Growth performance, nutrient digestibility, and ruminal fermentation of dairy calves fed starter diets with alfalfa hay versus corn silage as forage and soybean oil versus palm fatty acids as fat source

The present study was intended to evaluate the effect of forage source (alfalfa hay; ALF vs. corn silage; CS) along with a supplemental fat source (soybean oil; SO vs. rumen-inert palm fatty acids; PF) on growth performance, nutrient digestibility, and ruminal fermentation in dairy calves. Forty-eight new-born Holstein female calves (3 d old) were assigned to one of 4 treatments: (1) alfalfa hay with soybean oil (ALF–SO); (2) alfalfa hay with palm fatty acids (ALF–PF); (3) corn silage with soybean oil (CS–SO); (4) corn silage with palm fatty acids (CS–PF). Starter diets had equal amounts of forage (100 g/kg dry matter; DM) and fat source (30 g/kg DM). Calves were fed a constant amount of milk (d 1 to 63) and had ad libitum access to water and starters (d 1 to 83). The lowest and greatest starter intakes during the preweaning period occurred in ALF–SO and CS–PF, respectively. This coincided with forage × fat source interaction for average daily gain (ADG) during preweaning. The forage source affected total DM intake and ADG over the entire period, body weight (BW) at weaning, and final BW with greater values in calves that received CS compared with ALF. The concentrations of total short-chain fatty acids and butyrate were increased, whereas concentration of acetate and acetate: propionate ratio were decreased in the rumen of calves fed CS compared with ALF. Feeding CS increased urinary excretion of allantoin and, as a trend, total purine derivatives (PD) and estimated microbial protein synthesis in comparison with ALF. The fat source affected starter intake, ADG, and BW postweaning with the highest values in PF. The digestibility of neutral detergent fiber, crude protein and, as a trend, organic matter were higher in calves fed PF


INTRODUCTION
Providing forage in the starter diets of dairy calves is a common practice in commercial dairy farms. However, recommendation for forage incorporation level in starter diets of dairy calves may be influenced by forage source, forage particle size, starter physical form, milk feeding volume, and other factors (Phillips, 2004;Beiranvand et al., 2014;Mirzaei et al., 2016;Hosseini et al., 2019). Reduced total DMI may be a negative side effect of forage inclusion in starter diets of young calves (Phillips, 2004;Hosseini et al., 2019), attributable to their NDF filling effect in the rumen (Allen and Piantoni, 2014). Moreover, the lower energy content of forage compared with concentrate can reduce the energy density per unit of starter feed when forage is incorporated in starters compared with forage-free starters (Molaei et al., 2021). This can finally limit the total metabolizable energy provided to young dairy calves and may become an obstacle especially for early weaning programs where adequate energy content in starter feed is essential.
Fat supplementation can compensate for the low energy density per unit of feed when forage is included in starters (Ghasemi et al., 2017;Karimi et al., 2021). However, negative effects of supplemental fat on fiber and other nutrients' digestibility remain a concern (Ghorbani et al., 2020;Karimi et al., 2021). Furthermore, supplemental fat level and source and method of its delivery can have an influence on the extent of these negative effects on ruminal fermentation (Berends et al., 2018;Ghorbani et al., 2020;. The unfavorable effects of fat on ruminal fermentation are related to toxic effects of lipid metabolites on ruminal microbes, the physical coating effect of fat on fiber, and alterations of ruminal microbes through fat supplementation (Ikwuegbu and Sutton, 1982;Soliva et al., 2004). Young calves lack sufficient populations of cellulolytic bacteria in the early weeks of life; therefore, forage inclusion in starters should be done with caution in this critical period (Nocek and Kesler 1980). Moreover, supplemental fat has a potential to deteriorate the nutrient digestibility of the forage portion of starters due to negative effects on the development of the microbial community (Ikwuegbu and Sutton, 1982). Although establishment of ciliate protozoa occurs later than bacteria in young ruminants (Eadie, 1962), the negative effect of some fatty acids (FA) on protozoa numbers in the rumen may be partly responsible for reduced NDF digestibility in fatsupplemented diets. This can be due to the pivotal role of protozoa in fiber digestibility in ruminants (Sutton et al., 1983). However, data availability on the effect of supplemental fat on ruminal protozoa in young calves is rather scarce.
Inclusion of forage in starter diets of dairy calves has favorable effects on the stability of the ruminal environment, microbial development, and the health of animals (NASEM, 2021). In a recent recommendation, the National Academies of Science, Engineering, and Medicine (NASEM, 2021) stated that alfalfa hay (ALF) should be limited to no more than 10% of total DMI in young calves. Therefore, it seems vital to evaluate strategies to feed forage and fat concurrently in dairy calves to support the high energy demand for accelerated growth and, at the same time, keep the animal in a healthy condition.
Recently, Karimi et al. (2021) evaluated soybean oil (SO) in starter diets containing ALF as the sole source of forage. They concluded that feeding ALF with a fat supplement may not be recommended in early weeks of life. However, the amount of ALF included in that study was 150 g/kg DM, which was higher than the maximum level recommended by NASEM (2021). These results suggest a limitation of feed intake by gut fill when including too much forage in calf starter diets (Allen and Piantoni, 2014). The latter conforms with Aragona et al. (2020), who stated that dairy calves can consume substantial amounts of forage with concurrent suppression of total DMI. In a comparison of different forages, ad libitum intake for ALF was higher than ad libitum intake of oat hay (Castells et al., 2013). Consequently, limiting the amount of forage, especially of ALF, is deemed appropriate for young calves to keep their health and optimum growth performance.
In addition to suppressing total starter feed intake (Phillips, 2004) and decreasing BW gain (Leibholz, 1975), forage addition to young calves has also been blamed for decreasing nutrient digestibility (Leibholz, 1975) and shifting rumen fermentation in favor of acetate rather than butyrate production, thus, delaying rumen papillae development (Tamate et al., 1962;Nocek and Kesler, 1980). Moreover, the digestibility of NDF may be more negatively influenced when fat is included with an ALF diet (Karimi et al., 2021).
In contrast to ALF, corn silage (CS) has been shown to have favorable effects on young dairy calves at dietary inclusion proportions of 15%, which can be due to reduced dustiness of starters, increased moisture, and higher palatability . Nonetheless, application of CS as a forage source in young dairy calves is also seen to be critical based on the general concerns about forage addition described above. Additionally, to the knowledge of the authors, no study exists that evaluates fat supplementation in calves receiving CS as forage source in starter.
It can be supposed that an optimum forage level along with a suitable source of fat may have favorable effects on the energy content of starter feed to support maximum growth of young calves. Furthermore, it might be proposed that using an optimum content of fat and forage in starter diets of dairy calves may create the opportunity to eliminate or at least reduce negative effects of fat on nutrient digestibility. Although SO, as unsaturated fat source rich in linoleic acid, has been used extensively in dairy calves (Hill et al., 2015;Ghasemi et al., 2017;Ghorbani et al., 2020;Karimi et al., 2021;Yousefinejad et al., 2021), its effects in starter diets containing CS are unknown. Moreover, we additionally evaluated rumen-inert palm fatty acids (PF) rich in palmitic acid in the current study. A previous study on steers fed a high level of forage showed that supplementation of 40 g/kg of hydrogenated palm oil showed no negative effects on feed intake and growth performance but modified the FA profile and some quality traits of meat (Castro et al., 2016). The response of young calves to different fat sources has not been documented when calves received different forage sources. Thus, the aim of the present study was to assess the effects of 2 forage sources (ALF vs. CS) along with 2 fat sources (SO vs. PF) on growth performance, nutrient digestibility, ruminal fermentation, protozoa population, and urinary purine derivatives in young dairy calves. We hypothesized that rumen-inert PF would provide extra energy for growth with less suppressive effects on ru-minal fermentation and that a combination of PF with CS would affect the target variables most favorably to allow for maximum growth.

MATERIALS AND METHODS
The present study was conducted in a commercial dairy farm (Zarrin-Khooshe Agriculture and Animal Husbandry, Arak, Iran). Experimental design, management protocols, and procedures were approved by the Animal Care Committee at the University of Zanjan, Iran (ID 1353).

Animals, Experimental Treatments, and Management
A total of forty-eight 3-d-old female Holstein dairy calves with 40.7 ± 2.1 kg of initial BW were randomly assigned to experimental diets (n = 12 calves per treatment) in a 2 × 2 factorial arrangement with the factors forage source (ALF vs. CS) and fat source (SO vs. PF) of starter diets without blinding to investigators. Calves were separated from their dams immediately after birth, weighed, and moved to individual pens (1.2 × 2.5 m) bedded with sand, the latter being renewed every 24 h. Based on the routine protocol of calf rearing at the farm, all calves were fed 2.5 L of colostrum at each of the first 2 feedings (i.e., within 1.5 h of life and at 12 h after the first feeding). Colostrum feeding was continued for the first 2 d of life. The quality of colostrum was measured with a digital Brix refractometer (PAL-1, Atago Co. Ltd.); colostrum was discarded if the Brix scale was below 22. Calves received whole milk in amounts of 4.5 L/d from d 3 to 10, 7 L/d from d 11 to 40, 3.5 L/d from d 41 to 50, 2.5 L/d from d 51 to 60 and 1.5 L/d on d 61 and 62. Calves were weaned on d 63 and remained in the study until d 83 of age. Whole milk was sampled weekly and analyzed for fat, CP, lactose, and TS using an infrared spectrophotometer (Foss Milk-O-Scan, Foss Electric). The average composition of offered milk was 31.9 ± 0.10 g/kg fat, 30.9 ± 0.4 g/ kg CP, 48.8 ± 0.4 g/kg lactose, and 119.1 ± 6.9 g/kg total solids. Experimental diets were isoenergetic, isonitrogenous, and formulated to meet National Research Council (NRC, 2001) requirements. We evaluated 4 treatments in the current study: (1) starter diet containing 10% ALF with 3% SO (ALF-SO); (2) starter diet containing 10% ALF with 3% PF (ALF-PF); (3) starter diet containing 10% CS with 3% SO (CS-SO); and (4) starter diet containing 10% CS with 3% PF supplementation (CS-PF). To eliminate bias by particle size distribution , geometric mean particle size was similar in ALF (2.88 ± 0.11) and CS (2.93 ± 0.12 mm). The content of CP was 16.8 and 9.1% and the content of NDF was 49.3 and 47.8% for ALF and CS, respectively. The concentrate feed was mixed well with forages and the diet was offered as TMR throughout the study. The SO source (Naz Industrial Vegetable Oil Co.) had the following FA composition: C16:0 = 12.1%, C18:0 = 5.2%, C18:1 = 21.8%, C18:2 = 51.2%, C18:3 = 8.1%, and other FAs = 1.6%. The PF source (rumen-inert; Energizer RP-10, IFFCO) contained: C12:0 = 2.3%, C14:0 = 4.2%, C16:0 = 86.0%, C18:0 = 2.0%, C18:1 = 4.1%, and other FAs = 1.4%. Ingredients and chemical composition of the experimental calf starters are presented in Table 1. Starter feed was fed ad libitum to permit at least 10% waste or uneaten feed. The calves had free access to water throughout the experimental period.

Intake, Daily Gain, Feed Efficiency, and Nutrient Digestibility
Starter feed refusals were collected and recorded daily at 0730 h and fresh starter was fed at 0800 h. Measurements of BW were taken at 10-d intervals through the study starting from d 3 until d 83. Calves were weighed before the morning meal to minimize the effects of gastrointestinal tract fill on recorded BW. Starter feed offered and refused was weighed daily to determine the total starter DMI for each calf throughout the study. Average daily gain and feed efficiency (FE), defined as kg of BW gain/kg of total DMI (starter DMI + milk DMI), were calculated pre-and postweaning, and over the whole experimental period. Feed and leftover feed samples were combined over 21-d intervals and dried in a convection oven (60 C° for 48 h). Subsamples of dried feeds and uneaten feeds were mixed thoroughly and ground in a mill (Ogaw Seiki Co. Ltd.) to pass a 1-mm screen before chemical analysis. Standard methods of AOAC International (2002) were used for the determination of DM (method 2001.12), ash (method, 942.05), CP (method 991.20), and ether extract (method 920.39). The method of Van Soest et al. (1991) was used to determine NDF. For NDF analysis, samples were treated with a heat-stable α-amylase in the absence of sodium sulfite and not corrected for residual ash and protein. To determine apparent digestibility, fecal samples were collected manually from the rectum during 5 consecutive days (from d 78 to 82) at 6, 12, and 18 h after the morning meal. The collected fecal samples were dried at 72°C for 48 h in a forced-air oven, ground to pass a 1 mm screen in a Wiley mill (Ogaw Seiki Co. Ltd.), and then mixed thoroughly. Acid insoluble ash was used as an internal marker to estimate apparent total-tract digestibility coefficients of OM, CP, ether extract, and NDF according to Van Keulen and Young (1977).

Structural Growth Indicators
Structural growth measurements were taken at the start of the experiment (d 3), at weaning (d 63) and at the end of the study (d 83) according to the method described by Heinrichs et al. (2003) for dairy calves. They included withers height (distance from the base of the front feet to withers), hip height (distance from the base of the rear feet to the hook bones), heart girth (circumference of the chest), body length (distance between the points of shoulder and rump), and body girth (circumference of the belly before feeding).

Ruminal Sampling and Protozoa Counting
Ruminal fluid (approximately 20 mL) was collected (3 to 4 h after morning feeding) on d 34 (preweaning) and d 79 (postweaning) of the experiment using a stomach tube fitted to a vacuum pump. The first 10 mL was discarded to eliminate the potential saliva contamination and thereafter, ruminal pH was measured immediately (HI 8314 membrane pH meter, Hanna Instruments). Ruminal fluid samples were squeezed through 4 layers of cheesecloth. An aliquot (10 mL) was preserved with 2 mL of 25% meta-phosphoric acid and frozen at −20°C. After thawing at room temperature, experimental samples were analyzed for short-chain fatty acids (SCFA) concentrations using gas chromatography (model CP-9002, Chrompack) as explained in detail previously . A ruminal fluid subsample was thawed at room temperature and clarified by centrifuging (15,000 × g for 20 min), then decanted and analyzed for NH 3 -N concentration using a modified phenol-hypochlorite reaction (Broderick and Kang, 1980). Rumen protozoa were counted by use of a Neubauer counting chamber (0.1 mm depth; Hausser Scientific Co.) from aliquots of ruminal contents conserved in a solution of methyl green-formalin-saline according to Dehority (1993). Treatments were alfalfa hay-containing starter diet supplemented with soybean oil (ALF-SO); alfalfa haycontaining starter diet supplemented with palm fatty acids (ALF-PF); corn silage-containing starter diet supplemented with soybean oil (CS-SO); corn silage-containing starter diet supplemented with palm fatty acids (CS-PF).

Urinary Purine Derivatives and Urinary Nitrogen Excretion
The spot sampling technique was used to estimate microbial protein synthesis (MPS) in the rumen. This technique is based on the purine derivatives (PD) excretion obtained via urine in dairy calves during the postweaning period as explained by Kazemi-Bonchenari et al. (2022). Urine volume excretion rate per day was estimated by urinary creatinine excretion using the following model; BW × 26.8/creatinine concentration (mg/L) as reported by Dennis et al. (2018). Spot urine samples were collected on 3 consecutive days during the postweaning period from each calf in the morning (between 0900 and 1100 h) and afternoon (between 1500 and 1700 h). Samples (approximately 10 mL) were collected when calves urinated spontaneously. An aliquot of 5 mL of each sample was diluted immediately with 45 mL of 0.036 N sulfuric acid and stored at −20°C for analysis. The concentrations of creatinine, urea-N, uric acid, and allantoin were measured in thawed urine samples as described previously . Estimated daily urine output was used to calculate daily urinary excretion of allantoin + uric acid as total daily PD. The ruminal MPS was estimated from total daily PD output based on an equation of Chen and Gomes (1992): MPS (g/d) = 70 × PD (mmol/d)/(0.85 × 0.116 × 0.83 × 1,000) × 6.25; where 70 is the N content of purines coefficient (mg N/mmol), 0.85 is the efficiency of PD absorption, 0.116 is the ratio of purine-N to total N in mixed ruminal microbes, 0.83 is the average digestibility of microbial purines, and 6.25 is the N content of protein.

Statistical Analysis
All data points without any exclusion and calf as experimental unit were analyzed as a completely randomized design with a 2 × 2 factorial arrangement of treatments, with the factors forage source (ALF vs. CS) and fat source (SO vs. PF). The MIXED procedure of SAS (SAS 9.1, SAS Institute Inc.) was used with fixed effects of time, forage source, fat source, and their interactions and calf as a random effect. Power analysis for sample size estimation was performed (Morris, 1999) for the primary response variables, including intake, BW, and ADG, based on previously published values (Zhang et al., 2010;Miller-Cushon and DeVries, 2011). From the power test analysis, using α = 0.05 and power = 0.80, the projected sample size was 12 calves per treatment for starter intake, total DMI, ADG, and BW. Repeated measures analysis was used for intakes of starter feed, milk, and total DMI (recorded daily), ADG, FE, BW (recorded every 10 d), skeletal growth (d 3, 63, and 83), ruminal pH, urinary PD excretion, MPS, and protozoa population over time. Initial structural growth measurements were considered as covariates for skeletal growth analysis. Before analyses, all data were screened for normality using the UNIVARIATE procedure of SAS. A heterogeneous autoregressive type 1 covariance structure was used in the mixed model. If the primary test indicated statistical significance, differences among treatment means were determined using Tukey's multiple range tests. Effects were considered significant when P ≤ 0.05, and a tendency was considered when 0.05 < P ≤ 0.10. All data are contained within the manuscript.

Starter Intake, Daily Gain, Feed Efficiency, and Nutrient Digestibility
Starter intake during the preweaning period was affected by forage source (P = 0.033) and fat source (P < 0.01) with a trend for interaction between forage × fat source (P = 0.054) with CS showing greater intake than ALF and PF showing greater intake than SO (Table 2). An effect of fat source (PF > SO) was also evident in the postweaning period (P = 0.056) and, as a trend, over the entire period (P = 0.094). Because milk intake was not different among groups, total DMI (i.e., intake of milk DM + starter DM) also showed an effect of forage source (P = 0.021) and fat source (P = 0.016), with trend for an interaction between forage × fat source that pointed to highest DMI in CS-PF (P = 0.060).
The changes in starter intake were almost mirrored by changes in ADG, which tended to be affected by forage source (P = 0.063) and was affected by fat source (P = 0.011), with an interaction between forage × fat source in the preweaning period with greatest ADG in CS-PF and lowest ADG in ALF-SO (P = 0.047). An effect of fat source (PF > SO) on ADG was also evident in the postweaning period (P < 0.01). Over the entire period, ADG was affected by forage (P = 0.045) and fat source (P = 0.017), with a trend for an interaction between forage × fat source (P = 0.075), pointing to greatest ADG in CS-PF. Given the positive effect of CS and PF on starter intake and ADG, BW at weaning and final BW were greater in calves fed CS versus ALF (P = 0.059 and P = 0.031, respectively) and greater for PF versus SO (P = 0.014 and P < 0.01, respectively). Feed efficiency (FE) was not different among groups, except for a trend for higher FE in calves receiving PF compared with SO in the preweaning period (P = 0.098; Table 2). Nutrient digestibility was not influenced by different forage types. The digestibility of NDF (P = 0.015), CP (P = 0.022) and, as a trend, OM (P = 0.052) was higher when calves were fed PF compared with SO (Table 3).
Regarding the time effect and its interaction with variables, results showed that time effect was significant for starter intake (Figure 1), total DMI, ADG (P < 0.01), and, as a trend, FE (P = 0.083). Interaction of time and forage was significant (P = 0.007) when ADG changes were considered over the entire period of the experiment (Table 2).

Growth Indices
Withers height and heart girth were or tended to be greatest at weaning in CS-PF as supported by forage × fat source interaction (P = 0.013 and P = 0.052, respectively; Table 4). A greater withers height in CS-PF was still measurable at the end of the experiment (forage × fat source interaction, P = 0.032). Feeding CS compared with ALF tended to increase hip height at weaning (P = 0.077). Supplemental PF in comparison to SO increased hip height during preweaning (P = 0.042) and the entire period (P = 0.036; Table 4).

Ruminal Fermentation Profile and Protozoa Population
Ruminal pH tended to be lower in calves supplemented with PF compared with SO during the preweaning period (P = 0.091; Table 5). This coincided with lower ruminal ammonia-N concentration in calves fed PF compared with SO during preweaning and the entire period (P < 0.05). Furthermore, ruminal ammonia-N concentration tended to be affected by forage source in the preweaning period (CS < ALF; P = 0.051; Table  5).
Regarding total SCFA concentration, effect of forage source during pre-(P = 0.038) and postweaning (P = 0.014) and an effect of fat source during preweaning were accompanied by forage × fat source interactions during both periods (P = 0.048) with greatest SCFA concentration in the CS-PF group (Table 5). No interactions were found for individual SCFA between forage source × fat source. However, the concentration of ruminal butyrate was greater and that of acetate was smaller in calves fed CS compared with ALF during pre-and postweaning (P < 0.05 each), which coincided with a lower ratio of acetate: propionate in calves fed CS. The concentration of branched-chain (BC)-SCFA was greater in the ruminal fluid of calves supplemented with PF compared with SO during the preweaning period (P = 0.023; Table 5).
Protozoa populations were higher when calves were supplemented with PF diets compared with SO diets (P = 0.027). Forage level or its interaction with fat source did not have any effect on protozoa population (Table 6).

Urinary Purine Derivatives and Nitrogen Efficiency
Feeding PF compared with SO increased the urinary excretion of allantoin (P = 0.023) and PD (P = 0.038)   Table 2. Error bars represent SEM. and, hence, the estimated MPS (P = 0.038; Table 6). Feeding CS in comparison with ALF also increased the urinary excretion of allantoin (P = 0.042) and tended to increase PD (P = 0.054) and estimated MPS (P = 0.054). Urinary N-excretion was decreased by supplemental PF compared with SO (P = 0.044; Table 6).

DISCUSSION
The present study evaluated the inclusion of 2 forage sources (ALF vs. CS) in combination with 2 fat sources (SO vs. PF) in starter diets of young dairy calves on feed intake, nutrient utilization, and growth performance. Starter intake during the preweaning period was enhanced by CS versus ALF and PF versus SO. A trend for interaction of forage × fat source even indicated that both factors can superimpose on each other and cause a 50% change in starter feed intake (581 vs. 376 g/d) in the critical preweaning period. Effects of forage source, as well as supplemental fat source, in starter diets of young calves have been evaluated separately in previous studies. However, limited work is published on the interaction effect of forage and fat in dairy calves during the preweaning period. Regarding forage source, CS may be especially favorable due to lower dustiness of the finely ground grains in the starter feeds containing CS together with a greater palatability and increased moisture (Beiranvand et al., 2016). In dairy cows, increased feed intake in CS versus ALF-based diets was suggested to result from higher ruminal buffering capacity (Erdman, 1988), which has not yet been verified in dairy calves.
Regarding different fat sources, UFA as contained in SO have a greater hypophagic effect than saturated FA (Drackley et al., 1992;Harvatine and Allen, 2006), which can be explained by enhanced oxidation of unsaturated FA in the liver (Mashek et al., 2002) with subsequent negative effects on appetite (Allen et al., 2009) or by reduced nutrient digestibility (Ghorbani et al., 2020). In addition, Tsai et al. (2017) stated that lineoleic acid contained in SO has some inflammatory effect in young ruminants that can modulate intake and performance in preweaning calves. Several studies indicated that supplemental FA generally have a negative influence on the starter feed intake in dairy calves under 2 mo of age (Hill et al., 2015;Ghorbani et al., 2020). However, no negative effects on the feed intake of steers was found when supplementing diets using 4% hydrogenated palm oil; the only findings were a modified FA profile and some alterations of meat quality traits (Castro et al., 2016). From this, it appears that separate positive effects of PF versus SO on ruminal fermentation and nutrient digestibility, on one hand, and higher palatability of CS versus ALF, on the other hand, interacted to potently increase the starter intake of the CS-PF diet during the preweaning period of the present study. In addition, individual FA effects can also be considered as an explanation for the different responses when supplemental fat is added to starter diets of young calves (Hill et al., 2011).
According to the greater feed intake, the greatest ADG (678 g/d) during the preweaning period was found in calves receiving CS-PF, coinciding with greatest heart girth (100.3 cm) and highest withers height  Values within a row not sharing a common letter differ (P < 0.05).
(97.3 cm) at weaning. These results show that feeding starters containing CS with PF supplementation can provide favorable nutritional conditions for acquiring better gain in young dairy calves. This is partly related to an improved ruminal fermentation profile achieved by CS-PF (Table 4) which provided a high energy and microbial protein output for the growth of calves. Our result clarified that feeding CS along with PF provided the greatest ruminal SCFA concentration during the preweaning (95.7 mmol) and postweaning periods (121.6 mmol/L). Bergman (1990) stated that nearly 70% of required energy in ruminants can be supplied through SCFA produced in the rumen; thus, higher SCFA production with CS-PF diets has greater potential to improve growth performance in dairy calves. Because SCFA production depends strongly on fermentable OM in the rumen , the higher ruminal SCFA concentration in CS-PF indicated that more OM is provided to be fermented when calves received CS along with PF.    198 In addition, a lower digestibility of OM, NDF, and CP in calves supplemented with SO may further explain the lower growth performance of SO diets. The lower protein digestibility was additionally supported by lower ruminal concentrations of BC-SCFA because digested protein is the most likely source of BC-SCFA (Wang et al., 2017). The lower fiber digestibility found for SO-supplemented diets likely plays a crucial role, affecting the digestibility of OM and CP as well, and may result from toxic effects of fat on fiber-digesting bacteria (Soliva et al., 2004). Karimi et al. (2021) recently also identified that unsaturated FA supplementation was associated with reduced digestibility of OM and NDF in dairy calves, which was exacerbated when forage was included in the starter. This suggests that depression of NDF digestibility by fat supplementation may become an issue, especially, upon forage inclusion in dairy calf starters because cellulolytic bacteria are not fully developed in the early weeks of life (Ghorbani et al., 2020). Although we did not characterize the bacterial community in the current study, reduced activity of cellulolytic bacteria such as Fibrobacter succinogenes (Maia et al., 2010) and Ruminococcus flavefaciens (Maczulak et al., 1981) has been reported previously regarding supplemental fat inclusion in ruminant diets.
Lower protozoa numbers could also contribute to the reduced nutrient digestibility in SO-supplemented starter diets. Ruminal protozoa have a high ability for fiber digestion (Orpin, 1986;Newbold et al., 2015) and their aminopeptidase activity contributes to protein digestion (Forsberg et al., 1984). Protozoal numbers were lower when supplying SO-supplemented diets, which conforms to previous studies where ruminally unprotected unsaturated FA from SO reduced (Machmüller et al., 1998) or eliminated protozoa (Sutton et al., 1983) and altered ruminal fermentation (Machmüller et al., 1998).
The establishment of the ciliate protozoal community occurs later in young ruminants (Eadie, 1962) compared with cellulolytic bacteria, fungi, and archaea (Fonty et al., 1987) and is impeded by low ruminal pH (Hristov et al., 2001). Such pH effects on protozoa abundance can be excluded for the present study because ruminal pH tended to be higher in SO-supplemented diets (5.95) versus PF diets (5.79) preweaning. This also eliminates the possibility that the reduced intake of SO-supplemented diets had a link to ruminal pH, because reductions in feed intake are commonly due to severe reductions of pH (Khan et al., 2008;Kazemi-Bonchenari et al., 2017).
The feeding of CS versus ALF changed the ruminal SCFA pattern toward less acetate (46.0 vs. 51.3 mmol/L for CS vs. ALF) and more butyrate (13.7 vs. 9.4 mmol/L for CS vs. ALF). Butyrate, in turn, is considered favorable for ruminal development (Sakata and Tamate, 1978;Mentschel et al., 2001;Soltani et al., 2017). Greater starch content in CS rather than ALF may be partly responsible for the higher butyrate concentrations in the ruminal fluid of calves receiving the CS diets (Khan et al., 2008;Mirzaei et al., 2016;Rastgoo et al., 2020). This may be partly related to different microbial species fermenting the different substrate provided in starters (Vital et al., 2013;Wang et al., 2017). As such, it is well accepted that starch is the main energy source for ruminal development in young ruminants (Hu et al., 2018) because its higher availability can improve dairy calf growth performance (Makizadeh et al., 2020).
The higher starch content of CS diets may also explain the greater urinary excretion of allantoin and total PD in CS compared with ALF (Kmicikewycz and Heinrichs, 2015), which is indicative of higher MPS in the rumen (Chen and Gomes, 1992). A reduction of allantoin and total PD excretion through urine was detected, on the other hand, in calves fed supplemental SO, which suggests lower microbial activity when supplementing SO compared with PF. The provided OM to ruminal microbes is a key factor that affects MPS . As discussed earlier, PF versus SO and CS versus ALF increased total DMI in the current study, which, as expected, provided higher OM to ruminal microbes. These facts collectively indicate that the potential to increase MPS in young calves is greater for CS compared with ALF and PF compared with SO. This is in line with total SCFA production in the current study, which was greatest in calves fed CS with PF. In turn, the lower protozoa number in calves receiving SO-supplemented diets does not seem to have a direct link to lower estimated MPS in those calves. Although ciliate protozoa may contribute up to 50% to the bio-mass in the adult rumen (Newbold et al., 2015), it is well known that elimination of protozoa increases microbial protein supply by up to 30% due to decreased bacterial protein turn-over in the rumen. This is because protozoa prey on bacteria as their main protein source (Williams and Coleman, 1992) and, as a result, defaunation makes the rumen more efficient in terms of protein synthesis (Newbold et al., 2015).
Urinary nitrogen excretion did not change with forage source; however, supplemental SO increased urea-N compared with PF diets. Greater urinary nitrogen excretion is indicative of lower nitrogen efficiency in ruminants (Kohn et al., 2005). This indicates that SO supplementation not only had negative effects on ruminal fermentation and nutrient digestibility but also increased urinary N concentration, indicating lower nitrogen utilization efficiency. This means that more nitrogen was directed toward urinary excretion in SO-supplemented calves instead of being used for MPS. This might be due to lower NDF and OM digestibility caused by supplemental SO, which reduced nutrient availability for ruminal microbes and, hence, less nitrogen was captured in microbial protein.

CONCLUSIONS
The present study provides unique insight into the effects and interaction of 2 different forages and fat sources on zootechnical traits and readouts of digestibility and ruminal fermentation in starter diets of young dairy calves. Feeding CS proved superior to ALF because it was associated with higher ruminal production of microbial protein and butyrate and enhanced total DMI and BW at weaning and final measurement. Feeding rumen-inert PF proved superior to SO because the latter impaired digestibility of NDF, OM, and CP; decreased estimated MPS; increased ruminal ammonia-N and urinary-N excretion; and was associated with lower DMI and BW at weaning and final measurement. Most importantly, the effects of forage × fat source interacted for ruminal SCFA concentration, ADG and, as a trend, for starter intake in the critical preweaning period with highest values in CS-PF. The latter indicates that concurrent supplementation of CS with PF at the applied dosages is particularly favorable in young calves before weaning.