The study was conducted in a small dairy farm for raising heifers located in Campo Alegre, David City, Chiriquí Province, Republic of Panamá; at Latitude 8.44058˚ (8˚26'26'' North) and Longitude −82.43592˚ (82˚26'9'' West) and its altitude 50 m. The farm’s area was divided into eight pasture blocks fixed with water and plastic feeders to provide nutritional supplements. All paddocks offered a few scattered middle trees, but there were no shaded installations. A stockyard (18 m long × 10 m wide) was fixed in two blocks having 6 pens separated by 2.16 m between animals and between blocks 5.5 m to entry and managed animals classified by breed for physiological evaluation each three hours from 06 am to 06 pm.

The environmental conditions around the animals included pastures, dispersed trees, grazing areas, feed, and water recipients. Pasture blocks measured 8120 m² and were divided into two portions, where animals grazed three days by blocks. The pre-experimental period included the time since animals arrived at the new environment in Campo Alegre from December (2019) to March (2021). The experimental evaluation went from February to March (2021), in which daytime thermal environment and animal physiological responses were measured each three hours on one day/week for four weeks to relate daytime thermal trends and respiratory physiology based on heat stress according to psychometric indicators in the humid tropical climate [3] [5] [61] and microclimate around David City [79] .

All female calves were weaned at age of 75 days by cutting milk as part of the diet and then animals continued receiving grains, hay, and green forage up to age of five months. Then, animals were moved from the dairy farm from an altitude of 1100 m to a farm located in Campo Alegre, District of David, at an altitude of 50 m. Animals were raised from five to 25 months in this new stressful environment under heat stress until physiological evaluation during summer time.

The study was done from February to March 2021, since those months represented the major daily heat stress in the summer of Panamá. The psychrometric indicators (ambient temperature, relative humidity, direct solar radiation, enthalpy, wind speed, Temperature Humidity Index (THI) and adjusted THI by direct solar radiation were taken between 06 am and 06 pm each three hours to register daytime changes. At the mind time, physiological indicators (body temperature and respiratory) were measured each three hours to relate environmental and physiological changes under the influence of heat stress during summer time in the tropical environment based on procedures and integrated strategies to evaluate heat stress in dairy cattle [20] [80] - [82] .

The psychrometric periods were 06 am, 09 am, 12 md, 03 pm and 06 pm and at the mind time physiological indicators were taken around each daily period from −30 to +30 minutes around the hour set for each period. Environmental indicators come from daily meteorological data provided by the program Weather Clock and satellite assistance for coordinates 8˚26'38''N and 82˚26'11''W in David City on each day of measurements. The physical environment in the experimental field included the psychrometric parameters [5] [83] plus adjustments by solar radiation and wind speed [2] [23] with modifications indicated by Araúz et al. [3] in the tropical climate to define the environmental momentum thermal force for heat stress.

Psychrometric variables were direct solar radiation (Kcal/m² hr), wind speed (m/seg), enthalpy (Kcal/ kg dry air), dry bulb ambient temperature (˚C), wet bulb ambient temperature (˚C), and temperature humidity Index (% ˚C) and adjusted THI [5] [20] . Thermal body and respiratory physiological overload work were calculated according to physiological status at 06 am and changes showed each three hours across daytime under heat stress and applications of the standard coefficients were based on psychrometric daily critical point at 12 middle days as described by Araúz et al. [3] . The Basic Temperature Humidity Index for bovine was calculated as THI_bovine and adjusted THI_Bovinos estimated according to Mader et al., [20] . Heat Stress Index (HSI) was calculated according to Benezra [16] .

Six Holstein and six jersey (three pregnant and three none pregnant by breed) were used; which were weighted by scale and special weight tape for dairy breeds described by Araúz [4] . Pregnant Holstein heifers aged 27.3 months and weight 391.3 kg averaging 5.23 ± 0.6 months of gestation and none pregnant age 24.3 months and weight 373.17 kg. Pregnant jersey heifers aged 25.3 ± 0.4 months and weight 282.7 kg and nonpregnant aged 24.3 months and weight 263.7 kg. The body condition score of all animals ranged from 3.25 to 3.50 according to Edmonson et al. [84] .

The health program included vaccination against eight clostridiums each six months by applying 2 ml subcutaneously of Ultrachoice^TM 7 to protect animal against Clostridium chavoei, Clostridium septicum, Clostridium haemolyticum, Clostridium novyi-Sordellii, Clostridium perfringens Types A & D and Bacterin toxoid. Endoparasites were controlled using anthelmintic drugs (Fenbendazole at 5 mg for every kg body weight or 1 ml for every 20 kg of body weight (BW) every two months). Ectoparasites (Tick and flies) control was done using amitraz at 20.8% by spraying a solution of 1.0 ml at 20.8% diluted in 1 L of Water. Animals also received a subcutaneous injection of 1 ml of iverme for each 50 kg body weight equivalent to 200-mcg ivermectin per kg BW. The biology and reproductive status of the experimental animals are in Table 1 and Table 2 . The veterinarian declared all animals healthy across the entire experimental period.

The feeding program by animal included silage 6.8 kg, molasses 1.13 kg, grain mix 1.81 kg and green forages (Brachiaria decumbens). Minerals (50 g by animals) were mixed with the concentrate. The farm used pasture irrigation in all paddocks during summer to ensure an average of 5.2% body weight for forage consumption [47] . The feeding programs for calves and heifers were based on the Nutrient Requirements of Dairy Cattle [15] .

Rectal and skin temperature and respiratory frequency from 6:00 AM to 6:00 PM were measured each three hours. Skin temperature was taken using a laser infrared thermometer, NUBEE, Code NUB8380, FDA (USA) Code 1420571-000. Rectal and vaginal temperature was determined using a veterinarian thermometer and stethoscope respiratory auscultation were performed as indicated by Fraser et al. [36] [37] and Dukes, and Swenson [85] . Metabolic body size and surface area were estimated as indicated by Curtis [5] and Araúz et al. [3] . Blood samples were taken one week before the physiological evaluation by the vacumtainer sampling method [36] and blood analysis was done in a Mindray 3000 Plus Autoanalyzer at the Laboratory of Animal Physiology, Animal Bioclimatology and Dairy Science in the Department of Animal Husbandry, Faculty of Agricultural Sciences in Chiriquí, University of Panamá. Body caloric capacity and body caloric overload in the animals were estimated based on Curtis [5] and Araúz et al. [3] .

The experimental design and data were built according to Gill [24] and Wilcox [86] based on a factorial design. Normal distribution tests were done using the graphic histogram and double quantiles [24] . Statistical analysis was based on three factors (Factorial Design A₂ × B₂ × C₅); where factor A_i was Breed (a₁: Holstein and a₂: Jersey), Factor B_k was reproductive status (b₁: pregnant and b₂: open) and Factor C_l was daytime sub-periods (c₁: 06 am, c₂: 09 am, c₃: 12 MD, c₄: 03 pm, and c₅: 06 pm) and Dj were animals (j^mo: 1, 2, 3 by breed and reproductive status). Overall additive lineal model was

${\text{Y}}_{\text{ijkl}}=\text{u}+{\alpha }_{\text{i}}+{\beta }_{\text{k}}+{\left(\alpha \beta \right)}_{\text{ik}}+{\gamma }_{\text{l}}+{\left(\alpha \gamma \right)}_{\text{il}}+{\left(\beta \gamma \right)}_{\text{kl}}+{\left(\alpha \beta \gamma \right)}_{\text{ikl}}+{\text{e}}_{\text{ijk}}$

The analysis of variance, daytime trends, least squares means, regressions and correlations was done in the SAS program [87] according to Gill [24] and Herrera and Barreras [88] .

Physical environment and major heat stress risks for bovine occurred from 09 am to 03 pm since THI_bovine ranged from 79.36 to 86.28. The inclusion of direct solar radiation and wind speed increased THI at 09 am in the morning up to 652.1 W/m²hr at 12 md and then decreased to 202.6 W/m²hr at 03 pm (−68.91%). The caloric content of dry air changed as enthalpy reached at 12 md 100.18 Kj/kg Dry Air and then reduced to 87.8 KJ/kg (−12.36%) around 03 pm ( Table 1 ).

Daily environmental conditions around animals from 09 am to 06 pm showed enough power and caloric potential to develop heat stress in dairy cattle since THI ranged from 79.36 to 90.79. It represented a high physiological risk for ruminants to develop heat stress since the thermal neutral zone demands a THI less than 72 [2] [23] [51] . Animals were influenced by direct solar radiation and therefore adjusted THI ranged from 82.89 to 88.96 between 09 am and 03 pm; resulting in a severe degree of daily heat stress for bovines. In consequence, acute physiological alterations regarding thermoregulatory and respiratory activity, endocrine and metabolic processes are primarily reflected in dairy cattle [14] [41] [44] ; which can also affect reproduction [31] [39] [43] [53] [67] , health, milk production, biological efficiency and productivity in dairy cattle [21] [22] [34] [48] [49] .

The entire physical daytime environment was characterized by psychrometric indicators because homeotherms have an advanced nervous system which can detect thermic pressure and change vital systemic processes [32] . Daytime physical conditions during summer time showed a very high variation from 06 am to 06 pm, where dry bulb temperature, relative humidity, enthalpy and THI indexes indicated the presence of potential severe heat stress for dairy cattle; whose maximum strength occurred around 12 md ( Table 1 ) and it was basically maintained until 06 pm. Dry bulb temperature increased up to 12 md; which resulted in a thermal change +14˚C over 25.3˚C detected at 06 am (+55.34%). At the mind time, THI increases from 75.14 (06 am) to 90.79 (+20.83%); which indicates the potential thermal environment to develop severe heat stress in bovines. In addition, dry bulb temperature was slowly reduced around 03 pm (−4˚C) and at 06 pm (−4.9˚C). THI in the afternoon did not change enough to reduce potential heat stress for dairy cattle, since temperature humidity index at 03 and 06 pm averaged 86.28 and 80.83. Therefore, the thermal environment in the morning was lower than afternoon; but daytime heat stress forced body caloric load reaching middle hyperthermia.

Ϯ (THIDSRWS): Temperature Humidity Index adjusted by Direct Solar Radiation and Wind Speed.

The animal responses against some specific environmental conditions depend on homeostasis, homeokinesis and homeorrhesis as processes that integrate the regulation and physiology in homeotherms [50] [72] . However, chronic and acute adaptations developed as a function of acclimatization contribute to improving physiological and productive performances confirming adaptations and sensitivity in dairy cattle [33] [35] [53] . Therefore, factors such as breed, age, sex, body weight and type of diet can influence how dairy cattle will perform according to psychrometric conditions under heat stress, management and technical practices for growing and production.

First at all, pregnant Holstein and Jersey heifers’ weight 381.23 and 272 kg at 25 and 23.33 months and open animals’ weight 373.67 and 307 kg at 26 and 25 months. Hemoglobin, erythrocyte, and leukocyte were greater in pregnant animals compared to open Holstein and Jersey heifers because of pregnancy influence. All animals were declared healthy by the veterinarian ( Table 2 ).

TRCV: Total red cell volume; HGBN: Hemoglobin; OTC: Oxygen Transport Capacity.

The biological indicators included age, body weight and width skin by breed and reproductive status according to reproductive status ( Table 3 and Table 4 ). Body mass is related to metabolic heat production and to surface area [5] [13] [20] ; which determines the rate of heat flux by radiation, convection and conduction if skin temperature is greater than ambient temperature [9] . Body heat production and surface area are directly related to body weight; however, thermal body insulation is inverse to body weight [50] . Holstein heifers showed higher body weight and greater body surface area than Jersey animals because of their breed’s body weight differences.

Caloric body content based on rectal, vaginal and tegumentary temperature changed across daytime (P < 0.001) as daytime psychrometric trends and degrees of heat stress occurred as indicated by basic and adjusted THI for bovines. Daytime impact of the environmental heat stress represented a thermal variation of 99.17% in skin temperature, followed by vaginal temperature (66.59%) and rectal temperature (66.59%). It means, the thermoregulatory system is more effective in controlling the endogenous temperature than caloric content in the skin because of the influence of external factors and sweating-evaporating process under heat stress. Other studies showed similar results in grazing dairy cattle under middle and severe heat stress [52] [68] . Rectal temperature was the best thermal indicator based on the level of correlation between body caloric content and daytime thermal changes. The correlations between ambient temperature and rectal temperature in open and pregnant heifers were 0.73 (P < 0.001) for both groups and in open and pregnant jersey heifers were 0.87 (P < 0.01) and 0.65 (P < 0.01). The second best indicator of severe heat stress was the respiratory frequency and daytime temperature in Holstein (open: r = 0.66, P < 0.01: pregnant r = 0.84 P < 0.01) and in Jersey (open r = 0.62 P < 0.01; pregnant r = 0.66 P < 0.01). There were little differences in the rectal temperature between breeds (P < 0.01; Table 5 ); however, major differences occurred from 06 am to 06 pm (P < 0.001) and from 06 am to 12 md. Vaginal and skin temperature also showed significant changes across daytime hours (P < 0.001). Nevertheless, rectal temperature was the biothermal indicator used to describe body caloric alteration [3] based on correlations between environment temperature and respiratory indicators. Other study [38] found a high relationship between THI and alterations in physiology, blood items and milk production in crossbred dairy cows.

The psychrometric changes across daytime were consistent between 09 am and 03 pm, which were the hottest hours and provided the greatest heat stress degree based on THI, adjusted THI and HSI in the animals. Rectal temperature was more influenced by breed and daily sub periods once the degree of heat stress increased toward 12 md with a little recovery in the afternoon in both breeds.

The influence of psychrometric conditions and potential heat stress must consider the elevation of ambient temperature, the activation of sweating and the increment of respiratory thermolysis in dairy cattle [10] [21] [48] [49] . In addition, high relative humidity interacts with air temperature to increase the degree of heat stress in ruminants [5] [23] . Other physical conditions such as direct solar radiation can increase the degree of heat stress in bovine [66] . However, the strength of heat stress will depend on physiological state [3] [13] , dietary energy intake [53] , altitude [4] [16] , access to nutritional and feeding strategies [8] [19] [77] [81] [82] , use of artificial and natural shade [6] [80] , wind speed [20] , enviromental temperature and relative humidity and adjusted temperature humidity index by direct solar radiation and wind speed [2] [20] [75] . Maximum rectal, vaginal and skin temperature occurred around 12 md in both breeds. Rectal temperature alteration in open and pregnant Holstein heifers were +1.5 and +1.3˚C from 06 am to 12 md; and open and pregnant Jersey resulted in +0.8 and +1.0˚C ( Table 6 ).

Significant differences at 0.1% (***, P < 0.001), 1% (**, P < 0.01) and 5% (*, P < 0.05) and not significant differences at 5% (ns, P > 0.05). A: Breeds (Holstein, Jersey) B = Reproductive Status (Open, Pregnant) C (06 am, 09 am, 12 md, 03 pm. and 06 pm).

DFT: Daytime thermal factor; DTB: Daytime thermal balance. ˚C—rmpm: Celsius degree—respiratory movements per minute.

Daytime thermal factor (DTF) in pregnant Holstein and jersey heifers were 13 and 10 units according to changes in rectal temperature in the morning and recovery of caloric body in the afternoon. Meanwhile, open Holstein and jersey animals showed a DTF of 7.5 and 2.7 units; which lower the daily thermal balance since body caloric overload was smaller. Pregnant animals were more sensible to daily heat stress from 09 am to 12 md because of their greater body weight and thermal insulation. Therefore, pregnant animals showed longer caloric retention and higher hyperthermia; showing also a major caloric overload trough the afternoon ( Table 6 , Figure 1 and Figure 2 ).

Critical environmental conditions reached the highest heat stress degree at 12 md based on daytime and psychrometric properties. Holstein heifers were more sensitive than jersey heifers in the morning between 06 am and 12 md ( Figure 1 and Figure 2 ) as described by the mean (solid lines) and predicted trends (dot sequences). Kinetics of daily rectal temperature in Holstein heifers showed a slight difference between open and pregnant animals before and after 12 md ( Figure 1 ). Pregnant Holstein and Jersey heifers kept higher rectal temperature than open animals in both breeds in the afternoon; which indicates possible negative effects on gestation and fetus [2] [67] [71] [74] .

Pregnant animal presented lower thermal reduction in the afternoon as compared to open animals, which indicated a disadvantage for regulation of body temperature under daytime heat stress. The degree of daily caloric pressure was severe as indicated by psychrometric indicators and adjusted THI; therefore, physiological risks were maintaining since endogenous temperature was higher than 1.0˚C over 38.6˚C, which is the physiological rectal temperature [37] .

The process of acclimatization around 20 months in Holstein and jersey heifers under tropical heat stress benefited both breeds because no animal reached a rectal temperature greater than 39.5˚C at critical heat stress degree (12 m: Dry Bulb temperature 39.2˚C and RH 52%); even when skin temperature was between 37.0 and 37.6˚C. Jersey animals showed lower rectal temperature than Holstein heifers around 12 md since they had smaller body mass, less dry matter intake and less digestive heat production based on their metabolic body size [5] and its predictive dry matter intake [15] . Other advantages of Jersey heifers were less thermal body insulation and less skin surface to gain heat from direct solar radiation. Both breeds were not different on the skin width (P > 0.05), but they were different by weight (P < 0.01).

Open heifers in both breeds showed a higher rectal temperature in the morning since they had lower body weight and lower surface area. None pregnant heifers presented less body water content and therefore their thermal body capacities were lower [5] [21] [76] . Hence, the exposure to tropical heat stress will produce a greater thermal body load and greater heat shock, at least, across the morning when Dry Bulb Temperatures (DBT) and direct solar radiation are increasing from 06 to 12 md. After comparing pregnant and open acclimatized Holstein and Jersey heifers based on rectal temperature and respiratory activity responses against daytime heat stress in the morning, it was observed that gestation is a process that allowed systemic and body composition adjustments that helped the heifer mother and the calf to have a lower thermal body response under the influence of middle and severe heat stress.

Daytime trends for rectal temperature in Holstein and Jersey heifers showed two phases in open and pregnant animals according to type of thermal response based on body temperature and body heat load; showing a thermal alteration and resulting in a slight hyperthermia. Pregnant Hosltein and Jersey heifers showed lower thermal increments than open Holstein and jersey heifers between 06 am and 12 md. This thermal trend occurred when THI at 06 am, 09 am and 12 md were 75.14, 76.36 and 90.79. However, the major environmental thermal change and pressure occurred between 09 am and 12 md; where, THI increased at the rate of +4.81˚C%/hour; reaching the highest environmental heat shock.

The highest THI coincided with the highest rectal temperature in acclimatized pregnant Holstein (39.2˚C) and Jersey heifers (39.0˚C). The highest body temperature increased at a lower rate in pregnant Holstein and Jersey heifers; since they were heavier and showed a higher caloric heat capacity as described by Araúz et al., [3] [4] . As consequence, pregnant animals maintain a lower change in rectal temperature in the morning when comparing them to open heifers in both breeds.

Pregnant animals in both breed showed a higher rectal temperature than in open heifers, which was sustained in the afternoon. Thermal overload was influenced by the reproductive status; which indicates that heat stress can affect more in the afternoon because animals developed hyperthermia, hyperpnea and some fatigue trying to maintain normothermia.

Thermolisis is accompanied by skin artery and venous vasodilation, which increases blood irrigation to tegumentary tissues and increases epidermal temperature; which facilitates a greater thermal difference between the environment and the tegumentary surface [5] [85] . This influences caloric heat flux by sensitive and none sensitive heat losses to the environment to maintain thermal balance closer to normothermia under mild or severe heat stress. Water composition in the body is very important for thermolisis, which in ruminants is about 68.5% and ranges from 51% to 81% [51] [52] . The amount and proportion of water and dry matter in the body influence caloric heat capacity and it depends on the specific heat of water (1.0 kcal/kg˚C) and the specific heat of dry matter is 0.4 Kcal/kg˚C adjusted by total body mass [3] [4] . Therefore, caloric heat capacity and body temperature will determine caloric heat content; as well as body heat overload based on thermal alteration in dairy animals under heat stress [4] [27] [85] and thermolisis [7] .

The body temperature is one of the clinical signs used to recognize a healthy state in the homeotherms in animal production. However, this physiological indicator of body heat could be used to indicate de degree of daytime alteration in animals under heat stress, according to production, grazing and locomotion under solar influences and stressful managing practices [4] [82] .

Body temperature alteration across daytime in the none pregnant Holstein heifers showed de highest temperature overload averaging +1.35˚C every three hours, followed by pregnant Holstein heifers averaging +1.05˚C. Jersey animals showed the lowest thermal alteration; resulting +0.60˚C and pregnant heifers +0.78˚C. It seems that higher body mass represents an advantage because of greater water content and caloric capacity. However, once the heat stress increased in the daytime hours, weightier heifers were less efficient in maintaining normothermia because of greater body insulation, heat production, feed intake and greater surface area for solar radiation; which limited thermolisis, increased body heat retention, potentiated thermal alteration and developed higher moderate hyperthermia than jersey heifers.

Smaller heifers presented a better chance to maintain the thermal balance closer to normal temperature in comparison to heavier animals. The highest alteration of body temperature occurred at 12 md (open Holstein +1.5˚C and pregnant Holstein +1.3˚C), which is a relative alteration of 3.89 and 3.41% based on rectal temperature at 06 am. Hyperthermia was smaller in jersey heifers than in Holstein animals. Open jersey showed the maximum thermal body alteration at 12 md (+0.8˚C) and pregnant animals +1.0˚C; which corresponded to 2.09 and 2.63% over the physiological temperature taken at 06 am.

Body heat overload in dairy cattle under heat stress could be expressed in a short moment since caloric balance is dynamic according to sensitive and none sensitive mechanisms and ways for caloric energy flux [4] [5] [7] . In the present study, thermal body alteration showed the thermal stage of body heat overload, since rectal temperature was greater than 38.6˚C; indicated as the upper normal body temperature for bovines [4] [33] [52] . The body caloric capacities in pregnant and open Holstein heifers were 293.07 and 306.43 kcal/˚C and maximum overload heat alterations were 293.07 and 459.65 kcal according to maximum body temperature alteration. Pregnant and open jersey heifers showed a body caloric capacity of 235.21 and 216.23 Kcal/˚C and maximum caloric overload alteration were 211.68 and 129.74 kcal. Hyperthermy can be a different thermal condition in heifers according to their weight, reproductive status, physiological capacity to perform thermolisis and environmental caloric conditions. Alteration of body temperature demonstrated that acclimatized Holstein heifers were more sensitive to heat stress based on their capacity to maintain normothermia. Jersey heifers presented lower body thermal displacement; corresponding to 57.41% in none pregnant animals (Jersey vs Holstein) and 73.81% of thermal alteration in pregnant Holstein and Jersey.

The changes in daytime body temperature and THI across daytime behaved in parallelisms in both breeds and reproductive status according to the increasing heat stress degree. The correlations between rectal temperature and THI in open and pregnant Holstein heifers were 0.72 (P < 0.01) and 0.74 (P < 0.01). At the mind time, open and pregnant Jersey heifers showed a correlation 0.86 (P < 0.01) and 0.67 (P < 0.01). It means that rectal temperature increased as THI increased until 12 md. Both rectal temperature and THI showed curvolineal trends across daytime hours; however, rectal temperature in pregnant Holstein and Jersey stayed with little reduction in the afternoon, because hyperthermia was higher than in open Holstein and Jersey heifers.

This study demonstrated that acclimatization in Holstein and Jersey heifers helped them to develop chronic systemic accommodation and become less heat sensitive to severe heat stress since body thermal temperature was not greater than 39.4˚C under the highest caloric pressure. It represents a good physiological point to mitigate somehow the influence of heat stress in the tropics. In addition, acclimatization could be combined with other strategies as proposed by Beede and Collier [19] , West [8] , Baumgard et al. [9] and Gupta et al. [27] , in order to mitigate the negative influences on dairy cattle, even under the tropics. Finally, strategies including nutrition, feeding, environmental physical improvements (shade, forced ventilation), reproductive control and management are useful to reduce and/or prevent exposure of animals to direct solar radiation in order to maintain normal body temperature in dairy cattle and provide comfort and welfare under any condition related to heat stress. Nevertheless, better results will be observed if all those strategies are accompanied by acclimatization and systemic adaptations in dairy animals such as in pregnant and open Holstein and Jersey heifers under heat stress in the summer of tropical environment.