Energy requirement for primiparous Holstein × Gyr crossbred dairy cows

Our objective was to estimate the requirements of metabolizable energy (ME) and NE M of lactating and dry cows, the efficiency of ME utilization for milk production ( k l ) and tissue gain ( k g ), and the use of body energy mobilization for milk production ( k t ) throughout the lactation of primiparous crossbred Holstein × Gyr cows, using open-circuit respiration chambers. Twenty-nine primiparous Holstein × Gyr crossbred cows with an initial BW averaging 563 ± 40.1 kg and 2.5 ± 0.09 yr old were used throughout lactation and dry periods. The cows were kept nonpregnant throughout the study to eliminate possible confounding factors. Apparent digestibility assays, followed by calorimeter measurements, were performed 6 times throughout the lactation period. In the dry-off period, the cows were also evaluated but fed with restricted intake (DMI = 1.1% BW/d) to achieve heat production close to maintenance. After 21 d of diet adaptation, an apparent digestibility assay followed by calorimeter measurements was performed. Parameter estimates for lactation period were obtained by mixed models including lactation stage as repeated measures. For restricted feeding at dry-off and fasting period assays, the requirements were estimated by exponential regression. For whole lactation, the values of the ME requirement for maintenance (ME M ) and NE M were 0.588 and 0.395 MJ/BW 0.75 , respectively. The efficiencies of k l , k g , and k t were 0.672, 0.771, and 0.814, respectively. However, ME M and NE M were higher in early and mid lactation than late, whereas k l was higher in early than other lactation stages. Dry and nonpregnant cows had ME M of 0.434 MJ/BW 0.75 and NE M of 0.351 MJ/BW 0.75 for maintenance level, and ME M of 0.396 MJ/BW 0.75 and NE M of 0.345 MJ/BW 0.75 for fasting metabolism level, and efficiency of ME utilization for maintenance was 0.80. Our findings confirmed that F 1 crossbred Holstein × Gyr dairy cows have differences in energy requirement and efficiency throughout the lactation stages, suggesting the use of different values in each stage. The estimated values of energy requirement for maintenance and efficiencies for primiparous lactating crossbred Holstein × Gyr were similar to those reported in the literature in specific studies and requirements systems.


INTRODUCTION
The energy requirements of dairy cattle have 2 important key parameters: the metabolizable energy (ME) requirement for maintenance (ME m ) and the efficiency of utilization of the ME intake for milk production (k l ).In the current energy systems for dairy cattle, the net energy (NE) or ME requirements for maintenance and milk production, as a k l , were derived from indirect calorimetry trials mainly using Holstein or Holstein-Friesian cows in temperate climate conditions (AFRC, 1993;NRC, 2001;CSIRO, 2007;INRA, 2018;NASEM, 2021).
Accurate estimates of energy requirements for dairy cattle allow nutritionists to formulate efficient diets, meeting maintenance and production needs.This approach enhances animal performance, reduces nutrient loss, and optimizes feed resource utilization.Meeting animals' requirements effectively mitigate environmental impacts associated with livestock (Knapp et al., 2014).However, prevalent systems for balancing diets and estimating NE or ME requirements for dairy cows do not adequately consider crossbred animals in tropical conditions, which exhibit lower production than purebreds like Holstein and Jersey in temperate climates.Previous studies estimating the energy requirements of crossbred animals in tropical conditions have identified differences in requirements and energy utilization efficiency when contrasted with Bos taurus purebreds (Oliveira, 2015;Oss et al., 2016;Sguizzato et al., 2020).Therefore, further studies are needed to estimate energy requirements for Bos Taurus × Bos Indicus dairy cows across different genetic compositions and productive stages.
Crossbreeding of Holstein and Zebu cows can provide good udder health, workability, heat stress tolerance, and resistance to endo-and ectoparasites (Vieira Ventura et al., 2020).Although F 1 crossbred Holstein × Gyr cows produce less milk than crossbreds with a higher proportion of Holstein, they have reduced age at first calving and calving intervals in tropical conditions (Canaza-Cayo et al., 2018).Milk production in tropical conditions using F 1 Holstein × Gyr cows can maximize the use of heterosis, combining higher milk production from Holstein and adaptability to tropical conditions from Gyr (Carvalho et al., 2019).Therefore, the crossbreeding of Holstein and Gyr in the tropics ally high productivity and resistance to tropical challenges.
Bos taurus × Bos indicus dairy cows have lower maintenance requirements but lower energy efficiency for milk production than Bos taurus cows (Oliveira et al., 2015).Properly estimating their energy requirements and efficiency of its use is crucial for dairy farms relying on crossbred dairy cows.Despite the comprehensive understanding of energy requirements and efficiency in Bos taurus animals (NASEM, 2021), to our knowledge, estimation for F 1 Crossbred Holstein × Gyr considering changes during lactation is still not described.
This study aimed to estimate i) NE for maintenance (NE m ), ME for maintenance (ME m ), the efficiency of ME utilization for milk production (k l ), tissue gain (k g ), and the use of body energy mobilization for milk production (k t ) throughout the lactation (whole, early, middle, and late) of primiparous crossbred Holstein x Gyr cows; and ii) NE m , ME m and k l for nonlactating and non-pregnant Holstein × Gyr crossbred cows in the dry-off period.

MATERIALS AND METHODS
The study was performed at the Bioenergetics Laboratory of the Brazilian Agricultural Research Corporation (Embrapa), at the Multi-user Laboratory of Livestock Bioefficiency and Sustainability at Embrapa Dairy Cattle, in Coronel Pacheco, Minas Gerais, Brazil, between April 30, 2017, andMay 09, 2018.This location is characterized by a humid subtropical climate -Cwa according to the Köppen-Geiger classification (Kottek et al., 2006) with an altitude of 437 m.Weather data were obtained from a meteorological station located at the farm with maximum, minimum, and average temperature (°C) and humidity (%) of 26 ± 2.0, 17 ± 2.5, 22 ± 4.9 and 79 ± 4, 59 ± 6, 70 ± 10, respectively.All animals were born and raised on the experimental farm until the study.All animal care and handling procedures were approved by the Embrapa Dairy Cattle Animal Care and Use Committee (Juiz de Fora, Minas Gerais, Brazil; Protocol CEUA-EGL 9264220317).

Animals and Management
Twenty-nine primiparous Holstein × Gyr crossbred cows were included in the study.Thirty days before the experiment commencement, the cows were housed in a stall with access to Cynodon sp.pasture for calving.During this period, they had ad libitum access to water and the prepartum diet.Immediately after calving, the cows transitioned to the experimental management and sampling phase, being accommodated in a free-stall system based on the calving date.
At the beginning of the experiment, the animals had an initial BW averaging 563 ± 40.1 kg and were 2.5 ± 0.09 years old.The experiment duration covered the period 0 to 300 DIM and included restricted feeding at dry-off and a dry fasting period.The cows remained non-pregnant throughout the study and were not subjected to reproductive routines.

Diet and Intake Measurements
The experiment was carried out in 5 stages, encompassing 3 lactation periods: early (0-100 DIM), middle (101-200 DIM), and late (201 -300 DIM) lactation; and 2 evaluations after dry-off, consisting of 26 d over restricted feeding and a 3-d dry fasting period (Figure 1).Data from 0 to 9 DIM were considered as adaptation periods to the facilities, diets, milking routine, and feeders.
All animals were fed the same diet (TMR), with 3 distinct diets outlined in Table 1.Cows were fed twice daily immediately after milking at 0800 and 1600 h, allowing ad libitum intake, with 5 to 10% of orts permitted.The intake was measured daily in the morning feeding.Samples of 300 to 500 g of corn silage and orts were taken 3 times a week and pooled into 2-week periods for chemical composition analysis.Hay and concentrate were sampled once a week and pooled into 4-week periods for analysis.All samples were packed in plastic bags and frozen at −20°C until processing.Intake was measured by individual electronic feed bins and headgates (AF-1000 Master Gate, Intergado Ltd., Contagem, MG, Brasil).Additionally, 2 electronic water bins per pen (WD-1000, Intergado Ltd., Contagem, Minas Gerais, Brazil) were utilized.Individual feed and water intake were monitored (Chizzotti et al., 2015), and each feed bin was randomly assigned to a single cow.

Milk and Body Weight Measurements
Milk samples were collected twice a week, preserved at 5°C with bronopol, and analyzed within 24 h using the Bentley FTS system (Bentley Instruments, Chaska, MN) following the recommendations of the International Dairy Federation (IDF, 2000).Body weight was measured continuously through an automatic weight scale attached to the water bins (WD-1000, Intergado Ltd., Betim, Minas Gerais, Brazil).The daily BW represented the average of weights recorded each day.Outliers were excluded considering the average body weight  of each animal and accepting a variation between daily measurements of ± 50 kg.

Energy Balance Assays and Respirometry
Six total-tract nutrient digestibility assays were performed per animal.During lactation, 2 assays were conducted for each lactation period at 46 ± 8,79 ± 17,138 ± 15,179 ± 20,251 ± 15,and 283 ± 13 DIM,respectively.In each trial, groups of 8 cows were moved to a tie-stall facility located close to the free-stall, milking parlor, and respiration chambers.Samples of concentrate, silage, and orts were collected over the 5 d of the assay to evaluate DM and nutrient intake.Throughout the sampling period, cows were milked in their stalls at 0730 and 1430 h using a portable milking machine (Weizur do Brazil Ltda, Sorocaba, Brazil).Individual milk yield was determined by weighing, and a sample from each animal was collected at each milking for milk composition analysis.
In each trial, total feces were collected for 5 d.At the end of each collection day, each animal`s feces were weighed and sampled after homogenization.The samples were then dried in a forced ventilation oven (55°C) for 72 h and ground through a 1-mm screen (Wiley mill; A. H. Thomas, Philadelphia, PA) for laboratory analysis.Simultaneously with fecal sampling on d 2, 3, and 4 of each trial, spot urine samples were collected.Samples (200 mL) were collected through stimulation of the pudendal nerve by massaging the area below the vulva or during voluntary urination at 8-time points with 3 h intervals split up over 3 consecutive days.After daily sampling, 2 aliquots were made: the first 10 mL was acidified with 40 mL of sulfuric acid (H 2 SO 4 , one mL/L) for creatinine and purine derivatives analysis, and the second aliquot was used for gross energy analysis, both stored at −20°C.
Following digestibility assays, cows were placed in respiration chambers for 2 periods of 24 h.The cows were removed from the chambers at milking times except for restricted feeding at dry-off and fasting periods.In that case, 22 h of respirometry data were extrapolated for 24 h.To minimize the possible effect of the respiration chamber, cows were randomly assigned to each chamber.Four open-circuit respiration chambers were used according to the specifications and procedures described by Machado et al. (2016).Each animal was placed in an enclosed space where the air was continuously ventilated, allowing for the measurement of oxygen consumption (VO 2 ), carbon dioxide (VCO 2 ), and methane (VCH 4 ) production.The oxygen, carbon dioxide, and methane concentrations in the incoming and outgoing air were analyzed to calculate metabolic rates and derive information about energy expenditure and nutrient utilization.Cows were individually housed inside the chambers after the first milking of the day.Cows were weighed to measure BW directly before and after each respiration chamber measurement.The chambers were maintained under thermoneutral conditions for crossbred Gyr x Holstein cows (23 ± 1°C and relative air humidity of 65 ± 5%).The respiratory quotient (RQ) was calculated using the ratio of CO 2 produced to O 2 consumed.Methane emission was calculated according to Machado et al. (2016).To determine energy partitioning, the gross energy of feed, orts, feces, and urine samples collected during the digestibility assay were previously determined using an adiabatic calorimeter (IKA -C5000, IKA Works, Staufen, Germany).Other energy fractions were calculated from the quantification of gross energy intake (GEI), obtained by the difference between the dietary energy and that found in orts.

Maintenance and fasting procedure
After drying off, the late lactation diet was individually offered at a restricted feeding level of 1.1% kg of DM per kg of BW (Table 1).The restriction was enforced to ensure consistent body weight, promote stability in nutritional conditions, and enable a focused study of metabolic dynamics during this period.After a 21-d adaptation period, the cows were transferred to the tie-stall facility for energy balance and respirometric measurements during restricted feeding at dry-off and fasting evaluations.The assay for totaltract nutrient digestibility and respirometric measurement were conducted as previously described.Fasting measurements were implemented as a deliberate and controlled approach to isolate and accurately measure maintenance energy requirements and were carried out immediately after the restricted feeding at dry-off assay.Fasting minimizes the influence of digestive processes, nutrient absorption, and other metabolic activities associated with feeding.Cows were confined inside the chambers for 72 h without access to feed.The chamber was randomly changed every 24 h, and thermoneutral conditions were maintained.The duration of the fasting period was sufficient to reduce the RQ to 0.74 and methane production to a negligible amount (<0.22/kg of BW 0.75 daily).
Urine samples were analyzed for creatinine concentrations (kit no.500701, Cayman Chemical Co., Ann Arbor, MI) using a chromate microplate reader set at a wavelength of 492 nm (Awareness Technology Inc., Palm City, FL).Allantoin concentrations were determined based on the method described by Chen et al. (1992), and uric acid concentrations were measured using assay kit no.1045-225 (Stanbio Laboratory, Boerne, TX).Allantoin and uric acid were determined at 522 and 520 nm wavelengths, respectively, in a UV/visible spectrophotometer (Beckman Coulter Inc., Pasadena, CA).The daily volume of excreted urine was estimated based on urinary creatinine concentration, assuming a constant creatinine excretion rate of 29 mg/kg of BW/d, according to Valadares et al. (1999).Urinary excretion of allantoin, uric acid, and total purine derivatives (allantoin plus uric acid) was calculated by multiplying the concentration of each metabolite by the urinary volume.

Calculations and statistical analysis
Digestible energy (DE) was obtained by the difference between the gross energy (GE) consumed and the energy lost in feces.Subsequently, metabolizable energy (ME) was calculated by discounting urine and CH 4 energy losses.Energy lost as CH 4 was calculated considering 0.395 MJ/L of CH 4 (Brouwer, 1965).The metabolizability (q) of the diet was calculated by the relation between the intake of ME and GE.
The net energy for lactation (NE l , MJ/day), defined as the energy contained in the milk produced, was calculated based on the equation proposed by NRC (2001), which considers NE L as the sum of the heat of combustion of individual milk components, as follows: [3] The relationships between ME/DE, HP/ME, NE l /ME, and EB/ME were also calculated as indicators of energy use efficiency.
Metabolizable energy for maintenance, k l , k g, and k t throughout the lactation and for each period (early, middle, and late lactation) was estimated from multiple linear regression analysis proposed by Moe et al. (1971): where: MEI is the dietary ME intake (MJ/kg of BW 0.75 per day), NE l (MJ/kg of BW 0.75 per day), PEB is the positive energy balance (MJ/kg of BW 0.75 per day), and NEB is the negative energy balance (MJ/kg of BW 0.75 per day); PEB and NEB were zero if the cow was in negative or positive energy balance, respectively; β 0 is the intercept and, β 1 , β 2 , and β 3 are the estimates parameters describing the change in MEI with unit changes in EL, PEB, and NEB, respectively.According to Moe et al. (1971), β 0 represents the ME m , k l = 1/β 1 , k g = 1/β 2 , and k t = β 3 / β 1 .
The energy requirement of maintenance for restricted feeding at dry-off and fasting period assays were estimated by the following model (Moe et al., 1971): where HP is the heat production (MJ/kg of BW 0.75 per day), MEI is the dietary ME intake (MJ/kg of BW 0.75 per day), β 0 and β 1 are the fixed parameters, δ are random effects of cows.ME m (k m ) efficiency was obtained from the NE m to ME m ratio.
For lactation data, the following model was used: where Y ijklmn = dependent variable, μ = overall mean, EL i = fixed effect of milk energy output, PEB j = fixed For the maintenance data, ME intake, feeding stage (lactation, restricted feeding at dry-off, and fasting), and the interaction between ME intake and feeding stage were included in the model as a fixed effect.The cow was included in the model as a random effect.Values are presented as least squares means with standard errors of the mean.The following model was used: where Y ijk = dependent variable, μ = overall mean, MEI i = fixed effect of ME intake, F j = fixed effect of feeding stage, A k = random effect of cow, MEI i × F j = interaction between ME intake and feeding stage, and e ijk = residual error.Covariance structures for repeated measures of the feeding stage were selected by the lowest corrected Akaike information criterion.The normality of residuals was checked with normal probability and box plots and homogeneity of variances with plots of residuals versus predicted values.Outliers were removed from statistical analyses when studentized residuals were >2.5 or < −2.5.

RESULTS & DISCUSSION
Numerous studies have presented measures of energy requirements and efficiency parameters, predominantly using pure-breed animals, with Holstein and Jersey cows being among the most studied breeds (Agnew and Yan, 2000;Moraes et al., 2015;Guinguina et al., 2020).Traditionally, the energy requirement for dairy cows utilizes data from studies using Holstein or Holstein-Friesian cows under temperate climate conditions, characterized by moderate temperatures and distinct seasonal changes, and has limited consideration for possible differences in metabolic rates between pure Holstein cows and their crossbreeds (AFRC, 1993;NASEM, 2021).Holstein × Gyr crossbred dairy cows have a prominent contribution to milk production under tropical conditions, described by warm climate, high humidity, and absence of distinct seasonal changes.However, there is still a gap in knowledge regarding the energy requirements of Holstein × Gyr crossbred dairy cows (Oliveira, 2015).

Lactation period
The current study indicates distinct variations in energy requirement across different stages of lactation and disparities between the data derived from whole and individual lactation stages.Notably, cows in the early stage of lactation exhibited higher requirements of ME and NE than the other stages, aligning with expectations.We estimate whole lactation values of 0.588 and 0.395 MJ/BW 0.75 for ME m and NE m , respectively (Table 2).Values of ME m and NE m were higher in early (+27% and +41%) and middle (+27% and +41%) lactation when compared with late lactation values (Table 2).The heightened requirement is attributed to increased milk production, potential shifts in energy balance, and heightened energy needs for production during the initial stages of lactation.These dynamic factors contribute to fluctuations in energy requirements, as explained by Moe et al. (1972).Furthermore, the current study relies on respiration chamber data, which is considered a more precise method for estimating energy requirements compared with approaches such as estimates derived from body weight changes or comparative slaughter (Moraes et al., 2015).
In a meta-analysis of respiration chambers conducted by Dong et al. (2015), data from Holstein, Norwegian, and Crossbred cows (F 1 Holstein × Norwegian and Holstein × Jersey) were examined throughout the entire lactation period.The authors demonstrated that the values of ME m estimated from the linear regression of milk energy output adjusted to zero energy retention against ME intake were similar between the groups.Dong et al. (2015) documented values of 0.688 and 0.686 MJ/BW 0.75 for Holstein and non-Holstein dairy cows, respectively.These values diverge when compared with the results presented in the current study for whole lactation (0.588 MJ/BW 0.75 ) and late lactation stage (0.544 MJ/BW 0.75 ), and interestingly, align with our values for early (0.689 MJ/BW 0.75 ) and middle (0.672 MJ/BW 0.75 ) lactation (Table 2).This discrepancy prompts a thorough examination of factors contributing to the observed variation in ME m values between the studies.
The results from whole lactation in our study corroborate the findings of Moraes et al. (2015), that using multivariate and univariate analysis to analyze energy balance data from Holstein lactating cows and investigated potential changes in maintenance requirements and partial efficiencies of energy utilization, established value of 0.57 MJ/BW 0.75 to ME m .Likewise, the ME m presented in our study is within the range of estimates (0.49 to 0.67 MJ/BW 0.75 ) valued by Agnew and Yan (2000).Indicating no difference in the metabolizable energy requirement for maintenance between studies with purebred Holsteins and F 1 Holstein × Gyr cows.
The ME m values observed in our study also corroborate with those estimated by Oliveira (2015), applying a meta-regression using feeding trials data with lactating Bos taurus × Bos indicus cows in tropical conditions (Oliveira, 2015).The value obtained by Oliveira (2015) for ME m was 0.558 ± 0.103 MJ/BW 0.75 , which is within the range of whole lactation data in our study.However, the author concluded that crossbreed cows have lower ME m requirements than Bos taurus pure-breed (Holstein mainly) cows.The differences in energy requirements were justified due to the lower milk production.Additionally, the energy requirements for maintenance can represent a larger share of the total net energy due to the lower viscera/liver size and activity and lower body protein turnover of Gyr cows.These factors could contribute to lower endogenous energy expenditure in crossbred Holstein × Gyr dairy cows (Oliveira, 2015).
In our study, the whole lactation values of NE m ranged from 0.37 to 0.42 MJ/BW0.75 (Table 2), which is similar to the value adopted by NASEM (2021) 0.42 MJ/BW 0.75 (Moraes et al., 2015).Furthermore, our values were similar to other studies using crossbred and pure-breed Jersey cows (Carvalho et al., 2019;Morris and Kononoff, 2021;Piazza et al., 2023).Piazza et al. (2023) analyzed data from 3-breed crossbred cows of Viking Red, Montbeliard, and Holsteins breeds with purebred Holstein cows computed NE m of 0.41 MJ/ BW 0.75 .Carvalho et al. (2019) evaluated middle lactation F 1 Holstein × Gys cows by comparing 4 different nutritional plans (Ad libitum and feed restriction of 5, 10, and 20% DMI related to the Ad libitum period).They observed a NE m of 0.44 MJ/BW 0.75 , which is similar to the value estimated in this study (Table 2).Additionally, Morris and Kononoff (2021), evaluating maintenance energy requirements and efficiencies of lactating and dry Jersey cows, suggested that there are no differences when compared with Holstein animals.This statement is supported by Olson et al. (2010), who, analyzing data from Holstein, Jersey, and reciprocal F 1 crossbred cows, concluded that there were no differences in net energy requirement for production and that the energy balance was not significant between the genetics groups.
Estimates of the efficiency of utilizing dietary ME for tissue gain (k g ) and the efficiency of using body tissues for milk production (k t ) were similar to the k g and k t proposed by Moraes et al. (2015).Their values of k g were 0.75 and 0.70 compared with 0.77 using whole lactation data, and 0.79 and 0.75 for early and mid-lactation data, respectively, and their values of k t were 0.80 and 0.89 compared with 0.81 using whole lactation data, and 0.85 for the early stage of lactation (Table 2).Moreover, Moraes et al. (2015) suggest that there may be substantial differences in the estimation of efficiencies between European and North American databases rather than differences in the models used to estimate energetic efficiencies.This can be emphasized by the divergence in k g between our study and the results published by Kirkland and Gordon (2001), where the authors observed k g values of 0.14, 0.40, and 0.44 for early, middle, and late lactation stages.Differences in k g could be an outcome of the gain composition and different degrees of cow maturity at the beginning of lactation, and k g is theoretically affected by diet composition and how differences in the nutrient fractions containing dietary ME have the potential to alter the efficiency of dietary energy utilization.
Similarly, the estimates k t from our study suggest a high efficiency of utilization of body stores for milk production, which is in good agreement with the NASEM (2021).Constant efficiency of tissue utilization ranged between 0.82 and 0.84 in a study by Agnew et al. (2003) comparing different feeding systems.In contrast, Derno et al. (2019), evaluating 2 distinct groups with high and low metabolic efficiency throughout the lactation, found a broader range of 0.73 to 0.97.The values presented in our study ranged from 0.81 to 0.98 (Table 2), aligning with the findings of both studies.Two important points must be considered while estimating energy efficiencies.The first is that the tissue energy balance calculations are subjective to cumulative errors from measurements of ME intake, heat production, and milk energy output.Second, is the instability in estimating energetic parameters from indirect calorimetry and the inherent correlation between energetic efficiencies within a model (Moe et al., 1971;Strathe et al., 2011).
Evidence indicates that the efficiency of utilizing dietary ME for milk production (k l ) remains relatively constant in a wide range of conditions such as dietary composition, animal characteristics, genotype, and production level (Agnew and Yan, 2000).However, the findings in our current study, using crossbred Holstein × Gyr cows, introduce some complexity to this established theory.While our results (k l = 0.67; 0.65-0.69)align with values previously reported by Agnew and Yan (2000) (k l = 0.66), and NASEM (2021) (k l = 0.66), utilizing predominantly Holsteins cows, they deviate of the value reported by other authors (0.64, 0.63, and 0.64 as reposted by Dong et al., 2015;Moraes et al., 2015;and Guinguina et al., 2020.Furthermore, they deviate by being ~26% higher when compared with the value reported by Oliveira (2015) (k l = 0.53), derived from data involving Bos Taururs × Bos indicus animals.This discrepancy challenges the notion of a uniform k l value across different studies and warrants further investigation into the factors influencing these variations.Ferris et al. (1999), using Holstein dairy cows with high and medium genetic merit and different concentrate proportions, conclude that k l decreases with the increasing proportion of concentrate in the diet.Even our results showed the opposite; the k l demonstrated in this study decreased with lower concentrate proportion, and our value of k l for the whole lactation data were similar (0.67 vs. 0.67) compared with 37% of concentrate proportion included in the diet (Ferris et al., 1999).The decrease in k l in this present study followed the lactation stages.Cows at the final lactation stage had lower k l than cows at the early stage (Table 2).Our results are in line with studies of Kirkland and Gordon (2001) and Derno et al. (2019), who reported that cows have greater k l in early lactation stages and that the proportion of MEI partitioned to milk energy decreases as the animal progresses through lactation.
The value of 0.35 MJ/BW 0.75 for NE m was close to estimates of other studies.Estimates for nonlactating and non-pregnant Holstein cows of 0.31 MJ/BW 0.75 were reported (Flatt et al., 1965), which was adopted by NRC ( 2001) with 10% of addition for activities.Birnie et al. (2000) also evaluating fasting energy metabolism of nonlactating and non-pregnant Holstein cows with different body score conditions, reported a NE m of 0.35 MJ/BW 0.75 .Data provided by Morris and Kononoff (2021) showed a NE m of 0.42 MJ/BW 0.75 for dry Jersey cows, which is within our 95% confidence interval (Table 3).The authors concluded that derivation for NE m determined using lactating cow data did not differ from dry cows (Morris and Kononoff, 2021).The values from our study emphasize the statement that NE m for lactating and nonlactating F 1 Holstein × Gyr may not differ.
The efficiency of metabolizable energy utilization for maintenance (k m ) in this study was estimated at 0.80, ranging from 0.64 to 0.97 (Table 3).This value is comparable to the findings of Birnie et al. (2000), where k m was reported as 0.73.The k m assessment is crucial for understanding the relative efficiency of dietary energy compared with body total energy for meeting maintenance needs.Birnie et al. (2000) observed that the small effect of each kilogram of fat mass on fasting heat production (MJ/BW 0.75 ) and the lack of a significant relationship between fasting heat production (MJ/d) and fat mass suggest the limited relevance of fat tissue when considering the maintenance requirements of the cows.The authors conclude that the higher the condition score, the lower the fasting heat production (MJ/BW 0.75 ), and vice versa.This underscores the intricacies involved in evaluating the efficiency of utilizing metabolizable energy for maintenance, as highlighted by Morris and Kononoff (2021), acknowledging It is important to note that similarities between the values observed in this study and previously reported values can be related to the methods for energy partitioning measurements.Like our study, other reports and studies (NASEM 2021; Dong et al., 2015;Moraes et al., 2015;Carvalho et al., 2019) are mainly based on respirometry methods with controlled temperature and humidity during trials.Indeed, these are more appropriate to evaluate lactating cows.However, tropical environments have a high variation in several factors such as feeds and digestion, temperature and humidity, walking and grazing, which results in different behavior and energy partitioning (Maia et al., 2020).Thus, observed values in this study should be extrapolated for different tropical scenarios with caution.

CONCLUSION
We estimated NE and ME requirements for maintenance, efficiencies of ME utilization for milk production, tissue gain, and use of body energy mobilization for milk production throughout lactation and during the dry-off period of primiparous F 1 Holstein × Gyr cows.Differences in requirements and efficiency throughout lactation stages were detected, and values that can be used in nutrient requirements systems were suggested.Overall values were similar to those reported in the literature in specific studies or requirements systems such as NASEM (2021).However, several factors, such as parity, genetic proportions, and feeding systems (e.g., grazing versus feedlot), may affect energy requirements and its efficiency of use.So, extrapolating the values observed in this study to these before-mentioned scenarios should be made with care.Studies evaluating factors usually observed in the tropics are still needed.
Sacramento et al.: Energy Needs and Efficiencies in Crossbred Dairy Cows

Figure 1 .
Figure 1.Summary of the experimental periods and main samplings.
Sacramento et al.:  Energy Needs and Efficiencies in Crossbred Dairy Cows effect of positive energy balance, NEB k = fixed effect of negative energy balance, LS l = fixed effect of lactation stage, A m = random effect of cow, C n = random effect of chamber, EL i × LS l = interaction between milk energy output and lactation stage, PEB j × LS l = interaction between positive energy balance and lactation stage, NEB k × LS l = interaction between negative energy balance and lactation stage and ε ijklmn = residual error.Covariance structures for repeated measures of the lactation stage were selected by the lowest corrected Akaike information criterion.The normality of residuals was checked with normal probability and box plots and homogeneity of variances with plots of residuals versus predicted values.Outliers were removed from statistical analyses when studentized residuals were >2.5 or < −2.5.
Sacramento et al.: Energy Needs and Efficiencies in Crossbred Dairy Cows Sacramento et al.: Energy Needs and Efficiencies in Crossbred Dairy Cows the multifaceted influences that dietary composition, physiological state, and environmental conditions can have on these measures.

2
NE m = net energy for maintenance.3k m = efficiency of utilizing ME for maintenance.

Figure 2 .
Figure 2. Linear relationships between ME intake and heat production for primiparous F 1 Holstein × Gyr dairy cattle throughout lactation, maintenance, and fasting stages.

Table 1 .
Ingredients and chemical composition of diets fed to primiparous F 1 crossbreed Holstein × Gyr cows throughout lactation stages and dry-off period

Table 2 .
Sacramento et al.: Energy Needs and Efficiencies in Crossbred Dairy Cows Energetic parameters and 95% confidence intervals (in parentheses) of primiparous F 1 Holstein x Gyr crossbred cows in the whole lactation and each lactation stage

Table 3 .
Energetic parameters and 95% confidence intervals (in parentheses) of primiparous F 1 Holstein × Gyr crossbred cows during the maintenance and fasting period level 1 ME m = metabolizable energy for maintenance.