IntroductionTop

Material and methodsTop

Location of experiment and approval

The present study was carried out at commercial dairy farm (Zarrin-Khoosheh Dairy Farm), Arak, Iran. All the animal procedures were approved by the Animal Care and Use Committee of Arak University (IACUC Protocol #IR95-3108) outlined by the Iranian Council of Animal Care (1995).

 

Experimental design, treatments, and management

Forty-eight 3-day-old female Holstein dairy calves with 44.5 ± 1.8 kg of initial BW were randomly assigned to ex-perimental starter diets (12 calves per treatment) in a 2 × 2 factorial arrangements with the factors of forage source (AH vs. WS) and RUP:RDP ratio [low ratio (LR)=28:72; high ratio (HR)=36:64]. The crude protein content was constant across the treatments (20%, DM basis), and the figures for the RUP:RDP ratios were based on the crude protein content (Table 1). Immediately after birth, calves were separated from their dams, weighed, and moved to individual pens bedded with sand. Calves received 4 L/d whole milk in galvanized tin buckets twice a day at 0730 and 1630 h from d 3 to 10 and 7 L/d from d 11 to 48 of the study followed by 3 L/d from d 49 to d 52 of the study. Whole milk samples were weekly taken and analyzed for fat, CP, lactose and total solids using an infrared spectrophotometer (FOSS milk-o-scan; FOSS Electric, HillerØd, Denmark). The average composition of offered milk was 3.16 ± 0.09% fat, 3.03 ± 0.07% CP, 4.83 ± 0.04% lactose and 11.8 ± 0.16% total solids. All the calves were weaned on d 53 of the study but the experiment continued 20 days thereafter. Experimental treatments were: (1) AH with low RUP:RDP ratio (AH-LR); (2) AH with high RUP:RDP ratio (AH-HR); (3) WS with low RUP:RDP ratio (WS-LR); and (4) WS with high RUP:RDP ratio (WSHR). Experimental starter concentrates were formulated to meet the current NRC (2001) nutrients requirements. Ingredients and chemical composition of experimental diets are presented in Table 1. Starter concentrates were formulated to be iso-nitrogenous but different in RUP:RDP ratio by replacing corn gluten meal by soybean meal as stated by Amirabadi Farahani et al. (2017). The level of AH and WS was 70 g/kg of the basal diet. Because particle size could be a variation source of forage inclusion in starter diet of dairy calves (Mirzaei et al., 2015), similar geometric particle size has been considered for treatments when chopping AH and WS to prevent this variation (geometric mean particle size was 2.92 ± 0.13 and 2.95 ± 0.12 mm, for AH and WS, respectively). The concentrate feed was mixed well with forages and the diet was offered as total mixed ration to dairy calves throughout the study. Starter diet was fed ad libitum to permit at least 10% orts in a day. The calves had free access to water throughout the experimental period.

 

Table 1. The ingredients and chemical composition of experimental starter diet (g/kg of DM, unless otherwise stated)

¹VTreatments were: (1) alfalfa hay with low dietary RUP:RDP ratio (AH-LR); (2) alfal-fa hay with high dietary RUP:RDP ratio (AH-HR); (3) wheat straw with low dietary RUP:RDP ratio (WS-LR); (4) wheat straw with high dietary RUP:RDP ratio (WS-HR). 2Contained per kilogram of supplement: 700,000 IU vitamin A, 150,000 IU vitamin D, 1000 IU vitamin E, 1 g Mn, 100 g Ca, 2g Zn, 60 g P, 12 g Mg, 10 g Na, 2 g Fe, 45 mg Co, 150 mg Cu, 30 mg I, and 3 mg Se. 3Calculated from NRC (2001) 4Non-fiber-carbohy-drate was calculated as [DM- (NDF + CP + ether extract + ash)] (NRC, 2001).

 

 

 

Data recording for intake, gain, and efficiency

Starter feed refusals were collected and recorded daily at 0730 h and fresh starter feed was fed at 0800 h. Body weight (BW) was recorded every 10 d using an electronic balance which was calibrated before initiation of the study and every month thereafter. Calves were weighed before the morning meal to eliminate the effects of the empty/full status of the gastrointestinal tract on BW. Average daily gain (ADG, kg of BW/d) was calculated as the difference between BW taken every 10 d apart divided by 10. Feed efficiency (FE) was computed as g of ADG/kg of total DM intake (DMI; liquid feed DMI + starter feed DMI).

 

Chemical composition of diets and digestibility trial

At 10-d intervals, samples from feeds and orts were dried in a convection oven (60C° for 48 h). Subsamples of dried feeds and orts were mixed thoroughly and then ground through a mill (Ogaw Seiki Co., Ltd., Tokyo, Japan) to pass a 1-mm screen and stored at ‒20°C until chemical analysis. Diet samples were analyzed for CP (method 988.05; AOAC, 2002), ether extract (EE, method 920.39; AOAC, 2002), and NDF without sodium sulfite, but with the inclusion of α-amylase adopted by Van Soest et al. (1991). The non-fibrous carbohydrate component was computed as 100 – (CP + NDF + EE + ash) (NRC, 2001).

In the last five days of the experiment, fecal samples were collected from the rectum of each calf initiated on 08:00 h of first sampling day and continued by 12 h intervals (10 samples per each calf). Collected fecal samples were dried in a forced dried oven (60°C; 72 h), and then ground in a Wiley mill through a 1-mm screen. Aliquots of fecal samples were analyzed to determine total N, ash and NDF. Apparent total tract digestibility of nutrients (DM, NDF, CP, and EE) was measured by using acid insoluble ash as an internal marker based on Van Keulen & Young (1977).

 

Fecal scoring and behavioral data recording

Fecal scoring was as follows: 1=firm and well-formed; 2=soft and pudding-like; 3=runny and similar in consistency to pancake batter; and 4=liquid splatter and similar in consistency to pulpy orange juice (Heinrichs et al., 2007).

Behavioral data (standing, lying, eating, ruminating, and non-nutritional behavior were monitored by direct observations of all calves over the total time (min) devoted to each monitored feeding behavior twice on pre-weaning period (d 45 and 52 of the trial) as well as twice after weaning (d 66 and 73 of the trial). On behavioral recording days, calves were taken under observation 1 h after the solid feed offering and 3 h immediately after the morning milk feeding during the pre-weaning period. During the post-weaning recording times, calves were observed 1 h before and 3 h after the solid feed was offered at 08:00 h. Therefore, the total time for each calf’s behavior recording length was equal to 16 h (8 h before and 8 h after weaning).

 

Growth indices recording

Growth variables, including heart girth (circumference of the chest), body length (distance between the points of shoulder and rump), body girth (circumference of the belly before feeding), wither height (distance from the base of the front feet to wither) and hip height (length from the base of the rear feet to the hook bones) were taken at the start of the experiment (d 3), weaning (d 53) and last day of experiment (d 73).

 

Microbial protein yield measurement

The MPY was estimated based on the purine derivatives (PD) excretion obtained via spot sampling technique explained by Valadares et al. (1999). Because feeding milk into dairy calves could interfere with PD excretion during the pre-weaning period due to its PD content (Gonzalez-Ronquillo et al., 2003), the spot urine sampling technique was used for MPY estimation only in the post-weaning period as reported by Makizadeh et al. (2020) in the post-weaned calves. This technique was used for the estimation of daily urine output from creatinine concentration as explained in detail by Valadares et al. (1999). Urine volumes were estimated as BW × 26.8 / creatinine concentration (mg/L) in post-weaned dairy calves as reported by Dennis et al. (2017). Spot urine samples were collected on 3 sampling days during the post-weaning period from each animal during the morning (between 09:00 and 11:00 h) and during the afternoon (between 15:00 and 17:00 h) as explained in a recent work on dairy calves (Kazemi-Bonchenari et al., 2020). Samples were collected when calves urinated spontaneously (≈ 10 mL). An aliquot of 5 mL of each sample was diluted immediately with 45 mL of 0.036 N sulfuric acid then stored at –20°C for analysis. Later, urine samples were thawed at room temperature and analyzed to determine the creatinine (Kit No. 555-A; Sigma Chemical Co.), UN (using the assay described by Broderick & Kang, 1980), uric acid (Kit No. 68550; Sigma Chemical Co). Allantoin was measured using the high-performance liquid chromatography method described by Chen & Gomes (1992). Total excretion of allantoin and acid uric was calculated from estimated daily urine output and determined metabolite concentrations. The ruminal microbial N synthesis was calculated from daily urinary PD output using the following equation described by Chen & Gomes (1992): Microbial N (g N / d) = X (mmol/d) × 70 / (0.116 × 0.83 × 1000); where X is microbial purine absorbed (mmol /d), 70 is the N content of purines coefficient (mg N / mmol), 0.116 is the ratio of purine-N to total N in mixed ruminal microbes which is 11.6:100, and 0.83 is average digestibility of microbial purines (Chen & Gomes, 1992).

 

Blood sampling

The blood samples for the experimental period were collected on d 35 (pre-weaning) and 70 (post-weaning) 4 h after morning feeding (at 1200 h) from the jugular vein into 10 mL tubes, placed on ice and centrifuged at 3,000 × g for 20 min at 4°C. Serum subsamples stored at –20°C and subsequently were analyzed to determine concentrations of glucose (Kit No. 93008), albumin (Kit No. 9307), total protein (Kit No. 9304), and blood urea N (BUN) (Kit No. 93013) using commercial kits in accordance to the manufacturer’s instruction protocols (Pars Azmoon Co., Tehran, Iran). Beta-hydroxybutyrate (BHB) was measured with a commercial kit (Abbott Diabetes Care Ltd., Oxin, UK). Liver function indicator enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were measured using ELISA (Auto Analyzer Hitachi 717 Instruments, Inc., Tokyo, Japan) and commercial laboratory kits (Kits No. 92005 and 92004, for AST and ALT, respectively).

 

Statistical analysis

Data were subjected to ANOVA using the MIXED procedure of SAS (SAS 9.1, SAS Inst. Inc., Cary, NC, USA) during the pre-weaning (d 3 to 53), post-weaning (d 54 to 73) and overall (d 3 to 73 of the study) periods with time as repeated measures for starter intake, total DMI, ADG, and FE. The model included fixed effects of the time (sampling date), forage source (AH vs. WS), the RUP:RDP ratio (28:72 as LR and 36:64 as HR), and their interactions and calf as a random effect. Before analyses, all data were screened for normality using the UNIVARIATE procedure of SAS. Two variance-covariance structures (auto-regressive type 1 and compound symmetry) were tested and the covariance structure that minimized the Schwarz’s Bayesian information criterion was chosen. The fecal score was square-root transformed for better homogeneity of the distribution of residuals. The structural growth, digestibility, behavior, UN, and PD excretion, microbial protein yield, and blood metabolite variables were analyzed using a similar model, but without the effect of time. Initial BW was used as a covariate for weaning weight and final weight. All reported values are least square means, with the PDIFF procedure of SAS used to separate treatment means. The threshold of significance was set at p ≤ 0.05; trends were declared at 0.05<p ≤ 0.10.

 

 

Starter intake and feed efficiency

The results for starter intake, ADG, BW, and FE are presented in Table 2. The ADG in the post-weaning period was improved in HR diets compared to the LR diets (p=0.05). Accordingly, the FE was improved in the HR diets during the post-weaning period (p=0.04). The greatest BW was observed for WS-HR treatment among the experimental treatments at final measurement (p=0.05). Starter intake did not differ among experimental treatments neither pre-weaning nor post-weaning.

Fecal score, nutrients digestibility, and feeding behavior

Results showed that feeding WS improved fecal score during the pre-weaning period (p=0.03) and the entire period (p=0.02) compared to feeding AH diets (Ta-ble 2). Regarding the nutrients digestibility, results revea-led that digestibility of NDF was greater when WS was fed to dairy calves in comparison with AH diets (p=0.04). However, the digestibility of DM, CP, and EE were not influenced with experimental factors or their interaction.

Regarding the feeding behavior results showed that the time spent for rumination tended to be greater (p=0.06) in WS fed calves in comparison with AH diets. The time spent for standing, lying, eating, and non-nutritional be-haviors were not influenced with different treatments. No interaction was found for forage source and RUP:RDP ratios regarding the feeding behavior results in the present study.

Table 2. Least square means for starter intake, ADG, FE, fecal score, nutrients digestibility and feeding behavior in calves fed different forage source (AH vs. WS) and different rumen undegradable to degradable protein ratios (28:72 vs. 36:64).

¹Treatments: see Table 1. ²Statistical comparisons: Forage = forage sources (AH vs. WS); RUP; different ratios of RUP:RDP [low ratio (LR) = 28:72; high ratio (HR) = 36:64]; Forage × RUP= interaction between forage source and dietary RUP:RDP ratios. ³ kg of body weight gain/kg of total dry matter intake Means within a row with different superscript letters are statisti-cally different (p<0.05).)

 

Skeletal growth

With respect to the skeletal growth indices re-sults showed that the greatest wither height was found for the WS-HR treatment among experimental treat-ments (p=0.05; Table 3). Hip height was greater in final measurement when the HR diets were fed to dairy calves compared to the LR diets (p=0.04). Wither height at final measurement tended to be greater also for the HR diets compared with the LR diets (p=0.08). Heart girth, body length, and body girth were not influenced with different experimental treatments.

 

Table 3. Least square means for growth variables in dairy calves fed different forage source (AH vs. WS) and different rumen undegradable (RUP) to degradable protein (RUD) ratios (28:72 vs. 36:64).

¹Treatments: see Table 1. 2Statistical comparisons: Forage = forage sources (AH vs. WS); RUP; different ratios of RUP:RDP [low ratio (LR) = 28:72; high ratio (HR) = 36:64]; Forage × RUP= interaction between forage source and dietary RUP:RDP ratios. Means within a row with different superscript letters are statistically different (p<0.05))

 

 

Purine derivatives, microbial protein yield, and urinary nitrogen

The results for purine derivatives, microbial protein synthesis, and urinary nitrogen excretion are presen-ted in Table 4. The urinary allantoin concentration was greater in the calves received the HR diets compared to the calves fed LR diets (p=0.04). The purine deri-vatives and subsequently microbial protein yield were increased when calves were fed the HR diets in comparison with the LR diets (p=0.05). The lowest UN concentration was observed for the WS-HR treatment among other experimental treatments (p=0.05). Allan-toin excretion through urine tended to be greater in calves fed WS compared with AH fed calves (p=0.09). Uric acid was influenced neither with the forage source nor with RUP:RDP ratio.

 

Blood metabolites and liver enzymes

The greatest concentration of BHB among experi-mental treatments was found in the blood of calves fed WS-HR treatment (p=0.04; Table 5). The concentration of BUN was greater in the LR diets in comparison with the HR diets (p=0.03). The concentrations of glucose, total protein, and albumin in the blood of experimental animals was not affected by the different treatments (p > 0.05). Regarding the liver enzymes activity in the current study, results revealed that no changes were found for liver en-zymes concentrations in the blood of calves among expe-rimental treatments.

 

 

 

Effect of forage source

The intake of starters did not differ among experimen-tal treatments in the current study. However, previous works identified different starter intakes when calves were fed va-rious forage sources (Movahedi et al., 2016; Mirzaei et al., 2017). Mirzaei et al. (2017) stated that feeding corn silage increased intake in dairy calves compared to AH feeding due to the greater palatability of the starter with increased mois-ture in corn silage fed calves. AH and WS diets had constant moisture content in the current study, and similar intake was observed during the pre-and post-weaning periods. Mova-hedi et al. (2016) reported lower intake in dairy calves fed WS in comparison with AH fed calves while forage was available as free choice. The forage to concentrate ratio was 9.1:90.9 and 28.6:71.4 for WS and AH diets, respectively, in free choice feeding (Movahedi et al., 2016). Those results contrast with the constant forage to concentrate ratio consi-dered for AH and WS diets (7:93; Table 1). Recently, Poczy-nek et al. (2020) stated that the increase of NDF content in the starter feed up to 310 g/kg DM with replacement of corn by soybean hull or hay supplementation did not influence the starter intake and performance of calves at weaning. In general, the effect of forage feeding on starter intake in dairy calves may be influenced with different variables such as in-clusion level, particle size, starter feed texture, total dietary moisture, and grain source (Hill et al., 2010; Mirzaei et al., 2015). ADG and FE were similar in calves fed either AH or WS diets in the current study mostly due to the similar starter intake among diets.

The fecal score was improved in the current study du-ring pre-weaning period and the entire period of experi-ment when calves were fed with WS in contrast to fee-ding AH diets. The ruminal variables were not measured; however, previous works stated that ruminal conditions such as high passage rate could reduce fecal consistency (Kazemi-Bonchenari et al., 2017; Mirzaei et al., 2017). Thus, it may be assumed that WS can provide more a fa-vorable ruminal condition than AH to have better fecal score. Minimum forage content (5%, DM basis) was pro-posed to be included in starter feed that is necessary to avoid pH decline in dairy calves (Aragona et al., 2020). Assessing the behavioral results show that the time spent on rumination was greater in WS fed calves which is due to the greater NDF content supplied in this diet (20.85 vs. 23.45% NDF for AH and WS diets, respectively). The greater content of NDF could increase the time spent for rumination in the pre-weaning dairy calves (Poczynek et al., 2020). Greater NDF digestibility was also obtained in calves fed WS in the current study, which suggests that the ruminal environment was more favorable for cellu-lolytic bacteria activity in WS diets. It could be suppo-sed that greater saliva secretion could be induced by the greater rumination time when calves fed WS compared to AH feeding which can improve rumen function and subsequently improve animal gastrointestinal health sta-tus (Maekawa et al., 2002; Aragona et al., 2020). Our re-sults indicate that feeding WS compared to AH in limited amount (7% of starter diet, DM basis) is supposed to pro-vide more stable ruminal fermentation which consequent-ly caused better fecal score, more ruminating time, and greater NDF digestibility.

 

Table 4. Least square means for purine derivatives, microbial protein yield, and urinary nitrogen in dairy calves fed different forage source (AH vs. WS) and different rumen undegradable (RUP) to degradable protein (RUD) ratios (28:72 vs. 36:64).

¹Treatments: see Table 1. ²Statistical comparisons: Forage = forage sources (AH vs. WS); RUP; different ratios of RUP:RDP [low ratio (LR) = 28:72; high ratio (HR) = 36:64]; Forage × RUP= interaction between forage source and dietary RUP:RDP ratios. Means within a row with different superscript letters are statistically different (p<0.05)

 

 

 

Effect of RUP:RDP ratio

Regardless the similar intake in the calves fed different dietary RUP:RDP ratios, the ADG and FE improved in HR diets in the current study. The higher levels of avai-lable amino acids reaching into small intestine could positively influence the growth rate in young animals (Kazemi-Bonchenari et al., 2018). Furthermore, the greater microbial protein yield which was observed in the HR diets in the current study could also contribute to a greater availability of amino acid in the small intestine, which positively influenced growth rate of young calves. Accor-dingly, the HR diets increased hip height at final measure-ment which is in line with improved ADG and FE in this diet. Heinrichs et al. (2007) showed a positive correlation between growth rate and skeletal growth indices in growing dairy calves.

The urinary excretion of allantoin was greater in the HR diets compared to LR diets. Accordingly, total uri-nary PD excretion was greater in HR diets than in the LR diets. According to the greater PD excreted through urine in the HR diets compared with the LR diets, the MPY also was greater in this diet. The higher MPY obtained in HR diets can be attributed to the more efficient N metabolism which indicates higher capture of N toward mi-crobial protein synthesis. It should be further discussed here that in addition to the RUP:RDP ratio in dairy calves, the incorporated RUP source in the starter diet can also influence animal performance. However, some discre-pancies among previous studies (Hill et al., 2007; Kaze-mi-Bonchenari et al., 2016) for the effects of RUP:RDP ratios on dairy calves’ growth rate might be attributed to the differences in the source and level of RUP feeding and different amino acid profiles of experimental feedstuffs included in the starter diet.

The concentration of BUN was greater in the LR diets compared to the HR diets during the post-weaning period probably due to the greater ruminal NH3-N concentration found in these diets. Blood urea N may be a promising indicator of N capturing in the rumen and blood urea ni-trogen level is positively correlated with ruminal NH3-N concentration (DePeters & Fergusen, 1992). The MPY was improved in the calves received HR diet compared to calves fed LR diets indicating the more efficient capturing of the ruminal NH3-N produced in the rumen toward microbial utilization rather than converting to urea and excrete through urine. Hence, our results show that from the N perspective, HR diets were more efficient rather than LR diets in the current study because of reduction in BUN and then reduced urinary N excretion through urine. It is notable here that lower growth performance in animals fed LR diets compared to HR diets may be related to higher urinary N excretion through which can increase energy wastage through urea synthesis in the liver and kidneys that has been shown to reduce energy partitioning toward production in ruminants (Lapierre & Lobley, 2001; Kohn et al., 2005).

 

Table 5. Least square means for blood metabolites and liver enzymes in dairy calves fed different forage source (AH vs. WS) and different rumen undegradable to degradable protein ratios (28:72 vs. 36:64).

¹Treatments: see Table 1. ²Statistical comparisons: Forage = forage sources (AH vs. WS); RUP; different ratios of RUP:RDP [low ratio (LR) = 28:72; high ratio (HR) = 36:64]; Forage × RUP= interaction between forage source and dietary RUP:RDP ratios. Means within a row with different superscript letters are statistically different (p<0.05)

 

 

Interaction effects of forage source and RUP:RDP ratio

The greatest BW and the greatest wither height at final measurement were obtained for WS-HR treatment which can be partly due to the greater N efficiency in this diet. Our results suggest that feeding WS with high RUP:RDP ratio has potential to improve gain and efficiency which is in line with the previous finding in heifers (Koch et al., 2017), growing lambs (Dorri et al., 2021) or steers (Wickersham et al., 2008). Our results confirm previous works (Wickersham et al., 2008; Koch et al., 2017) which indicated that partial incorporation of LQF in mature ruminant’s diet can improve ruminal N metabolism probably through increased urea recycling in to the rumen. Partial dietary inclusion of WS as a LQF in the starter diet of young calves would be more efficient when greater dietary RUP:RDP ratio was supplied for dairy calves. Koch et al. (2017) postulated that additional fiber in the heifers’ diet depressed microbial protein f low and N utilization, but providing greater undegradable protein in the diet was instrumental in reestablishing N balance. The suggested mechanism for the positive impact of greater RUP level on microbial protein yield in animals fed with LQF is providing the greater available N in the rumen through higher rate of urea recycling (Wickersham et al., 2008).

Blood glucose concentration was constant among experimental treatments that is mostly because of similar intake among experimental treatments. However, blood BHB concentration was influenced with the interaction of forage source and RUP:RDP ratio with the greatest blood BHB concentration was found for WS-HR treatment. It has been stated before that the blood BHB in young ruminants is an indicator of more developed rumen in pre-ruminant animals which could positively influence animal growth performance (Soltani et al., 2017). Therefore, in addition to the more efficient N metabolism in WS-HR treatment, the greater blood BHB concentration in this diet can also be an indicator of higher developed gastrointestinal activity. Total protein and albumin concentrations were not differed across experimental treatments probably because of the adequate crude protein supplied in all experimental treatments. Similar liver enzymes concentrations among the experimental treatments in the current study indicate that liver function was not negatively influenced with different forage sources or dietary RUP:RDP ratios in dairy calves.

 

 

 

 

The authors thank Dr. Moshayedi, manager in-chief and Mr. Sadeghi, technical manager of the Zarrin-Khoosheh commercial dairy farm. Great thanks to Mr. Namdari (calf rearing technician) for valuable helping throughout the experiment. Thanks to Amin Mirjafari, Sahar Avakh, and other students (Arak University) for helping in urine sampling times in this experiment.