72 for the first four days, with the initial three days designated as an adjustment period and the fourth day marked as the pre-treatment (PrTr) phase, serving as the baseline measurement. Moderate heat stress in dairy cattle is reported to begin at THI-72 [34]. Accordingly, heat stress at either THI-75 or THI-80 was applied on day 5, referred to as the treatment (Tr) period (simulated heat stress phase). From day 6 to day 8, the chamber’s thermal conditions were returned to THI-72 as part of the recovery phase, denoted as the post-treatment (PoTr) days. Thus, three distinct thermal conditions, represented by THI-72, THI-75, and THI-80 at their different periods, were examined in this study. The THI value was determined using the suggested formula, with the humidity consistently fixed at 60%, while the temperature was adjusted according to the desired conditions. The THI values used for the current heat stress study were selected based on the 30-year (1986–2015) THI trend in Bangladesh. The THI patterns were computed using 3-hourly temperature and humidity data collected from 34 meteorological stations throughout Bangladesh, employing the equation proposed by Mader et al. [33]. As shown in Figure 1. the highest average THI values were observed during the monsoon, ranging from 78.5 to 81.4. This value decreased to a range of 74.1 to 80.5 in the pre-monsoon and further dropped to 67.9 to 72.3 in the post-monsoon period. Therefore, in the present study, we examined the heat stress effect against these two THI values of 75 and 80. The experimental layout is illustrated in Figure 2.

2.4. Experimental Cows

Six multiparous 50% Holstein-Friesian crossbred dairy cows in their 3rd parity and 5 to 10 days post-calving were purchased from the local market. The experiment was conducted from October to November, 2023. At the start of the experiment, the cows had an average milk production of 6.4 ± 0.8 kg/day and were at 70.8 ± 3.5 days in their lactation stage. A diet comprising Napier grass, rice straw, and commercial concentrate feed in pellete form (Aftab Bahumukhi Farms Ltd., Dhaka, Bangladesh) was formulated according to NRC [35]. The feed ingredients and composition of the mixed diet are shown in Table 1. The average daily nutrient requirement of each cow was estimated to be 5698 gTDN and 1067 gCP. Each cow daily received 27 kg of a mixed diet on a fresh basis. The diet was offered four times at 9:00 h 13:00 h, 17:00 h, and 21:00 h. Milking was performed twice a day at 8:00 h and 16:00 h. A total of four trials were conducted, with each trial involving three cows. Two consecutive trials were performed under target stress conditions (THI-75 or 80), with the cows alternating between trials to maintain consistent exposure to the conditions.

2.5. Measurement of Physiological Parameters

2.5.1. Rectal Temperature

The RT of each cow was measured with a digital thermometer with an accuracy of ±0.1 °C (Thermalert TH-5, Physitemp Instruments Inc., Clifton, NJ, USA), twice daily (7:00 h and 19:00 h), by inserting the thermistor probe (ESO-1) into the rectum to a depth of around 2.2 cm, kept until reaching the stable temperature, which required 2 min/cow.

2.5.2. Respiration Rate

The RR was assessed twice daily (7:00 h and 19:00 h) by observing the flank area of each cow according to the published protocol [36], commencing with an exhale. Briefly, a stopwatch was employed to measure the time required to count 10 complete breaths. A complete breath consists of both an inhalation and an exhalation. The cow was considered to be exhaling when the flank appeared sunken, and inhaling when the flank appeared to be filled with air. The following equation was used to calculate breaths per minute: 10 ÷ number of seconds required for 10 complete breaths × 60. The RR was expressed in breaths/min. The time required for determining RR is less than 1 min/cow.

2.5.3. Heart Rate

The HR of the individual cow was assessed twice daily (7:00 h and 19:00 h) using a stethoscope and listening over the left-hand side of the cow’s chest behind the elbow for 1 min. The HR was expressed as beats per minute.

2.6. Determination of Feed and Water Intake

The FI and WI were determined using an intelligent feeder developed by the authors. The intelligent feeder consisted of a stainless bucket positioned on a weight balance that was connected to a controller. The real-time data collected by the controller were transmitted to the application software through a cloud database. During the experimental period, each cow received a specified diet divided into four meals per day. After each meal, the stainless steel bucket was cleaned and refilled with fresh water, with the same procedure repeated before each meal.

2.7. Recording Milk Production and Composition

The daily total MY for each cow was recorded after each milking session using a digital weighing scale. The milk composition, especially MF and MP percentages, was determined by an automated milking machine (Julie X, Scope Electric Instruments, Razgrad, Bulgaria).

2.8. Collection of Blood and Analysis

Blood was collected once daily at 19:00 h from the jugular vein of each cow. For analyzing hematology, the blood was collected in a heparinized tube to prevent clotting. Immediately after blood collection, hematological parameters, including RBC, WBC, Platelets (Plt), Hemoglobin (Hb), Neutrophils (Neu), and Lymphocytes (Lym), were determined during the experimental period using an automatic blood analyzer (3-part Hematology Analyzer with Standard Reagent, Medsinglong Co., Ltd., Jianxi, China). For biochemical analysis, the blood was collected in a clot activator tube and kept at room temperature for 20–30 min to clot. Then, serum was separated by centrifugation at 2000 rpm for 10 min and collected from the upper portion of the tube. Biochemical parameters, including S.Pro, S.Glu, S.Cho, S.Cre, and S.Tg were measured immediately after serum preparation using an automatic biochemistry analyzer (Medsinglong Co., Ltd., Jianxi, China) on a daily basis. Commercial clinical chemistry reagents (HUMAN Diagnostics, Wiesbaden, Germany) were used in accordance with the manufacturer’s instructions.

2.9. Statistical Analysis

The data generated in the study were inserted into an Excel sheet (Excel 2016, Microsoft, Redmond, WA, USA) and were analyzed with the add-in statistical package as per the requirement of the study. The results are expressed in mean ± SD. One-way analysis of variance (ANOVA) with Duncan’s Multiple Range Tests (DMRT) among means was carried out. All tests of significance were two-tailed, and p < 0.05 was considered significant. The correlation matrix among variables was determined in Excel and pairwise association was generated by a web-based server Heatmapper (http://www.heatmapper.ca/; accessed on 10 November 2024). The correlation thresholds used in this study were based on a review article [37] and categorized as follows: r > 0.6: strong positive, r > −0.6: strong negative, r = 0.4 to 0.6: moderate positive, r = −0.4 to −0.6: moderate negative, r < 0.4: weak positive, and r < −0.4: weak negative.

3. Results

3.1. Effect of THI-75 on Physiological and Production Parameters

The effects of heat stress (THI-75) on various physiological and production parameters of dairy cows are presented in Figure 3. The exposure of cows at THI-75 during the Tr period revealed significant changes in RT (Figure 3A), HR (Figure 3B), and RR (Figure 3C) compared to comfort conditions (THI-72 during PrTr). The mean RT, HR, and RR at THI-72 were found 38.6 ± 0.4 °C, 76.7 ± 1.5 beats/min, and 29.7 ± 1.2 breaths/min, respectively, which were significantly increased to 39.2 ± 0.3 °C, 79.3 ± 1.5 beats/min, 31.7 ± 1.5 breaths/min after increasing THI to 75. After one day of removing heat stress (PoTr1 phase: THI value returned to 72), the RT, HR, and RR also decreased to 38.8 ± 0.2 °C, 77.3 ± 1.2 beats/min, and 30.3 ± 0.6, respectively, which were close (p > 0.05) to the baseline value observed at THI-72 during PrTr phase (Figure 3A–C). As compared to THI-72, FI (Figure 3D) and WI (Figure 3E) altered significantly during the simulated heat stress phase (Tr) at THI-75. The mean FI and WI at THI-72 were recorded at 25.5 ± 0.5 and 36.7 ± 2.1 kg/day, respectively. When cows were exposed to THI-75, the FI was significantly decreased to 23.8 ± 0.8 kg/day whereas the WI increased to 39.3 ± 1.5 kg/day. However, both parameters returned to baseline values (p > 0.05) by day 2 of the recovery phase (PoTr2). The MY was also affected by heat stress (Figure 3F). As compared to baseline MY (6.3 ± 0.1 kg/day), a significant reduction in MY (5.9 ± 0.1 kg/day) was observed during the simulated heat stress period (Tr) at THI-75. Within one day of commencing the recovery phase, MY returned to the baseline value (p > 0.05). The milk composition was also altered during the heat stress phase. Although MF percentage was not affected significantly (Figure 3G) as compared to the baseline value (4.2 ± 0.05%), the percentage of MP was significantly decreased to 3.3 ± 0.2% than that of baseline measurement (3.7 ± 0.1%) during PrTr phase under THI-72 (Figure 3H). Milk production and composition returned to baseline levels (3.6 ± 0.1%, p > 0.05) by the second day of the recovery phase (PoTr2) following the withdrawal of heat stress from the climate chamber.

3.2. Effect of THI-75 on Hematological and Serum Biochemical Parameters

The hematological profiles and serum biochemical parameters of cows at different THI levels during the PrTr, Tr, and PoTr period is presented in Table 2. In stressed cows, significant reductions were observed in Plt counts, Hb levels, and Lym counts, compared to cows in the PrTr phase at THI-72. Additionally, RBC, WBC, and Neu counts remained unchanged (p > 0.05) in stressed cows. Regarding serum biochemical parameters, it was observed that S.Pro, S.Cho, S.Glu, and S.Tg levels were significantly decreased with a significant elevation of S.Cre during the stress condition (THI-75), compared to the baseline phase under THI-72 (PrTr). However, all the parameters returned to baseline levels within 24 h of the recovery phase (PoTrD1). In contrast, S.Cre required 48 h (PoTrD2) to approach the baseline.

3.3. Effect of THI-80 on Physiological and Production Parameters

The impact of simulated heat stress under THI-80 on various physiological and production parameters of dairy cows is shown in Figure 4. The average RT (40.6 ± 0.3 °C) (Figure 4A) of stressed cows during the Tr phase was significantly higher (38.5 ± 0.2 °C) than that of the baseline phase at THI-72 (PrTr). After decreasing the THI value to 72 during the recovery phase, RT gradually declined and approached baseline levels by day 3 (PoTrD3), as shown in Figure 4A. The HR (Figure 4B) and RR (Figure 4C) during the simulated heat stress phase (Tr) were 84.0 ± 1.0 beats/min and 35.0 ± 1.0 breaths/min, respectively, which were significantly higher than that of baseline measurement (77.3 ± 2.1 beats/min and 28.3 ± 1.5 breaths/min) under THI-72 (PrTr phase). Following the withdrawal of heat stress, the HR and RR returned to baseline levels (p > 0.05) at day 2 (PoTrD2) and 3 (PoTrD3) of the recovery phase, respectively. In contrast to cows maintained during the PrTr phase under THI-72, FI in stressed cows decreased significantly from 26.3 ± 1.5 to 22.7 ± 1.0 kg/cow/day (Figure 4D), while WI increased from 38.3 ± 0.6 to 45.7 ± 0.6 kg/cow/day (Figure 4E). During the recovery phase, PoTr period, the FI and WI of the cows approached baseline levels by day 2 (PoTrD2) and 3 (PoTrD3), respectively. In terms of production performance, the baseline record of MY was found 5.9 ± 0.3 kg/cow/day during the PrTr phase which declined to 5.0 ± 0.3 kg/cow/day under simulated heat stress conditions at THI-80 (Tr phase) (Figure 4F). The initial percentages of MF (Figure 4G) and MP (Figure 4H) were 4.4 ± 0.2% and 3.7 ± 0.1%, respectively, in the PrTr phase. These values significantly decreased to 3.7 ± 0.2% and 3.0 ± 0.1% during the Tr phase under THI-80. Both parameters returned to near-baseline levels (p > 0.05) by day 3 of the recovery phase (PoTrD3).

3.4. Effect of THI-80 on Hematological and Serum Biochemical Parameters

The hematological profiles of cows at different THI levels during the PrTr, Tr, and PoTr periods are presented in Table 3. There was a significant reduction in Plt counts, Hb levels, and Lym counts in stressed cows (Tr) compared to cows kept at baseline conditions at THI-72 (PrTr). RBC and WBC counts remained unchanged (p > 0.05) in stressed animals. However, significant increments were observed in Neu counts. In serum biochemical parameters, it was observed that S.Pro, S.Cho, S.Glu, and S.Tg levels significantly declined during the stress condition as compared to the baseline measurement. In contrast, S.Cre levels were significantly increased at heat stress exposure. Notably, after the cessation of heat stress, the parameters required 72 h in the recovery phase (PoTrD3) to revert to baseline levels.

3.5. Comparison Among Different THI Patterns in Terms of Heat Stress Impact

The comparison among THI-72, the baseline thermal condition used in this study, and THI-75 and THI-80, the two simulated heat stress conditions is compiled in Figure 5. The comparison includes cow physiological markers, behavioral responses, daily MY and composition (particularly fat and protein content), and stress-associated hematological and serum biochemical parameters. In comparison to THI-72, physiological parameters including RT, HR, and RR, increased by 1.6%, 3.0%, and 9.2%, respectively, in cows exposed to THI-75, and by 5.3%, 9.1%, and 20.7% at THI-80 (Figure 5A–C). All these parameters showed significant differences across THI levels. With the increase in THI, FI in cows significantly decreased (Figure 5D). The FI declined by 8.0% at THI-75 and 17.7% at THI-80 compared to the baseline measurement under THI-72. The reduction in FI at THI-80 was approximately twice that observed at THI-75. An opposite trend was observed in WI, which increased significantly with rising THI levels (Figure 5E). Compared to THI-72, WI was 4.9% higher at THI-75 and 21.8% higher at THI-80, indicating a four-fold increase in WI response under elevated THI conditions. The MY and MP content showed significant decreases with increasing THI levels (Figure 5F,H). A 2.5% reduction in MY was observed at THI-75, while a 17.3% reduction was recorded at THI-80 compared to baseline conditions (THI-72). In contrast, MF percentage showed an 8.1% decrease at THI-75 and a 14.6% decrease at THI-80. While the reduction in MF at THI-75 was not statistically significant, the decrease at THI-80 was significant, as depicted in Figure 5G. Among the hematological parameters, blood Hb levels decreased by approximately 1.5-fold with increasing THI levels; however, no significant difference was observed between THI-75 and THI-80 (Figure 5I). Neu count increased significantly, while Lym count decreased significantly as THI increased (Figure 5J–K). Neu count increased by 14.6% at THI-75 and by 40.4% at THI-80 compared to baseline conditions (THI-72). In contrast, Lym count decreased by 25.8% at THI-75 and by 39.1% at THI-80. Biochemical parameters, including S.Pro, S.Cho, S.Glu, and S.Tg levels, showed significant reductions of 6.4%, 8.3%, 13.8%, and 10.7%, respectively, at THI-75. These values were further significantly reduced at THI-80, with decreases of 15.8%, 16.4%, 34.5%, and 14.4%, respectively, compared to baseline conditions at THI-72 (Figure 5L–O). However, S.Cre levels showed a significant increase under heat stress, rising by 17.5% at THI-75 and further increasing to 25.6% at THI-80 compared to baseline levels (Figure 5P).

3.6. Association Among Heat Stress Related Variables at Different THI

Figure 6 presents a correlation matrix depicting the pairwise associations among physiological, behavioral, hematological, and biochemical parameters analyzed at THI-72, THI-75, and THI-80. Here, we described only the association that exhibited either strong positive (r > 0.6) or strong negative (r > −0.6). A total of 171 pairwise correlations were observed. At THI-72, THI-75, and THI-80, the number of strong positive associations was 14, 35, and 30, respectively, and strong negative interactions numbered 5, 35, and 17, respectively. The highest number of weak positive correlations (54) was observed at THI-80, whereas the maximum number of weak negative correlations (70) occurred at THI-72. Moderate positive correlations were most prevalent at THI-80 (18) followed by 13 each at THI-72 and THI-75. Conversely, moderate negative associations were higher at THI-72 (17) compared to THI-75 (13) and THI-80 (12).

Under THI-72, two strong positive interlinked associations were observed: one among Neu, Lym, and S.Glu, and another among S.Pro, S.Cho, and S.Cre. Among physiological markers, strong positive correlations were identified between RT and WI, HR and RR, HR and Plt, and RR and WBC. The behavioral parameter WI showed a strong positive correlation with S.Tg, while the production trait MF exhibited interactions with S.Cho and S.Cre. Among hematological parameters, Plt demonstrated a strong positive correlation with Hb. In contrast, the production trait MY displayed strong negative interactions with the physiological markers RT and HR, as well as with the behavioral parameter WI. Additionally, HR interacted with RBC, and the hematological parameter Lym showed a strong negative correlation with the biochemical parameter S.Cho.

The increase in THI from 72 to 75 was reflected in the correlation pattern shown in Figure 6. Four strong positive interrelated associations were identified: one between HR, RR, WI, and S.Tg, another between FI, MY, MF, and WBC, and a third between the biochemical parameters S.Pro, S.Cho, S.Glu, and S.Cre. A fourth association was observed among the hematological parameters RBC, WBC, and Neu. In addition, several other strong positive correlations were observed, including those between FI and RBC, WBC, Neu, and S.Cre; MF and RBC, S.Glu, and S.Cre; MP and RBC, WBC, and Neu; Plt and Hb; and Lym and S.Tg. A significant number of variable pairs exhibited strong negative interactions. The variables FI, MF, MP, RBC, and WBC showed correlations with all the physiological parameters (RT, HR, and RR), whereas MY and Neu interacted only with HR and RR. The two behavioral parameters were interlinked in a strongly negative manner. WI demonstrated a negative dependence on MY, MF, MP, RBC, WBC, and Neu. The biochemical parameter S.Tg negatively correlated with FI, MY, MF, RBC, WBC, and Hb. The hematological parameter Lym showed a strong negative correlation with Plt, while Neu was linked exclusively with Plt.

At THI-80, six strong positive interrelations were identified. The first was among physiological parameters and WI, while the second involved most biochemical parameters, including S.Cho, S.Pro, S.Glu, and S.Cre. The third, fourth, and fifth interrelations were observed among hematological and biochemical parameters: RBC, WBC, and S.Tg; Plt, Neu, and S.Pro; and WBC, Hb, and S.Pro, respectively. The sixth interrelation was observed among mixed parameters, including FI, MY, and S.Tg, as well as Neu with RR and S.Cho. Additionally, the hematological parameters Neu displayed a strong positive correlation with RR and S.Cho; RBC and WBC showed a strong positive association with FI, while Lym exhibited a correlation with MF; and Hb demonstrated a strong positive interaction with Plt. In contrast, RT and HR displayed strong negative correlations with FI, RBC, and WBC, while HR showed a strong negative association solely with S.Tg. The physiological parameter RR exhibited a strong negative interaction with MP and RBC, whereas the behavioral parameter WI was linked with RBC. The production parameter MF displayed a strong negative association with Hb, while MP interacted with Plt, Neu, S.Pro, and S.Cho. Finally, the hematological parameter Lym demonstrated a strong negative correlation with S.Glu and S.Cre.

4. Discussion

Livestock are exposed to various stressors including climatic variables such as ambient temperature, relative humidity, wind speed, solar radiation, and others that affect production performance, FI, WI, blood composition, biochemical properties, and health [38]. The current study aims to investigate the effect of heat stress on the physiological, hematological, biochemical, and production parameters of Holsten-Friesian crossbred dairy cows in Bangladesh. The average ambient temperatures have been continuously increasing particularly during the summer season due to global warming, further aggravating damage to livestock [39]. THI is recognized as one of the most reliable heat stress indexes. However, the upper comfort zone at which the onset of heat stress occurs in dairy cattle depends on breed type, physiological status, parity, lactation stage, geographical location, and many other factors [8,9]. Several studies have reported varying THI threshold levels for Holstein-Friesian crossbred cows based on physiological and production parameters, with thresholds ranging from THI-74 to 84 [40], THI-74 [41], THI-75 [31,32], and THI-80 [42]. In this study, dairy cows were exposed to three distinct thermal conditions represented by THI-72, THI-75, and THI-80. The experimental cows were maintained in an environment with a THI of 72 throughout the PrTr and PoTr periods, whereas during the Tr phase, they were exposed to either THI-75 or THI-80. Various physiological stress markers, behavioral markers, production traits, hematological profiles, and biochemical parameters were observed and compared across these three treatment periods.

Rectal temperature is commonly used as a parameter for assessing thermal balance in cows [18] and is also used to evaluate the influences of the thermal environment on growth, lactation, and reproduction in dairy cows [43,44,45]. In the current study, we observed increases in RT with rising THI levels. The elevated THI in the climatic chamber may have hindered heat dissipation, leading to increased RT at higher THI levels [46]. Previous studies have shown that RT in crossbred dairy cows in Bangladesh is significantly higher during heat stress periods compared to non-heat stress periods [3,29]. Animals experiencing heat stress need to dissipate heat from the body to maintain normal core body temperature [11]. Therefore, increased HR is observed [47,48], which causes enhancing blood flow in the body surface [49,50] to transfer core body temperature to the environment [51]. Besides this, the evaporative cooling mechanism is initiated [52], which includes elevated RR and increased sweating [53,54], leading to greater body fluid loss. In turn, it raises water requirements to regulate dehydration and maintain blood homeostasis [46,55]. An increase in RR leads to greater expulsion of CO₂ through the lungs, resulting in respiratory alkalosis due to a reduction in blood carbonic acid concentration [56]. Therefore, the animal must compensate for higher blood pH by excreting bicarbonate in the urine to maintain the ratio of carbonic acid and bicarbonate [57,58]. As a result, normal blood homeostasis of dairy animals becomes compromised, resulting in tachycardia and tachypnea in stressed cows. Consistent with these findings, our study demonstrated a significant rise in RR and HR as the baseline THI in the PrTr phase increased from 72 to the stressed phase THI levels of 75 or 80. The increased HR in crossbred dairy cows under tropical climates was also revealed in a previous study [59], which supports our present findings.

Studies have demonstrated that behavioral responses especially FI and WI of dairy cows significantly altered under heat stress conditions as reviewed by [10,14]. These changes occurred in dairy cows due to their physiological demand to reduce thermal load [13,17,60]. Heat stress induces discomfort and reduces the appetite of cows [14]. Additionally, the digestion of roughage generates metabolic heat, prompting animals to lower their FI as a physiological adaptation to minimize further heat production [61,62,63]. Our findings revealed a reduction in FI during the stress period, with a 6.5% decrease at THI-75 and a 14% decrease at THI-80, indicating a THI-dependent response. Compared to our findings, a diverse FI reduction (16%, 24.8%, and 32.3%) was revealed by previous studies [47,53,64]. Additionally, the study indicates that for each 1 °C increase in air temperature above the thermal neutral zone, there is a corresponding reduction in FI of 0.85 kg [65]. In another study, it is depicted that dairy cattle under heat stress consume roughly 12% less dry matter [62]. In contrast, water consumption increased significantly under heat stress conditions, driven by rapid water loss from the animals’ bodies and an increased need for hydration to maintain normal internal body functions [46,61]. The activated thermoregulation mechanisms during heat stress lead to increased WI [46,55]. In comparison to the 19% increase in water consumption observed in the present study, a 23% and 25% increase in water consumption is reported in Holstein dairy cows [47,66]. Several factors such as breed, environmental conditions, diet, or experimental methods may be responsible for these differences in FI and WI. Heat stress, arising from the interaction of hot and humid environmental conditions, has a detrimental impact on both milk production and composition in dairy animals, particularly those of high genetic merit [36]. A recent meta-analysis of 34 studies reported a 16.9% reduction in MY as a result of heat stress [14], which supports our present findings showing a 14.7% reduction in MY at a THI value of 80. Milk composition, particularly MF and MP content, declines when the THI exceeds 72 [67]. A decrease of 39.7% in MF, 18.9% in solids-not-fat (SNF), and 16.9% in MP has been reported in a review literature [68]. Another study shows that the protein and fat content in milk decreased from 2.96 and 3.58% to 2.88 and 3.24%, respectively, due to an increase in THI from 68 to 78 [15]. These findings are well aligned with our study, where we observed a significant reduction in both MY, MF, and MP content when cows were exposed to a THI value of 80. It is revealed that environmental heat loads exceeding 35 °C trigger stress response systems in lactating dairy cows [6]. In response, cows reduce FI, leading to a negative energy balance, which is a primary factor contributing to the decline in milk production [19]. This reduction in milk synthesis may result from both the indirect effect of reduced dry matter and nutrient intake, as well as their impaired utilization, and the direct effect of tissue hyperthermia on mammary function [68,69,70]. The decreases in MF observed in cows during heat stress are primarily attributed to metabolic and physiological adjustments that prioritize thermoregulation and homeostasis over lactation. These adaptations divert energy and nutrients from MF synthesis to support the animal’s response to elevated temperatures [17,18,71]. Likewise, the reduction in MP has been linked to a decreased availability of amino acids, as oxidative stress, immune activation, and gluconeogenesis divert these amino acids away from MP synthesis [58]. This explanation may also pertain to our findings regarding the reduction in MY, as well as in MF and MP percentages under heat-stressed conditions.

In the present study, cows in the stressed phase at THI-80 showed significantly lower Plt counts, Hb levels, and Lym counts, but higher Neu counts compared to cows in the PrTr phase at THI-72. These changes may be attributed to the hemodilution effect resulting from increased water consumption for evaporative cooling [72,73]. Several studies have shown that Plt, Lym, and Hb levels decrease under heat-stressed conditions [24,66,74], which is in agreement with the present study. The variability in Neu counts among heat-stressed cows remains a topic of debate, as stated in a recent review [75]. One hypothesis suggests that Neu counts may increase in Zebu cattle under heat stress, indicating that breed differences could be a significant factor influencing Neu response to heat stress. Since our experimental cows possess a Zebu genetic background, this breed-specific trait may have contributed to the observed increase in Neu counts under heat-stressed conditions. In addition, increased Neu counts suggest an immune response potentially aimed at countering inflammation or other stress-induced conditions [76]. We observed in the current study that the levels of S.Pro, S.Cho, S.Glu, and S.Tg in blood serum were significantly decreased, while S.Cre levels have significantly increased during heat stress conditions (THI-75 or 80). Glucose and total cholesterol are the key metabolites of energy metabolism. S.Glu reduction can be explained by reduced energy intake which is further modulated by the upregulation of thermoregulatory processes and the downregulation of gluconeogenesis. On the contrary, the reduction in S.Cho level during heat stress could be due to lipid utilization by other peripheral tissues [77]. Heat stress reduces S.Tg levels primarily due to decreased FI, lowering the energy level required for triglyceride synthesis. Additionally, heat stress redirects fat metabolism towards the production of non-esterified fatty acids and ketone bodies as immediate energy sources, further dropping S.Tg concentrations [17,18,71]. The mechanism behind the decrease in S.Pro levels in stressed cows may be due to catabolism of proteins into amino acids, which are then converted into glucose through gluconeogenesis [78,79]. The decreased levels of S.Glu, S.Pro, S.Cho, and S.Tg during heat stress have also been revealed in another study [25]. It is reported that heat stress causes mild injury to the body and increases the risk of pathological events, namely acute kidney injury [19]. The increased S.Cre level in our study might be due to this reason. In accordance with our study, other studies [23,25] also reported an increased level of S.Cre in heat-stressed cattle compared to the comfort environment. Generally, heat stress negatively affects the physiology, feeding behavior, and milk production and its composition in dairy animals, especially animals of high genetic merit [18,66]. However, 50% of Holstein-Friesian crossbred dairy cows in our study showed a considerable adverse alteration in physiology, FI, and milk production parameters. The blood parameters as well as serum biochemical properties were also undesirably affected.

The correlation analysis showed that the maximum percentage (71%) of weak associations at THI-72 which reduced to 44% at THI-75 and 54% at THI-80, suggesting that interaction between the parameters analyzed in the study shifted due to an increase in THI. The number of strong interrelated associations between variables was observed higher at THI-80 (6 correlations) than those at THI-72 (2 correlations) and THI-75 (4 correlations). This finding indicates that an increase in THI increases the dependency of variables with each other. Although the percentage of strong positive associations at THI-75 (21%) and THI-80 (18%) was very close, a huge difference between THI-75 (21%) and THI-80 (10%) was observed when strong negative interactions were considered. For example, RT increased and MY decreased significantly at THI-80 compared to THI-72 and THI-75, expecting a strong negative correlation (Figure 5). However, correlation analysis revealed a weak association between RT and MY at THI-80, similar to the findings at THI-72, whereas a strong negative correlation was observed at THI-75. This outcome may be attributed to the limited number of animals used in the study, which was insufficient to account for variations in individual physiological differences. Additionally, a single chamber with the capacity to rear three cows at a time was used in this study. As a result, conducting experiments under two heat stress conditions with six cows required four separate trials (Figure 2). This experimental design introduced variability in factors such as lactation stage and physiological status among the cows, which may have influenced the results. To address this limitation in future studies, constructing multiple chambers to simultaneously conduct experiments under different conditions would be beneficial.

5. Conclusions

In Bangladesh, there has been a growing trend of rearing Holstein-Friesian and Jersey crossbred dairy cows in recent years. The current study indicates that Holstein-Friesian crossbred dairy cows exposed to heat stress (THI 75 and 80) exhibit elevated physiological stress markers, alongside reduced FI, MY, and milk quality. Additionally, declines in hematological and biochemical markers highlight the vulnerability of these cows to heat stress. Based on these findings, it can be assumed that the effects of heat stress may be even more detrimental to high-yielding (>15 kg/cow/day) crossbred dairy cows, particularly those reared in commercial farms in Bangladesh. Animal scientists and breeders should consider these findings to ameliorate this situation. Further studies are also required to assess the effect of heat stress on high-yielding crossbred dairy cows and other genotypes available in Bangladesh. Additionally, mitigation strategies such as providing shade, using cooling systems like sprinklers, increasing shed height for ventilation, reducing concentrate feed, and ensuring ample water supply can be implemented to alleviate heat stress in dairy cows.

Author Contributions

Conceptualization, M.M.R., A.S.M.S., and S.A.M.H.; methodology, M.U.H., S.A.M.H., M.U., K.M.A.-N., and M.M.R.; software, M.M.R.; validation, M.U.H., S.A.M.H., and M.M.R.; formal analysis, M.A.S. and S.A.M.H.; investigation, M.M.R.; resources, M.M.R., A.S.M.S., and S.A.M.H.; data curation, K.-A.-J.E., M.U., S.N.T., and S.Z.S.; writing—original draft preparation, M.U.H., M.U., and S.A.M.H.; writing—review and editing, A.S.M.S., S.A., and M.M.R.; visualization, M.M.R.; supervision, M.M.R.; project administration, M.M.R.; funding acquisition, M.M.R., A.S.M.S., and S.A.M.H. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The animal study protocol was approved by the Animal Research Ethics Committee of Bangabandhu Sheikh Mujibur Rahman Agricultural University (Ref. No. FVMAS/AREC/2022/01).

Data Availability Statement

All data incurred in this study are included in this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Tables

Figure 1. Dairy cattle associated temporal THI distribution in Bangladesh over the 30-year period (1986–2015). The data shown in the graph include all the temperature and humidity data recorded every three hours at 34 weather stations of Bangladesh Meteorological Department. Pre-monsoon: March-May; Monsoon: June to October. Post-monsoon: November to December. The red colored dotted line indicates the comfort THI level (72).

Figure 2. Schematic diagram of the experimental layout. (A) Pictorial view of the climatic chamber. (B) Schematic diagram of the climatic chamber’s interior layout. (C) Timeline of each trial. (D) Experimental layout.

Figure 3. Changes in physiological and production parameters of dairy cows under heat stress conditions at THI-75. (A) Rectal temperature (°C); (B) Heart rate (beats/min); (C) Respiration (breaths/min); (D) Feed intake (kg/day); (E) Water intake (kg/day); (F) Milk yield (kg/day); (G) Milk fat (%); (H) Milk protein (%). The values are mean ± SD of six replicates. Different lowercase letters (a or b) on the bars indicate the significant differences (p [less than] 0.05). PrTr and Tr represent pre-treatment and treatment days. PoTrD1, PoTrD2, and PoTrD3 represent the post-treatment day 1, day 2, and day 3, respectively. The numbers 72 and 75 represent the THI-72 and THI-75, respectively.

Figure 4. Changes in physiological and production parameters of dairy cows under heat stress conditions at THI-80. (A) Rectal temperature (°C); (B) Heart rate (beats/min); (C) Respiration (breaths/min); (D) Feed intake (kg/day); (E) Water intake (kg/day); (F) Milk yield (kg/day); (G) Milk fat (%); (H) Milk protein (%). The values are mean ± SD of six replicates. Different lowercase letters (a, b, c, or d) on the bars indicate significant differences (p [less than] 0.05). PrTr and Tr represent pre-treatment and treatment days. PoTrD1, PoTrD2, and PoTrD3 represent the post-treatment day 1, day 2, and day 3, respectively. The numbers 72 and 80 represent the THI-72 and THI-80, respectively.

Figure 5. Comparison among effects of different THI levels on physiological markers, behavioral responses, MY and composition, and stress-associated hematological and serum biochemical parameters of cows. (A) Rectal temperature (°C); (B) Heart rate (beats/min); (C) Respiration (breaths/min); (D) Feed intake (kg/day); (E) Water intake (kg/day); (F) Milk yield (kg/day); (G) Milk fat (%); (H) Milk protein (%); (I) Blood Hb (g/dL); (J) Neutrophil (×109/L); (K) Lymphocyte (×109/L); (L) Protein (mg/dL); (M) Cholesterol (mg/dL); (N) Glucose (mg/dL); (O) Triglyceride (mg/dL); (P) Creatinine (mg/dL). The values are the mean ± SD of six replicates. Different lowercase letters (a, b, or c) on the bars indicate the significant differences (p [less than] 0.05).

Figure 6. Correlation matrix showing pairwise association among different variables related to heat stress response in dairy cows at THI-72 (A), THI-75 (B), and THI-80 (C). Correlation strength is classified as r [greater than] 0.6: strong positive; r [greater than] −0.6: strong negative; r = 0.4 to 0.6: moderate positive; r = −0.4 to −0.6: moderate negative; r [less than] 0.4: weak positive; and r [less than] −0.4: weak negative. RT: Rectal temperature; HR: Heart rate; RR: Respiration rate; FI: Feed intake; WI: Water intake; MY: Milk Yield; MF: Milk fat; MP: Milk protein; RBC: Red blood cell; WBC: White blood cell; Plt: Platelet; Neu: Neutrophil; Hb: Hemoglobin; Lym: Lymphocyte; S.Pro: Serum protein; S.Cho: Serum cholesterol; S.Glu: Serum glucose; S.Cre. Serum creatinine; S.Tg: Serum triglycerides.

Table 1

Feed ingredients and composition of the mixed diet.

Item	Amount (g/kg DM)
Ingredients (Roughage)
Rice straw	383
Napier grass	344
Ingredients (Concentrate)
Commercial concentrate feed 1	253
Others supplements
Common salt	10
Commercial vitamin–mineral premix 2	10
Chemical composition of the mixed diet
TDN	556.3
CP	105.1

¹ Commercial concentrate was labeled as 12% moisture, 21% crude protein, 10% crude fiber, 4% crude fat, 75% total digestible nutrient (TDN), 1.5% calcium, 0.8% phosphorus. ² Vitamin A, Vitamin D3, Vitamin E, Vitamin B2, Vitamin B6, Vitamin B12, Vitamin K3, Nicotinic Acid, Folic Acid, and Mintrex PSE as claimed by the manufacturer.

Table 2

Hematological and biochemical profiles of cows during stress (THI-75) and comfort (THI-72) conditions.

THI	72	75	72
Period	PrTr	Tr	PoTrD1	PoTrD2	PoTrD3
Hematology
RBC (×1012)/L	7.75 ± 0.87 a	7.05 ± 0.40 a	7.51 ± 0.76 a	7.68 ± 0.83 a	7.84 ± 0.93 a
WBC (×109)/L	14.48 ± 1.52 a	13.51 ± 0.62 a	14.05 ± 1.36 a	14.36 ± 1.46 a	14.64 ± 1.61 a
Plt (×109)/L	349.89 ± 15.95 a	241.89 ± 58.60 b	320.02 ± 22.41 a	335.64 ± 17.31 a	350.09 ± 15.03 a
Neu (×109)/L	3.29 ± 0.23 a	3.45 ± 0.48 a	3.61 ± 0.25 a	3.38 ± 0.23 a	3.16 ± 0.23 a
Hb (g/dL)	13.29 ± 2.32 a	8.79 ± 0.81 b	12.31 ± 1.82 ab	13.01 ± 2.18 a	13.66 ± 2.52 a
Lym (×109)/L	12.00 ± 0.16 ab	8.70 ± 0.17 d	11.06 ± 0.34 c	11.73 ± 0.21 b	12.36 ± 0.09 a
Biochemical parameters
S.Pro (mg/dL)	9.02 ± 0.58 a	8.50 ± 0.36 b	8.74 ± 0.39 ab	8.77 ± 0.46 ab	9.10 ± 0.68 a
S.Cho (mg/dL)	162.37 ± 4.26 a	150.71 ± 2.69 b	158.44 ± 3.21 ab	161.34 ± 2.98 a	164.56 ± 4.59 a
S.Glu (mg/dL)	56.45 ± 2.17 a	47.65 ± 2.92 b	52.56 ± 2.36 ab	57.53 ± 3.16 a	56.81 ± 2.01 a
S.Tg (mg/dL)	1.88 ± 0.08 a	1.69 ± 0.06 c	1.76 ± 0.11 b	1.90 ± 0.12 a	1.89 ± 0.09 a
S.Cre (mg/dL)	1.61 ± 0.06 c	1.93 ± 0.05 a	1.82 ± 0.04 b	1.63 ± 0.09 a	1.59 ± 0.10 a

The values are mean ± SD of six replicates. Different lowercase letters (a, b, c, or d) in the same parameter (row) differ significantly (p < 0.05). PrTr and Tr represent pre-treatment and treatment days. PoTrD1, PoTrD2, and PoTrD3 represent the post-treatment day 1, day 2, and day 3, respectively. The numbers 72 and 75 represent the THI 72 and THI 75, respectively. RBC: Red blood cell; WBC: White blood cell; Plt: Platelet; Neu: Neutrophil; Hb: Hemoglobin; Lym: Lymphocyte; S.Pro: Serum protein; S.Cho: Serum cholesterol; S.Glu: Serum glucose; S.Cre. Serum creatinine; S.Tg: Serum triglycerides.

Table 3

Hematological profiles of cows during stress (THI-80) and comfort (THI-72) conditions.

THI	72	80	72
Period	PrTr	Tr	PoTrD1	PoTrD2	PoTrD3
Hematology
RBC (×1012)/L	7.19 ± 0.87 a	6.11 ± 0.94 a	6.53 ± 0.74 a	6.88 ± 0.50 a	7.15 ± 0.90 a
WBC (×109)/L	13.92 ± 1.52 a	11.95 ± 2.04 a	12.71 ± 1.51 a	13.34 ± 1.18 a	13.85 ± 1.65 a
Plt (×109)/L	341.33 ± 15.95 a	235.33 ± 58.60 b	279.79 ± 39.89 ab	314.06 ± 49.90 ab	346.67 ± 24.38 a
Neu (×109)/L	2.73 ± 0.23 c	4.22 ± 0.42 a	3.65 ± 0.32 ab	3.11 ± 0.35 bc	2.79 ± 0.33 c
Hb (g/dL)	12.73 ± 2.32 a	8.23 ± 0.81 c	9.96 ± 1.00 bc	11.49 ± 1.24 ab	12.87 ± 1.56 a
Lym (×109)/L	11.44 ± 0.16 a	7.14 ± 0.97 c	8.80 ± 0.66 b	10.30 ± 0.87 a	11.29 ± 0.36 a
Biochemical parameters
S.Pro (mg/dL)	9.15 ± 0.62 a	7.65 ± 0.43 c	7.86 ± 0.39 c	8.31 ± 0.25 b	9.12 ± 0.52 a
S.Cho (mg/dL)	166.32 ± 3.18 a	137.44 ± 2.31 b	143.45 ± 3.04 b	158.44 ± 2.65 a	167.81 ± 3.65 a
S.Glu (mg/dL)	54.13 ± 1.12 a	36.19 ± 1.31 d	43.21 ± 1.18 c	49.25 ± 2.06 b	55.36 ± 2.11 a
S.Tg (mg/dL)	1.91 ± 0.03 a	1.62 ± 0.02 c	1.73 ± 0.02 b	1.90 ± 0.06 a	1.92 ± 0.07 a
S.Cre (mg/dL)	1.68 ± 0.04 c	2.07 ± 0.03 a	1.98 ± 0.03 a	1.72 ± 0.07 b	1.65 ± 0.08 c

The values are mean ± SD of six replicates. Different lowercase letters (a, b, c, or d) in the same parameter (row) differ significantly (p < 0.05). PrTr and Tr represent pre-treatment and treatment days. PoTrD1, PoTrD2, and PoTrD3 represent the post-treatment day 1, day 2, and day 3, respectively. The numbers 72 and 80 represent the THI-72 and THI-80, respectively.

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