Has the soybean oil supplementation effect on milk production, feed efficiency, and grazing behavior of Girolando cows under moderate heat stress in western Amazonia?

The effect of soybean oil supplementation in the diet of early lactating Girolando cows under moderate heat stress were studied in a grazing trial. Dry matter intake (DMI), milk yield, feed efficiency, and grazing behavior (during daytime and nighttime) were evaluated in two groups of eight crossbred Girolando (Holstein × Gyr) cows grazing palisade grass ( Urochloa brizantha syn. Brachiaria brizantha ) with initial means of 53±33 days in lactation and 22.6±3 kg of milk day -1 . They were arranged in a 2×2 crossover trial with two supplements (with or without soybean oil), two genetic groups and two periods. During the experimental period, the mean of temperature and humidity index (THI) was 75.11, suggesting moderate heat stress. Total DMI was based on fecal production estimated by LIPE® and the diet in vitro DM digestibility (IVDMD). Grazing behavior was assessed for 48 hours by acoustic data Under moderate heat stress, oil supplementation did not change grazing behavior, dry matter intake, or milk yield in early lactating Girolando cows .


Introduction
In Rondônia, as throughout the Brazilian Amazon, there are areas characterized by a predominance of smallholders, where dairy farms using crossbred herd in pasture-based systems play an important role in the strengthening of dairy market chain (Soler et al., 2014). In this region, the climate is characterized by high air temperature coupled with high relative humidity and solar radiation (Alvares et al., 2014), which causes heat stress in livestock, especially dairy cows. Extended periods of high air temperature compromise the ability of dairy animals to dissipate excess body heat, which affects feed intake, milk production, and reproductive efficiency (Correa-Calderón et al., 2022).
Heat stress directly and indirectly affects the productive, physiological, and behavioral parameters of livestock.
Changes in grazing and rumination patterns, such as excessive dislocation and frequent water intake, can be signs of heat stress (Correa-Calderón et al., 2022). When DMI is reduced, management interventions that modify the diet composition to either promote a greater consumption or compensate for the low DMI may be possible. Nutritional recommendations to mitigate negative effects of heat stress on intake and metabolism have focused on ensuring constant availability of feed and adjustments to ration formulation to maintain energy density without lowering effective fiber. Supplemental fat has been advocated as a possible means of reducing heat stress on lactating dairy cows due to high energy content and low heat increment of fat relative to other nutrients. It reduces thermogenesis because fat generates less heat increment than dietary carbohydrate or protein (Wang et al., 2010).
Supplementing corn oil to grazing dairy cows has been shown to increases milk yield without affecting voluntary intake or diet digestibility (Girón et al., 2016), and Holstein cows supplemented with fish oil also have an increased milk yield (Pirondini et al., 2015). However, studies on the effect of oil supplementation on grazing crossbred dairy cows are limited.
The strategy of crossbreeding Bos taurus × Bos indicus has been used widely as a method to improve milk production under grazing conditions in the tropical and subtropical areas of Latin America. Brazilian breeders have sought to combine the desirable characteristics of Gyr, a Bos indicus breed which is resistant to hot temperatures and tropical diseases, with Holstein cows, a Bos taurus breed, this crossing originates the Girolando cattle (Dalcin et al., 2016).
In the present study, our hypothesis was that soybean oil supplementation reduces the negative effects of heat stress on forage intake patterns, including decrease in grazing and rumination time, which strongly affects milk production. The intake, milk yield, grazing behavior (during nighttime and daytime) and feed efficiency were accessed using lactating cows grazing palisade grass with or without soybean oil supplementation.

Materials and Methods
All management practices applied to the experimental animals were approved by the Ethics Committee on Animal Use (CEUA) of Embrapa Rondônia (process number 03-2014).
The trial was carried out on an experimental field of Empresa Brasileira de Pesquisa Agropecuária (Embrapa) located in Porto Velho, Rondônia state, Brazil (8º47'38''S, 63º50'46''W). The predominant climate in this region is Am, which is characterized by a dry season from June to August and annual rainfall exceeding 2,250 mm (Alvares et al., 2014).
The trial was carried out from February to March 2015, when the daily average air temperature and precipitation were 25.75 °C and 275.1 mm, respectively. The daily means of the environmental parameters registered by the weather station located 500 m from the experimental field were used for the estimation of the temperature-humidity index (THI) with the following equation by Thom (1959): THI= Tdb+ 0.36Tdp + 41.2, where Tdb is the dry bulb temperature (°C) and Tdp is the dew point temperature (°C).
The average THI during the experimental period was 75.11; during the nighttime (from 18:00 to 5:59) and daytime (from 6:00 to 17:59), the average THIs were 73.69 and 76.52, respectively.
Eight ½H×G (n = 4) and ¾H×G (n = 4) lactating multiparous cows were used for this experiment. At the start of the experiment, these cows had an average live weight (LW) of 471±53 kg, spent an average of 53±33 days in lactation, and produced an average of 22.6±3 kg of milk day -1 . Cows were distributed in a 2×2 crossover trial with two supplementation (with or without oil supplementation) and two 20-day experimental periods, with 15 days for diet adaptation and 5 days for data collection, totaling a 40-day trial period.
Cows were assigned to a treatment according to their genetic group and their average daily milk yield at the beginning of the experimental period, to control for these variation factors within the experimental design. During the total experimental period, cows grazed on palisade grass (Urochloa brizantha cv. Marandu), managed with an intermittent stocking with 2 days of occupation and 28 days of resting. The mean stocking rate was 2.5 animal unity (AU; 1 AU = 450 kg of body weight), and the forage available for grazing was 68 kg DM.AU -1 .day -1 . Cows had free access to water and mineral mixture.
The animals were mechanically milked twice daily (at 7:30 and 13:30), when they received the experimental rations.
During the last five days of each experimental period, individual milk yield (kg cow -1 day -1 ) was calculated as a sum of the total milk from the morning and afternoon milking, weighed by a portable electronic scale (Zarna Instruments Company©, India) with a 40 kg capacity and 10 grams accuracy. Research, Society and Development, v. 11, n. 14, e289111435663, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i14.35663 In the soybean oil supplementation group (+SO), the concentrate ration contained 7% of soybean oil (based on the DM), unlike the concentrate ration without soybean oil (-SO). The proportion of ingredients in the experimental rations (Table   1) were balanced for 25 kg of daily milk production and similar levels of metabolizable energy (ME) using the Cornell Net Carbohydrate and Protein System (CNCPS) software v6.5 (Cornell University, Ithaca, NY, USA).
The individual amount of concentrate ration offered to each cow was adjusted before starting each experimental period based on their last mean of milk yield. One kilogram of ration was given to the cow for every three kilograms of milk produced above the daily production amount of eight kilograms. The experimental rations were offered individually in two daily equal meals during the milking. Soybean oil was added and manually mixed to the +SO ration just before each meal.
Marandu palisade grass pasture samples were obtained by hand-plucking at the grazing for intake measurements. At  Table 1). Total DMI (DMItotal, kg day -1 ) was estimated by: DMItotal = TFP(1-IVDMD) -1 , where TFP is the total fecal production (kg fecal DM day -1 ) of each cow estimated by the LIPE ® (Produtos de Pesquisas Simões Saliba, Florestal City, MG, Brazil) marker supplied to animals. Pasture DMI (DMIpasture, kg day -1 ) was calculated from the difference between DMItotal and the intake of the DM concentrate ration (DMIration, kg day -1 ).
Grazing behavior was accessed by bioacoustic methodology (Souza et al., 2019) using MP3 audio recorders adapted to the cow's halters to register the sounds of grazing, ruminating, and resting on the 17 th and 18 th day of the experimental period. The audio was continuously recorded for 48 hours, and the time spent for each activity during the nighttime (from 18:00 to 5:59) and the daytime (from 6:00 to 17:59) was quantified by the Audacity ® software (Free Software Foundation, Boston, MA, USA).
Statistical analysis of the data was performed using the MIXED procedure in Statistical Analysis System (SAS) with the restricted maximum likelihood method (RELM). The model used for the grazing behavior data (time spent grazing, rumination, and resting) was: yijklmn = μ + τij + βj + ρk + σl + δm + σδlm + σδlm + eijklm; where µ is the general mean, τ is the random effect of each individual animal, β is the effect of the sequence, ρ is the period (experimental period 1 and 2), σ is the ration type (soybean oil and control), δ is the day period (repeated measures corresponded to the daytime and nighttime), and e is the unexplained random error.
The model used for the parameters of the DMI (total and pasture), milk yield, and feed efficiency was: yijklm = μ + τi + βj + ρk + σl + eijkl ; where µ is the general mean, τ is the random effect of each individual animal, β is the effect of the sequence, ρ is the period (experimental period 1 and 2), σ is the ration type (soybean oil and control), and e is the unexplained random error. The means were compared by Tukey's test at 5% significance.

Results and Discussion
The mean THI during the experimental period was 75.11. According to Armstrong's classification, a THI between 72 and 78 suggests that cows are subjected to mild or moderate heat stress (Armstrong, 1994). Under heat stress, animals reduce Research, Society and Development, v. 11, n. 14, e289111435663, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i14.35663 5 their feed intake and time spent grazing and ruminating. It is well reported that heat stress limits animal feed intake, which directly and indirectly affects the production performance and feed behavior in bovines (Sammad et al., 2020).
DM available in pasture during the experimental period was 3,830 kg DM ha -1 , and the post-grazing residue was 2,688 kg DM ha -1 , which is above the 2,250 kg DM ha -1 considered the minimum for not limiting DMI by grazing cattle (NRC 2001). The mean of DMIpasture (in %LW) was 2.13%, which is within the variation found in previous studies; in studies by Fukumoto et al. (2010) and Sousa et al. (2008), the means of pasture DMI (in %LW) were 2.0% and 2.4% for Brachiaria pasture grazed by lactating Holstein × Gyr cows daily supplemented with eight and two kilograms of concentrate rations, respectively. Supplementation with soybean oil had no significant effect on milk yield, DMI, and feed efficiency (Table 1). During the experimental period, averages of individual milk yield and DMItotal were 19.26 kg day -1 and 15.32 kg DM day -1 , respectively.
The daily intake of TDN did not differ between cows supplemented with or without soybean oil.
The addition of soybean oil in the diet of lactating cows did not change DMItotal or DMIpasture, and also the time spent with grazing and ruminating. This was likely due to the level of EE (6.7%, DM basis) in the diet ( Table 1) that was lower than that of 8% suggested as an intake limitation for lactating dairy cows (NRC, 2001). The reduction of DMI after dietary fat inclusion (above the level of 8%, DM basis) may be a result of an increase in ruminating time due to negative effects on rumen digestion; a slowdown of rumen emptying due to a metabolic effect of long chain fat acid. In both situations, a satiety effect due to rumen replenishment could occur (Martínez Marín et al., 2013). Cows supplemented with soybean oil had a significantly higher intake of ether extract (EE) and a significantly lower intake of ADF and TC (Table 2). This difference was expected due to the proportion of these nutrients in the SO+ ration ( Table   2). The daily intake of DE, ME, NE, and TDN did not differ between cows supplemented with and without soybean oil. Soybean oil supplementation had no significant effects on grazing behavior parameters; however, the cows spent more time grazing and ruminating at night than during the day (Table 3).
The cows in our study spent more time grazing and ruminating at night and resting during the day (Tables 3); during the daytime, the pasture was sunny with a THI of 76.52, compared to 73.69 at nighttime. Under these conditions, animals may prefer to feed at night when the air temperature is mild (lower THI) and avoid grazing during the warmer times of the day (Schmeling et al., 2022).
In contrast, a study by Oliveira et al. (2011) found that lactating Holstein vs. Zebu cows grazing Brachiaria decumbens in Santo Antônio da Alegria City (Bahia State, Brazil) allocated a greater percentage of the daytime grazing compared to other behavioral activities, as ruminating and resting. These authors suggest that this behavior was caused by once-a-day milking before dawn, which intensified grazing. Additionally, the cows in that study had access to a shaded area, which would affect the preference for grazing during the daytime. In another study, we also found that, during the daytime, dairy heifers spend more time grazing in a silvopastoral when compared to heifers in pasture with full sun condition (Souza et al., 2019).
After 24 hours of visual observations, the Oliveira et al. (2011) found that lactating Holstein×Zebu cows spent 718.59, 388.09, and 333.32 minutes grazing, ruminating, and resting, respectively. Cows spent 50% more time ruminating and resting (Table 3) in this study, because we assessed behavioral parameters by a bioacoustics method for 48 hours. Adjusting for a 24 hour period, the cows in this study spent 371.34 minutes grazing, which was 50% lower than that found in Oliveira et al. (2011) and below the 480 to 540 minute range considered as the minimum required for feed intake (Phillips and Rind, 2001).
Time spent by animals in grazing activities, such as grazing, rumination, and resting, is related to climate conditions, pasture traits (forage allowance and sward structure), and the physiological state of the animal, and thus relate to animal performance (Pérez-Prieto et al., 2011).

Conclusion
Under moderate heat stress, soybean oil supplementation to a diet of grazed palisade grass did not change milk yield, pasture intake, or grazing behavior of lactating crossbred cows.
We suggest carrying out further studies with a larger number of animals and testing different levels of inclusion of soybean oil in the diet of crossbred Holstein x Gir cows in lactation under grazing and heat stress.