Effect of shading on the physiological and reproductive parameters in Nelore cows

This study aimed to evaluate the physiological and reproductive parameters of Nelore cows under various microclimatic conditions. The experiment was performed in Central Brazil, which has a tropical climate with dry winters and rainy summers. The experimental groups were divided into an area with shading (5.8 trees/ha; n = 42) and an area with little shading (0.4 trees/ha; n = 42). The physiological (heart rate, respiratory rate, rectal temperature, surface body temperature, and vaginal temperature) and reproductive (estrus detection, conception rate, and rate of pregnancy loss) parameters were then assessed. The rectal temperature (p = 0.002); surface body temperature on the flanks, forehead, and shoulder (p < 0.001); and vaginal temperature (p < 0.001) of cows in the group with shading were significantly lower than those of cows in the group without shading. Estrus was higher (p = 0.08) in the shaded group; however, no effect was observed on conception and pregnancy loss rates. In conclusion, shading (5.8 trees/ha) provided better thermal comfort for Nelore animals in the tropical climatic region. However, this did not affect their performance after fixed-time artificial insemination.


Introduction
Reproduction is a consequence of health, genetic improvements, nutrition, and animal welfare (Brown-Brandl et al. 2005).Heat stress negatively affects animal welfare, growth, reproduction, and health (Brown-Brandl 2018).Under heat stress, animals reduce their feed consumption to reduce heat production, which directly affects their reproductive performance (Souza et al. 2021).Reproductive losses in bovine females exposed to high heat intensity range from difficulties in estrus detection (Barbosa and Damasceno 2022) to early embryonic losses (Rivera and Hansen 2001).However, adaptation mechanisms differ between bovine breeds.For example, zebu breeds (Bos indicus) have a better ability to regulate body temperature in response to heat stress than Bos taurus breeds (Hansen 2004).
The Nelore breed (Bos indicus) is widely distributed in Brazil (comprising 80% of the cattle population), and because of its adaptability to the hot climate (Barbosa et al. 2014), it is conducive to a better reproductive performance.Despite their resistance to heat, some studies have shown that increasing thermal comfort during periods of high temperature can improve the performance of these animals (Oliveira et al. 2019;Silva et al. 2020).One way to improve the thermal comfort of cattle in pastures is to provide adequate shading.Shading is known to change the microclimate (2-9 ℃ reductions in air temperature under trees, Junior et al. (2021)) and to reduce radiant heat load in animals.Consequently, it reduces body heat and facilitates thermoregulation, thus favoring animal welfare and productivity (Salla et al. 2009).
This study aimed to evaluate the reproductive and physiological parameters of Nelore cows under different native shading conditions during the breeding season in Central Brazil.We hypothesized that increased shading by approximately 5.8 trees/ha for 30 days before the breeding season and during the entire season would increase thermal comfort and pregnancy rates.

Experimental area
The experiment was performed on a commercial property located in the transition between the Brazilian Savanna and Pantanal, in Mato Grosso do Sul, Brazil (20° 29′ 01″ S, 55° 48′ 25″ W).According to the Köppen international classification, the climate of Anastácio/MS presents the following subtypes: Cfa-humid subtropical, mesothermal, with a mild winter and hot summer, significant precipitation in all months of the year, average temperature of the coldest month > 10°, and the average temperature of the hottest month > 22 °C, and Aw-tropical, megathermal, with the winter season poorly defined or absent, strong annual precipitation with summer rains and average temperature of the coldest month > 18 °C (Alvares et al., 2013).According to data from INMET, from 1991 to 2020, the Anastácio region had an average temperature of 24 °C and an average annual precipitation of approximately 1400 mm, with the rainiest months from December to March and the driest from June to September (INMET).
Throughout the experimental period, the cows had unrestricted access to water in drinkers and mineral salt (containing 9% phosphorous) in pans.
The amount of shade in each area was calculated as described by Silva (2006), considering the type of tree canopy and its dimensions, the values of longitude and latitude, and the time of measurement.The less shaded area with isolated trees measured 66 ha with a 0.30 ha shaded area (27 trees), equivalent to 0.41 trees/ha.The shaded area was 70.65 ha, with 10.07 ha shaded area (408 trees), equivalent to 5.78 trees/ha.
The black globe humidity index (BGHI) was calculated based on data obtained from a meteorological station at the State University of Mato Grosso do Sul (UEMS/INMET).To estimate the black globe temperature (BGT), the following formula (Abreu et al. 2011) was used: where BGT is the black globe temperature in °C and DBT is the dry bulb temperature in °C.
The BGHI was calculated as described by Buffington et al. (1981): where Tgn is the black globe temperature in °C and tpo is the dew point temperature in °C.

Animals
The experiments were performed according to the approved guidelines and with the authorization of the Ethics Committee (Brazil) under protocol no.649/2014 CEUA/UFMS.
An average stocking rate of 1.23 animal units (AU)/ha was adopted.In both areas, non-lactating, non-pregnant, multiparous females of the Nelore breed (n = 84) were used, with half in each experimental paddock.The mean age of the animals was 72 ± 12 months.The average weights of the animals in the shaded and less shaded areas were 443 kg and 428 kg, respectively (p > 0.05).The body condition score (BCS), assessed on a scale of 1-5 (Ferguson et al. 1994), was 3.1 in the shaded area and 2.9 in the less shaded area (p > 0.05).Before separating the groups, a gynecological examination using ultrasonography was performed to confirm the health of the reproductive system and the general health of the animals.The division of groups (shaded and less shaded areas) was performed 30 days before initiating the hormonal protocol for fixed-time artificial insemination (FTAI).
A chalk marker was used in the sacral-caudal area on D9 to observe the estrus intensity on the day of insemination as described by Nogueira et al. (2019), wherein HEATSC1 referred to little or no chalk removal and consequently no estrus detection; HEATSC2 referred to partial chalk removal (< 75%), with low estrus detection; and HEATSC3 referred to almost complete (> 75%) or complete chalk removal, with high estrus detection.
Artificial insemination (AI) was performed at 48-54 h after implant removal using a single inseminator.As it was a commercial farm, inseminations were performed with six bulls as indicated by the mating system of the genetic improvement program to which the property belongs (National Association of Breeders and Researchers, Associação Nacional de Criadores e Pesquisadores, ANCP); thus, both groups received semen from the same bulls.
Pregnancy was diagnosed 30 and 60 days after AI using transrectal ultrasonography (Pie Medical 401347, Aquila, Esaote, Europe B.V.).The conception rate was calculated as the number of animals with a positive pregnancy relative to the number of inseminated animals, and the pregnancy loss rate was calculated as the number of pregnancies at 60 days relative to the number of pregnancies at 30 days after AI.

Physiological parameters
Physiological parameters (heart rate, HR; respiratory rate, RR; rectal temperature, RT, in Table 1; and surface body temperature, ST, in Table 2) were measured during the experiment on days 0, 9, and 11 in all experimental animals (n = 84), when the females were taken to the management corral and allocated to the individual containment trunk, where physiological parameters were measured.RR was measured by visually counting the animals' flank movements for 15 s and multiplying the result by 4, whereas HR was measured using a stethoscope (Littmann, USA) for 15 s and multiplying the result by 4. RT was measured using a veterinary digital thermometer (Brasmed, Brazil) and ST Six animals from each group received an iButton thermometer (iButton Temperature Loggers, model DS1922L, Maxim Integrated Products, Inc., San Jose, CA, USA) to measure the vaginal temperature (VT) during the FTAI protocol, without the need for animal containment (Fig. 2).The thermometers used in this study were newly purchased and already calibrated according to the manufacturer's parameters.According to the manufacturer, the temperature sensor was 17.35 mm in circumference and 5.89 mm in diameter, weighed 3.3 g, and had a temperature range of − 10 to 65 °C and an accuracy of ± 0.5 °C.They were coupled to an intravaginal implant without progesterone as described by Burdick et al. (2012).This device remained in the females for 9 days during the same period and under the same hormonal protocol as in the cows that were inseminated.

Statistical analysis
Statistical analyses were performed using PROC GLIM-MIX (SAS® OnDemand for Academics).The design was completely randomized, and an analysis of variance between the shaded area and less shaded area groups was used for comparison.In each experimental group, an analysis was performed considering the fixed effects of treatment on the reproductive variables (pregnancy rate, pregnancy loss, and detection of estrus) and physiological parameters (ST, TR, RR, and HR).VT, using iButton, was repeatedly analyzed over time to select the covariance structure that best explained the residual correlation.The Akaike information criterion (AIC) was used to select the model with the lowest AIC value.p < 0.05 was considered significant and values between 0.06 and 0.10 were considered to indicate a statistical trend.

Results
Table 1 shows the meteorological data (rainfall, minimum and maximum temperatures, and relative humidity) obtained from the INMET meteorological system during the experimental period (October to December).According to the data, atypical rainfall was recorded during the experimental period, generating higher precipitation than reported in previous years.During the study period, the mean value of the wet-bulb globe temperature (BGHI) was 81.41 ± 8.97 (maximum, 94; minimum, 75).The RT of Nelore cows managed in the shaded area was lower than that of cows managed in the less shaded area (p = 0.0221); however, the RR (p = 0.5503) and HR (p = 0.1477) were not significantly different between the groups (Table 2).
The rectal temperature was slightly lower in the group managed with more shade, but the other parameters did not vary between the groups (Table 2).
The ST was higher in animals managed in the less shaded paddock when measured on the flank, forehead, and shoulder (Table 3).VT was automatically measured every 60 min over 9 days using the iButton device.VT (Fig. 3) varied significantly between groups (p < 0.0001) and time of day (p < 0.0001).The VT peak occurred at 15:00 in the shaded area and at 17:00 in the less shaded area.The minimum VT occurred at 07:00 and 08:00 in the shaded and less shaded areas, respectively.
Estrus detection tended to be higher in females managed in shaded areas than in those managed in the less shaded areas (Table 4).Conception and pregnancy loss rates did not vary between the groups (Table 2).

Discussion
The present study demonstrated that the homeothermy of Nelore heifers was maintained at the classic high temperatures (exceeding 40 °C) recorded during summer in Central Brazil.However, this required the activation of thermoregulatory mechanisms, which were reduced in areas with greater shade availability.According to Primavesi (1989), the thermoneutral zone for zebu cattle is in the range of 10-27 °C, and the thermoregulatory mechanisms in zebu cattle begin to fail when the ambient temperature is greater than 35 °C.In our study, the average room temperature was 28.5 °C, and considering that the rectal temperature and respiratory rate remained within the normal range for the species (Stöber 1993), there was no failure of thermoregulatory mechanisms, as described by Pires et al. (2019).
In the present study, ST, RT, and VT were lower in animals managed in areas with greater shade (5.78 trees/ha), demonstrating greater thermal comfort, corroborating the findings of Oliveira et al. (2019).
The surface temperature of the skin in Nelore cattle is influenced by coat and skin characteristics such as color, length, and density.These morphological traits are associated with animal adaptations (Baena et al., 2019).Kennedy (1995) reported that the ease of metabolic heat dissipation in Bos indicus is attributed to its ability to redirect blood from the internal organs to skin capillaries, which increases direct heat dissipation by non-evaporative heat loss and indirect heat loss by sweat stimulation.Approximately 70-85% of the maximum heat loss occurs through evaporation and transpiration, whereas the rest is lost by breathing.As the air temperature approaches the skin temperature, evaporation becomes the main route of heat exchange with the environment (Hansen, 2004).In the present study, animals with more access to shade had a lower ST at all evaluated points; however, no variation in HR or RR was observed between the groups.According to Barbosa et al. (2014), when HR and RR do not follow the variation in ST, an efficient exchange of heat through evaporation (sweat) is confirmed, indicating a mechanism optimized by the adaptive characteristics of the skin and coat in these animals.
We observed that the VT was higher in cows managed in less shaded areas throughout the experimental period and at all times of the day.It is important to emphasize that in the present study, the heifers were synchronized so that all of them were in the same stage of the estrous cycle, providing more reliability to the data, as the physiological parameters of the female changed significantly between the follicular and luteal phases of the cycle (Vicentini et al., 2020).Additionally, all animals managed in the less shaded area also maintained the maximum temperature for a longer period and required a longer time to reach the minimum temperature than the group managed in the shaded area.Oliveira et al. (2019) showed that VT increases as the BGHI increases, and corroborating the results of this study, they found that heifers managed in paddocks with greater shade availability had lower VT.In line with the findings of the present study, these studies revealed a delay in the increase in VT relative to the peak heat of the day.The reduction in VT at night is caused by the increase in the animal-environment thermal gradient, which increases heat dissipation (Settivari et al.,   2007); the lower the environmental temperature, the greater is the gradient.As previously discussed by Oliveira et al. (2019), physiological heat loss in response to alterations in the animal-environment thermal gradient is not immediate, as regulatory mechanisms need to be activated to reduce the body temperature.This phenomenon is consistent with that described by Gaughan et al. (2008), indicating that cattle can accumulate heat during the day and dissipate heat at night.However, if adequate cooling does not occur at night, cattle may begin the following day with an accumulated heat load, resulting in prolonged heat stress.Under the conditions of the current study, we observed that cows managed in less shaded areas tended to have a lower estrus intensity.Heat stress is well known to reduce the concentration of estradiol, the hormone responsible for estrus behavior in cattle (Wolfenson and Roth, 2019).Although estradiol concentrations were not measured in the present study, the biological mechanisms of thermoregulation in Nelore cows may have interfered with estradiol concentrations and consequently with estrus behavior, as observed in native zebu cows from Thailand, which were evaluated under BGHI up to 83.2; however, they did not show changes in RT.They demonstrated smaller diameters and vascularization of the pre-ovulatory follicle, as well as a lower ovulation rate and estradiol concentration as the BGHI increased (Jitjumnong et al. 2020).
Contrary to expectations, considering the differences in estrus expression, the conception rate and pregnancy loss did not differ between the groups.This was unexpected because the results of a recent study by our research group showed that increased shading in integrated systems resulted in an increase of > 50% in embryo production in vitro (Silva et al. 2020).Follicular development from the preantral to dominant phase takes approximately 5 months in ruminants, with 3 months required for the development of preantral follicles to the antral phase and two estrous cycles between antrum formation and ovulation (Webb et al. 2004).Therefore, throughout folliculogenesis, several critical points are observed, which may change according to environmental factors such as heat stress.In the present study, the cows were separated into experimental paddocks 30 days before the start of the hormonal protocol (41 days until AI); therefore, there was sufficient time for antral development until ovulation.In this sense, the high antral follicular count of zebu females compared with that of taurus females (Batista et al. 2014) may be part of the adaptive mechanism of this breed for the tropical climate.A larger number of follicles may be necessary to ensure a greater chance of selecting the most resistant follicles or oocytes.
The main difference between the study by Silva et al. (2020), in which the effect of shading on reproduction was clear, and the present study was based on the principle of the biotechnique used.When recovering immature oocytes by follicular aspiration guided using ultrasonography, follicles undergoing apoptosis caused by heat stress can be aspirated, which could have reduced the efficiency of in vitro embryo production.The FTAI ovulatory follicle is the most resistant to stressful processes and can develop adequately for establishing fertilization and pregnancy.However, the effect of shading on the conception rate after FTAI needs to be confirmed in a study with a larger number of inseminated animals.
In this study, the improvement of thermal comfort and, consequently, animal welfare was clear in the shaded area; this result alone should be sufficient to motivate greater investments in properly shaded pastures for livestock production.
In conclusion, Nelore cows can maintain vital physiological parameters within the normal range with a BGHI ranging between 75 and 94; however, in poorly shaded areas, thermal comfort was lower, and some signs of the failure onset in the thermoregulatory process, such as the onset of vaginal temperature recovery (reduction) after the peak of the day, required longer times; furthermore, a lower estrus intensity was observed.Areas with native natural shading of approximately 5.8 trees/ha promoted thermal comfort but did not improve the TFAI pregnancy rate.

Fig. 1
Fig. 1 Image of the experimental areas (Google Earth): shaded area (A) and less shaded area (B).In both areas, paddocks I, II, and III are shown, and in (A), the forests to which the animals had free access (bI, bII, and bIII) are indicated.The shaded area (A) was 70.65 ha,

Fig. 2
Fig. 2 Experimental protocol performed using Nelore cows under different shading conditions in Central Brazil

Fig. 3
Fig. 3 Vaginal temperature (°C) of Nelore cows managed in less shaded and shaded areas throughout the day.The values refer to the average temperature across the 9 days of evaluation

Table 4
Estrus intensity in Nelore cows managed in less shaded and shaded paddocks