Total Replacement of Millet Silage with BRS-716 Biomass Sorghum Silage in Crossbred Dairy Heifers Diets: Nutrient Intake and Digestibility, Microbial Protein Synthesis, Ingestive Behavior, and Growth Performance


 This study addressed the different proportions of millet and sorghum silage biomass BRS 716 (BRS-716 silage) in the diet of crossbred heifers ½ to ¾ Holstein x Zebu on their nutrient intake, and digestibility, microbial protein synthesis, nitrogen balance, ingestive behavior, and growth performance. Five experimental diets with 0, 25/75, 50/50, 75/25, and 100% of BRS-716 silage in compared to millet silage were evaluated. The roughage: concentrate ratio in the total dry matter (DM) of the diets was 75:25. The diets were evaluated in ten heifers with an initial body weight of 264.95 ± 19.4 kg (± SEM), following the experimental design in two 5 x 5 Latin squares, simultaneously. The increase in the proportion of BRS-716 silage in the diet of crossbred dairy heifers did not change the dry matter intake (DMI), total digestible nutrients, indigestible neutral detergent fiber (iNDFI), and metabolizable energy. The means observed for DMI, and iNDFI were 7.85 kg/day, and 1.18 kg/day, respectively. For the excretion of purine derivatives, and microbial synthesis, there was no significant effect (P > 0.05), the mean microbial crude protein synthesis was 503.37 g/day. The different proportions of the BRS-716 silage, and millet silage did not change the final body weight (P = 0.89), weight gain (P = 0.08), feed efficiency (P = 0.50), and the body measurements of heifers (P > 0.05). The final body weight and average daily gain of heifers were 278.46 kg, and 0.795 kg/day, respectively. The millet silage, and BRS-716 silage in the diet of crossbred Holstein x Zebu heifers, used exclusively or combined in different proportions, allow dry matter intake, and nutrients, digestibility, nitrogen balance, microbial crude protein synthesis, ingestive behavior, and performance similar body.


Introduction
In Brazil, there is a growing need for the search for forage alternatives with high productive potential and adapted to climatic conditions, aiming at the sustainability of production systems with ruminant animals (Borges et al., 2019;Queiroz et al., 2021). In this sense, millet (Pennisetum glaucum (L.) R. Br.) is widely used in the world (Assis et al., 2017), and in Brazil, about ve million hectares have been cultivated annually (Calegari et al., 2014;Carvalho et al., 2018). Due to the deep root system, capable of extracting nutrients and moisture from the soil, millet cultivation has stood out for its ability to grow under conditions of water stress (mean rainfall 400 mm). Furthermore, it is adapted to environments with high temperatures (up to 40°C), and presents an annual dry mass (DM) yield ranging from 10 to 20 t/ha (Assis et al., 2017), with good nutritional value (Carvalho et al., 2018).
In 2014, EMBRAPA Corn and Sorghum (Sorghum bicolor L. Moench) launched the biomass BRS 716 sorghum for the purpose of energy cogeneration through direct biomass burning, required by the thermoelectric, and sugar-cane industries. Some studies (Almeida et al., 2019;Castro et al., 2015) demonstrated the high productive potential of BRS 716 biomass sorghum, which presents high growth characteristics (up to six meters in height) and productivity of 50 t/ha DM, in two cycles. Furthermore, to being tolerant to pests, diseases, water de cit, and lodging. Due to these characteristics, this species is promising as a source of nutrients for ruminant animals in several regions of the world, including those with a semi-arid climate ( Considering the important adaptability and dry mass yield of these two forage species, millet and biomass sorghum, to semi-arid conditions, it is of great relevance, the evaluation of their forage potential in the form of silage to feed ruminant animals. In addition, forage plants with high growth can rapidly change cellular components (Monção et al., 2019(Monção et al., , 2020, modifying nutrient intake, and animal performance.
Based on the above, the objective was to compare different proportions of millet silage, and BRS-716 silage in the diet of crossbred Holstein x Zebu heifers, raised in a semiarid region, on their nutrient intake, and digestibility, microbial crude protein synthesis, nitrogen balance, ingestive behavior, and growth performance.

Animals, experimental design, and treatments
The study included ten crossbred heifers (264.95 ± 19.4 kg of body weight, BW; mean ± SD; averaging 14 mo old), with blood levels ranging from ½ to ¾ Holstein x Zebu were allotted to 10 outdoor pens (3 m feedbunk length × 2 m width) having compacted soil surface and equipped with concrete feedbunks and automatic waterers. All animals had the same origin and were selected from a 30-heifers herd. Prior to the onset of the experiment, animals were vaccinated against clostridiosis (Vac Starvac, Basso Pancotte, Nova Alvorada, RS, Brazil), injected with A, D, and E vitamins supplement (ADE Injectable and Emulsi able P zer, Zoetis, Morris County, NJ, USA), and for deworming purposes also subcutaneously injected with 3.5% ivermectin (Ranger LA, Vallée S. A., São Paulo, SP, Brazil) with dosages adjusted according to BW.
The total experimental period lasted 105 d, divided into ve periods of 21 days, including a 17-d initial adaptation, and four for data collection, and samples. Animals were assigned to an experimental design was in two Latin squares 5 x 5, simultaneous, being ve animals, ve treatments and ve experimental periods each. Each pen was considered as the experimental unit.
The diets were formulated according to the NRC (2001) for heifers with an average of 265 kg of body weight (BW) and the roughage: concentrate ratio in the ve experimental diets was approximately 75:25 on the basis of dry matter. The diets were supplied twice a day, at 07:00 h and at 14:00 h, in a complete diet system (Total mixed ration-TMR), homogenized in the trough. The leftovers were collected and weighed daily, in the morning, before the rst feeding, to adjust consumption and the quantity supplied was calculated based on the leftovers, which represented 5% of the total dry matter supplied.
Urea was used to correct the crude protein (CP) contents of the roughage fraction of the diets, using a single concentrate in the ve experimental diets. To ensure the maintenance of the roughage: concentrate ratio in the total DM of the diets and that they were kept isoproteic, the DM contents, and CP of the roughages were analyzed weekly.

Feed analyses
On the 18th, 19th and 20th day of each experimental period, samples of the feed supplied, leftovers (refusals) and feces were collected in the morning and stored in a freezer, -20ºC. Afterwards, the samples were thawed, dried in a forced-air oven at 55•C for 72 h, and ground with a Wiley mill (MA340, Marconi, Piracicaba, Brazil) to pass a 1-mm screen. The DM of 1-mm ground samples was determined with an oven at 105•C for 24 h (method 934.01; AOAC, 1990), and organic matter (OM) was determined by difference after heating at 600°C in mu e for 2 h (Method 942.05; AOAC, 1990). Nitrogen content was determined using a micro Kjeldahl apparatus (TE-036/1 model, Tecnal, Piracicaba, SP, Brazil) according to (AOAC, 1990). The CP content was calculated by multiplying N content by 6.25. Ether extract (EE) was measured using a Soxhlet apparatus (TE-044 extractor, Tecnal, Piracicaba, SP, Brazil) based on extraction with petroleum ether for 6 h (method number 920.39; AOAC, 1990). Lignin (cellulose solubilization with 72% w/w sulfuric acid) and NDFap were determined according to Van Soest et al. (1991). For NDFap procedure (TE-149 ber analyzer, Tecnal Laboratory Equipment Inc., Piracicaba, Brazil; INCT-CA F-002/1), a heat-stable α-amylase (A3306, Sigma Chemical. Co., St. Louis, MO, USA) was used without sodium sul te nor ash-and protein-corrections. Acid detergent ber (ADF) without ash and protein corrections was obtained as described in Goering and Van Soest (1970). The indigestible neutral detergent ber (iNDF) (INCT-CA F-008/1) and non-brous carbohydrates, following the recommendations described in Page 6/26 Detmann et al. (2012).The TDN was calculated according to Weiss et al. (1992). The ingredients and chemical composition of experimental diets are in Tables 1 and 2. ¹DM-dry matter; ²NDFap -Neutral detergent ber corrected for ash and protein; ³iNDF -Indigestible neutral detergent ber; 4 acid detergent ber assayed without ash and protein corrections; 5 total digestible nutrients calculated according to Weiss et al. (1992).

Ruminal kinetics
After pre-drying, the silage samples were ground in mills equipped with sieves with 2mm sieves and placed in non-woven fabric bags in the amount of approximately 3.0g of dry matter (DM)/bag, in order to maintain a ratio close to 20 mg DM/cm 2 of bag surface area. The incubation periods corresponded to times of 0, 3, 6, 12, 24, 48, 72, 96, 120, and 144 hours, with the bags being placed at different times to be removed all at the same time from the rumen. Two cannulated crossbred steers were used, with an average body weight of 580 ± 60 kg and mean age 8 years. The animals were adapted for 14 days to the diet containing 4 kg of concentrate (25% CP and 65% TDN), divided into two meals, morning and afternoon, in addition to the provision of roughage based on sorghum silage (50% millet silage and 50% BRS-716 silage). All the proportions of the silages used during the experiment were evaluated (100% of millet silage; 75% of millet silage and 25% of BRS-716 silage; 50% of millet silage and 50% of BRS-716 silage; 25 % millet silage and 75% BRS-716 silage and 100% of BRS-716 silage).
After the incubation period, the nylon bags were washed in running water until it was clean, then drying. The DM determination was made in an oven regulated at 55ºC, for 72 hours. In situ dry matter degradability data were obtained from the difference observed between the weights performed before and after ruminal incubation and expressed as a percentage.
As it is a rst-order asymptotic growth model, which was reparametrized by subdividing the asymptote value into two fractions, "a" and "B", the DM degradation rates were calculated using the equation The nonlinear parameters "a", "b" and "c" were estimated using least squares iterative procedures. The effective degradability (ED) of DM in the rumen were calculated using the model: ED = a + (B x c / c + k), where: k corresponds to the rate of passage of particles in the rumen, of according to the AFRC (1993). For the ED of NDF, the model was used: ED = B P *c/(c + k), where B P is the standardized potentially degradable fraction (%).

Intake and digestibility of nutrients
Feed intake was monitored from d 1 to 105. Feed delivery was adjusted daily and fed to appetite allowing ad libitum intake and orts below 5% of daily intake. Feed bunks were cleaned and orts weighed daily before morning feeding. Feed offered and orts were sampled weekly and frozen at -20•C for further DM determination. The DMI was calculated daily per pen by subtracting orts from offered feed (on a DM basis). To estimate the daily of metabolizable energy intake (MEI) was taken into account the DMI. The fecal dry matter production was estimated using indigestible neutral detergent ber (iNDF) as an internal indicator. Samples of feed, leftovers and feces, ground in a knife mill with a sieve with 2 mm diameter sieves, were incubated in two crossbred adult cattle, weighing 480 ± 30 kg, mean age 8 years, cannulated in the rumen, during 288 hours, following the methodology (INCT-CA F-009/1) presented by Detmann et al. (2012). The digestibility coe cient of all nutrients was calculated using the following equation: [quantity ingested -quantity excreted in the feces]/quantity ingested. Based on the digestibility coe cients, the value of total digestible nutrients was calculated.
Nitrogen balance and microbial synthesis Spot urine samples were obtained on the 18th day of each experimental period, approximately four hours after feeding in the morning, during spontaneous urination. 10 mL aliquots of this sample were ltered and immediately diluted in 40 mL of 0.036 N H 2 SO 4 for further analysis of creatinine. These aliquots were stored in plastic asks, identi ed and frozen for further analysis and quanti cation of urea, total nitrogen, creatinine, uric acid and allantoin.
Blood samples were collected on the rst and last day of each experimental period, via puncture of the jugular vein, using 5mL test tubes (Vacutainer ™) with EDTA (anticoagulant). Immediately, centrifugation was carried out at 5,000 rpm for 15 minutes and, subsequently, plasma samples were taken, which were packed in eppendorf and stored at -15°C for further analysis of urea.
The concentrations of urea, creatinine and uric acid in the urine and urea in the plasma were estimated using commercial kits (Bioclin, Belo Horizonte, Minas Geras, Brazil). The conversion of urea values into urea nitrogen was performed by multiplying the values obtained by the factor 0.4667.
The urinary contents of allantoin and uric acid were estimated by colorimetric methods, as speci ed by Chen and Gomes (1992), and the total nitrogen content estimated by the Kjeldhal method (Detmann et al., 2012). The balance of nitrogen compounds (Nitrogen balance; g/day) was calculated as: The microbial crude protein synthesis e ciency was calculated as follows: microbial crude protein synthesis e ciency = {(0.629 x AP) x 6.25)/TDN intake}, where: AP = absorbed purines (mmol/day); TDNI -total digestible nutrients intake; 0.629 represents the absorbed purine without considering the contribution of the endogenous fraction.

Determination of ingestive behavior
Ingestive behavior followed the method described in Monção et al. (2020). Visual observations for each pen (n = 1) were recorded every 5 min during the 24-h cycle on d 19 and 20 of each experimental period. Different groups of two observers each were assigned every 5-h interval. Each observer was responsible for recording the ingestive behavior of animals in 5 pens (5 animals). Pen observations were conducted sequentially, always following the same order per observer. Eating, ruminating, and total chewing times (min/d) were calculated by the number of observations multiplied by 5. Total chewing time was the sum of eating and ruminating times. Ingestive variables were also expressed as min/kg DMI. To allow this, DMI was determined by subtracting orts from offered feed (on DM basis).

Growth performance, and biometric measurements
At the beginning and at the end (21 th) of each experimental period, after a 16-hour fast of solids, the body weight of animals was evaluate, we used a mechanical scale (mechanical scale, Valfran, Votuporanga, São Paulo, Brazil). Moreover, measurements were made of the thoracic perimeter, withers and croup height and body length. The measurements were made according to the methodology of Hoffman (1997), with the animals in a forced station, that is, front and rear members perpendicular on a at oor, forming a rectangular parallelogram. Feed e ciency was calculated by dividing weight gain (kg/day) with DM intake (kg/day).

Statistical Analyses
Data were evaluated by analysis of variance using the MIXED procedure of SAS, version 9.0 (SAS Inst. Inc., Cary, NC, USA). Data normality (Shapiro-Wilk test at 5% probability) was veri ed by the UNIVARIATE procedure in SAS. The statistical model used for analyses was Y k(ijl) = µ + P i + A j + Q l +T k(ijl) + PI + Ql + e k(ijl) , where Y k(ijl) is the observation concerning the treatment "k", within period I, animal j and Latin square (Q) l; µ is a constant associated with all observations; Pi is the effect of period i, with i = 1, 2, 3 and 4; Aj is the animal effect j, with j = 1, 2, 3, 4, and 5; Ql is the Latin square effect l; T k(ijl) is the treatment effect k, with k = 1, 2, 3, 4, and 5; PI is the initial body weight as a covariable and e k (ijl) is the experimental error associated with all observations (Y k (ijl) ), which is independent and by hypothesis has a normal distribution with mean zero and variance δ 2 . The treatments (T k(ijl) ) were considered to be xed effects; animals (Aj), experimental period (Pi), initial body weight and the error term (e k(ijl) ) were random effects.
The ruminal degradability of DM and NDF was conducted in a randomized complete block design in subdivided plots, with ve treatments (plots), 10 incubation times (subplots) and ve replications. The variation of the animals' body weight was the blocking factor. Ruminal fermentation variables were analyzed as repeated measures using the PROC MIXED, according to the following model: Y ijklm = µ + A i +P j +B k + α kl + ω ijkl + Tm + T ×A mi + ε ijklm , where ω i jkl ≈ N (0, α² ω ) and ε ijklm ≈ MVN (0, R), and Y ijklm = observation on animal l, given treatment i, at period j, in block k, in time m; µ, A i , P j , B k , and α kl were previously de ned; ω ijkl = the residual error associated with cows within experimental period; T m = the xed effect of the sampling time (m = 1 to 10); T × A mi = the xed interaction effect between the time and treatment; ε ijklm = random residual error; α² ω = the estimated variance associated with experimental units (cows within period); MVN = multivariate normal; and R = the variance-covariance matrix of residuals due to repeated measurements. Variancecovariance matrices were evaluated [UN, UN1, CS, CSH, AR(1), ARH(1), TOEP, TOEPH, FA(1), and ANTE(1)] and chosen by the Bayesian method. The covariance matrix that best t the data according to the corrected Bayesian information criterion (BIC) was variance components (UN). When determined to be signi cant by the F test, the means of the treatments were compared by decomposing the sum of squares into orthogonal linear contrasts and quadratic effects at 5% probability, with subsequent adjustments to the regression equations. Outliers were identi ed and deleted if the absolute values of Studentized residuals exceeded ± 3. The mean values were considered to be different when p < 0.05.

Ruminal kinetics
The increase in the proportion of BRS-716 silage linearly reduced the readily soluble fraction (fraction "a"), potential degradability and effective degradability of DM at different passage rates 2, 5 and 8%/h (P < 0.01) and linearly increased the indegradable fraction of DM (Table 3). For each percentage unit of inclusion of the BRS-716 silage, there was a reduction of 0.10, 0.11, 0.06, 0.07, and 0.08 percentage points (pp) respectively.

Intake and digestibility of nutrients
Diets with different proportions of BRS-716 silage and millet silage for crossbred dairy heifers did not modify the dry matter intake (DMI), crude protein intake, NDF intake, ether extract intake, non-brous carbohydrates intake, total digestible nutrients intake, indigestible neutral detergent ber intake (iNDFI), and metabolizable energy intake ( Table 4). The means observed for DMI and iNDFI were 7.85 kg/day and 1.18 kg/day, respectively. SEM -Standard error of the mean. Five observations (pen) per treatment. p-Probability.
The DMI and nutrients when expressed as a percentage of BW, was not modi ed due to experimental diets, mean of 3.12% BW for DMI. There was a quadratic effect on the digestibility of the ether extract, with an increase in the proportion of BRS-716 silage in the diet. The greater digestibility of the ether extract was veri ed in the proportion of 56.5% inclusion of BRS-716 silage. As for the other variables related to apparent digestibility, no differences were found between the diets (P > 0.05).

Nitrogen balance and microbial synthesis
The different proportions of BRS-716 silage and millet silage in the diet of crossbred dairy heifers did not change the daily nitrogen intake (N; mean of 132.42 g). For the N excreted in the feces, a linear reduction from 0.03 g to 1% inclusion of BRS-716 silage was observed (Table 5).

Body weight and biometric measurements
The nal body weight (P = 0.89), weight gain (P = 0.08), feeding e ciency (P = 0.50) and the body measurements of the heifers has not been modi ed (P > 0.05) by the replacement millet silage with BRS-716 silage in the diets ( Table 7). The nal body weight and average daily weight gain of heifers were 278.46 kg and 0.795 kg/day, respectively. for crossbred lactating cows. The authors concluded that although the BRS-716 silage has a higher content of components brous (NDF; 725.6 vs. 664.0 g/kg), its ber is of satisfactory quality for adequate consumption. Furthermore, the authors observed that the replacement of forage sorghum silage with BRS 716 silage reduced DMI and dry matter digestibility, but did not alter the mean of milk yield (13.42 kg/day) and feed e ciency.
The biomass sorghum begins to have its forage potential evidenced, because, in addition to presenting high dry matter yields per hectare, it allows the obtaining of silages of good nutritional value. Almeida et al. (2019) evaluated the agronomic performance and chemical composition of six biomass sorghum hybrids for cellulosic ethanol production, observed that mutant sorghum hybrids are associated with reduced lignin content, making these genotypes more promising for enzymatic conversion processes of biomass. This ber characteristic is common for both purposes, as there is a preference for lower lignin contents, as lignin affects in the ber digestibility for ruminants and high percentages of lignin require higher concentrations of enzymes for the processing of biomass, making this economically unviable.
Even with the linear reduction of fecal nitrogen excretion, the nitrogen balance remained positive and similar between diets, as well as animal performance. The decrease in nitrogen excretion may result in less environmental impact and greater economic return on the production system by decreasing the use of nitrogen inputs. The nitrogen balance quanti es protein retention or loss by the animal, and is related to feed intake, when the nitrogen balance is positive it means that probably the protein requirements have been met (Gonçalves et al., 2014). Furthermore, despite the higher proportion of brous components and less energy availability with BRS-716 silage, the TDN intake and DMI were similar, justifying once again the similarity in body performance results of crossbred Holstein x Zebu heifers.
The similarity for allantoin and uric acid excretions is due to the diets being isoproteic. According to Braga et al. (2012), the total excretion of purine derivatives in the urine results from protein catabolism in ruminants and are important variables for estimating the amount of synthesized microbial protein.
Microbial protein synthesis was similar between diets, due to similarities in nitrogen and energy intake between experimental diets. The mean microbial e ciency veri ed in the diets was 130.5 g MCP/kg TDN, corroborating the NRC (2001) which proposes the value of 130 g MCP/kg TDN intake. Microbial e ciency can allow an increase in the availability of microbial protein to be absorbed in the intestine, thus meeting the requirements of growing animals .
The variations in the NDF, ADF and iNDF contents between experimental diets with different proportions of millet silage and BRS-716 silage were not su cient to modify the ingestive behavior of heifers, which presented similar nutrients intake and digestibility values. It should be noted that the brous fraction is the component of the diet of main relevance under the activities of ingestive behavior, especially for diets with a high participation of roughage feeds. In the growing phase of the heifers, provide a balanced diet to ensure gain enough weight to body development and early coverage is essential. In this study, the average daily weight gain of 0.80 kg/day is within the desirable range, demonstrating the potential use of millet silage and BRS-716 silage for this animal category.

Conclusion
Millet silage and BRS-716 silage in the diet of crossbred Holstein x Zebu heifers, used exclusively or combined in different proportions, allow dry matter intake and nutrients, digestibility, nitrogen balance, microbial synthesis, feeding behavior and performance similar body.