J. Dairy Sci.

98:7131–7141
http://dx.doi.org/10.3168/jds.2014-9144
© American Dairy Science Association®, 2015.

Rumen fermentation, blood metabolites, and growth performance

of calves during transition from liquid to solid feed: Effects
of dietary level and particle size of alfalfa hay
M. Nemati,* H. Amanlou,* M. Khorvash,† B. Moshiri,‡ M. Mirzaei,§ M. A. Khan,# and M. H. Ghaffari†1
*Department of Animal Science, Faculty of Agricultural Sciences, University of Zanjan, Zanjan 313-45195, Iran
†Department of Animal Science, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran
‡Ghiam Dairy Complex, Isfahan 833111-4761, Iran
§Department of Animal Science, Faculty of Agriculture and Natural Resources, Arak University, 38156-8-8349 Arak, Iran
#Animal Nutrition and Health Group, Grasslands Research Centre, AgResearch Limited Palmerston North 4442, New Zealand

ABSTRACT rations differing in forage PS; thus, providing calves

with MPS can improve calf performance and reduce
This study evaluated the effects of particle size (PS) their nonnutritive oral behaviors.
and dietary level of alfalfa hay (AH) on rumen fermen- Key words: alfalfa hay, particle size, dairy calf
tation parameters, blood metabolites, eating behavior,
and growth performance in dairy calves during tran-
INTRODUCTION
sition from liquid to solid feed. Sixty newborn dairy
calves (41 ± 2.5, kg of body weight) were used in a 2 Physical and metabolic development of the reticu-
× 2 factorial arrangement with the factors dietary AH lorumen in young calves is the prerequisite to their
level (medium, 12.5%, or high, 25%, on DM basis) and transition from a preruminant milk diet to a solid diet
PS (fine = 1 mm or medium = 3 mm, as geometric based on grain forage (Drackley, 2008). Although ru-
means) of AH. Hence, the dietary treatments were (1) men development in young calves is affected by many
medium level of AH with fine PS (M-FPS), (2) medium factors (Baldwin et al., 2004), it is mainly dictated by
level of AH with medium PS (M-MPS), (3) high level the amount and nature of the solid feed offered (Khan
of AH with fine PS (H-FPS), and (4) high level of AH et al., 2011). Development of the rumen epithelium is
with medium PS (H-MPS). Particle size of AH did not triggered by the VFA produced during microbial fer-
affect total DMI (TDMI) during the preweaning period, mentation of ingested solid feed in the rumen (Baldwin
although TDMI was greater for calves fed MPS than in et al., 2004). Development of rumen musculature, ru-
those fed FPS during the postweaning and overall peri- men movements, and the capacity of the rumen are
ods. Calves fed MPS spent more time eating solid feed influenced by the physical stimuli provided by the bulk
and ruminating and less time on nonnutritive oral be- of the ingested material (Beharka et al., 1998).
haviors compared with FPS calves. The dietary level of Cereal-based starter diets generally contain higher
AH did not affect behavioral parameters. Average daily levels of fermentable carbohydrates than forages, which
gain of calves was not affected by dietary treatment provide greater amounts of VFA to stimulate rumen
before weaning. During the postweaning and overall pe- papillae when they ferment in the rumen (Lesmeister
riods, average daily gain was greater in calves fed MPS and Heinrichs, 2004; Khan et al., 2008). Ingestion of for-
than in those fed FPS at the 25% AH level, but this age provides bulk in the rumen and thereby promotes
effect was absent with 12.5% AH. Furthermore, the ru- rumen musculature and helps maintain the integrity
men pH values on d 35 and 70 of the study were greater of the rumen epithelium in developing calves (Beharka
for MPS than for FPS, regardless of the dietary level of et al., 1998; Beiranvand et al., 2014b). Forage provi-
AH. Effects of AH level, PS, and their interaction did sion to young calves is generally discouraged because
not affect blood glucose concentrations in developing forage is less energy intensive and digestible compared
calves. These results indicate that feed intake, feeding with starter diets, and could, therefore, delay rumen
behavior, rumen fermentation parameters, and blood epithelial development and suppress growth in young
β-hydroxybutyrate concentration may be affected by ruminants (Stobo et al., 1966; Hill et al., 2008, 2009).
Furthermore, the guidelines of National Research
Received November 23, 2014.
Council (NRC, 2001) do not include any recommenda-
Accepted April 21, 2015. tions for fiber provision for developing calves. However,
1
Corresponding author: [email protected] recent studies have demonstrated beneficial effects of

7131
7132 NEMATI ET AL.

feeding forage (grasses and legumes) early in life on (12.5% on DM basis) and a high dietary level of AH
rumen fermentation (Castells et al., 2013; Terré et al., (25% on DM basis) with a fine (1 mm; geometric mean)
2013), rumen pH (Laarman and Oba, 2011), rumen de- or medium PS (3 mm; geometric means). Calves were
velopment, feed consumption, and growth performance assigned to 1 of 4 treatments: (1) medium level of AH
(Khan et al., 2011; Castells et al., 2012; Montoro et al., with fine PS (M-FPS), (2) medium level of AH with
2013; Beiranvand et al., 2014b). Furthermore, Castells medium PS (M-MPS), (3) high level of AH with fine
et al. (2013) reported increased expression of a VFA PS (H-FPS), and (4) high level of AH with medium
transporter (mono-carboxylate transporter 1) in the ru- PS (H-MPS). The ingredient and chemical composi-
men epithelium of calves that had access to forage com- tion of the experimental diets are shown in Table 1.
pared with calves fed pelleted starter without forage. All the diets were formulated and to be isoenergetic
Previous studies (Castells et al., 2013; Beiranvand et and isonitrogenous according to the Cornell Net Car-
al., 2014a,b) have shown enhanced concentrate intake bohydrate and Protein System version 5.1 (Fox et al.,
and growth performance in young calves supplemented 2000). For the first 16 d, all calves received the starter
with forage. Particle size (PS) of the dietary forage diet without forage provision. From d 16 onward, AH
affects the rumen environment, with high amounts of was mixed with finely ground concentrates and pro-
long and coarse forage resulting in decreased intake vided as a TMR. Starter containing AH (TMR) was
because of gut fill in dairy cows (Mirzaei et al., 2015). fed ad libitum to permit at least 10% orts (i.e., the por-
This may also apply to dairy calves, especially because tion of the starter not consumed over a 24-h period),
of the limited capacity of their reticulorumen (Beharka and TMR feed refusal from each individual calf was
et al., 1998; Godden et al., 2009). collected at 0800 h. Calves had ad libitum access to
We hypothesized that PS of alfalfa hay (AH) in the water and starter throughout the study. Before the ex-
diet with rapidly degradable starch, as in barley grain, periment, AH was chopped (Golchin Trasher Hay Co.,
may have the potential to modify rumen conditions and Isfahan, Iran) to obtain fine (1 mm, geometric mean)
to improve the performance of dairy calves. Hence, 2 and medium (3 mm, geometric mean) PS. At least 6
levels of PS (fine and medium) were combined with 2 representative samples were collected from each PS and
levels of AH (12.5 and 25% of dietary DM) in forage- used for analyzing PS distribution of AH by the Penn
supplemented diets to investigate their interaction on State Particle Separator (PSPS; Table 2). Geometric
feed intake, rumen fermentation parameters, feeding mean PS was calculated as described by the American
behavior, and growth performance in dairy calves. Society of Agricultural Engineers (ASAE, 1996; method
S424.1).
MATERIALS AND METHODS
Sampling
The experiment was conducted on a local dairy farm
(Ghiam Esfahan Plantation and Domesticated Co., Intakes of calf starter (forage and starter feed, as
Isfahan, Iran). All the calves were cared and managed TMR) and total DMI (milk, starter feed, and forage)
according to the guidelines of Iranian Council of Ani- were measured daily and BW were recorded weekly
mal Care (1995). during the preweaning (wk 1 to 7), postweaning (wk
8 to 10), and overall periods (wk 1 to 10). Amounts of
Calves, Management, and Treatments feed offered and refused were recorded daily for each
individual calf. Preweaning, postweaning, and overall
Sixty 3-d-old Holstein male dairy calves (41 ± 2.5 kg means of total DMI (milk solids, starter feed), ADG,
of BW) were separated from their dams, weighed, and and feed efficiency (kg of BW gain/kg of total DMI)
moved to individual pens (1.2 × 2.5 m) bedded with were also calculated.
sawdust that was renewed every 48 h. The animals were Body measurements at weaning (d 51) and at the
fed 3 to 4 L of colostrum within 2 to 6 h after birth. end of the study (d 71), including body length (distance
Colostrum feeding continued for the first 2 d of life. between the points of shoulder and rump), body girth
Calves were fed whole milk (4 L/d), at a rate of 10% (the measurement of the distance around the belly over
of BW, containing 3.22 ± 0.11% fat, 2.96 ± 0.07% CP, the part of the back before morning feeding), withers
4.92 ± 0.05% lactose, and 11.77 ± 0.15% TS in steel height (distance from base of the front feet to the with-
buckets twice daily at 0900 and 1700 h from d 3 to 47, ers), heart girth (circumference of the chest), hip height
followed by morning feeding (2 L/d) from 47 to 50 d of (distance from base of the rear feet to hook bones), and
age before being weaned on d 51 of age. hip width (distance between the points of hook bones),
The calves were randomly assigned (n = 15 per treat- of the calves were performed according to the method
ment) in a 2 × 2 factorial arrangement to a medium described by Khan et al. (2007).
Journal of Dairy Science Vol. 98 No. 10, 2015
 ALFALFA LEVEL AND PARTICLE SIZE IN CALVES 7133
Table 1. Ingredients and chemical compositions of experimental diets Table 2. Distribution of alfalfa hay with fine and medium particle
with different (12.5 and 25%) levels of alfalfa hay (AH) sizes (PS; mean ± SD) measured as DM retained on sieve (%)

Concentration in the diet1 Alfalfa hay

Item 12.5% AH 25% AH Item Fine PS Medium PS

Ingredient (% of DM) 19.0 mm 0 1.0 ± 0.2
Alfalfa hay 12.5 25.0 8.0 mm 6.5 ± 0.6 26.0 ± 1.9
Barley grain 57.5 45.0 1.18 mm 50.5 ± 1.2 35.1 ± 0.9
Soybean meal (45% CP) 25 20.8 <1.18 mm 42.9 ± 2.1 37.8 ± 2.3
Extruded soybean 1.6 5.0 GMPL1 (mm) 2.1 ± 0.1 2.9 ± 0.1
Calcium carbonate 1.0 1.0 1
Energy booster 1002 0.4 1.2 GMPL = geometric mean particle size calculated as described by
Vitamin and mineral premix3 1.5 1.5 ASAE (1996; method S424.1).
Salt 0.5 0.5
Chemical composition (% of DM)
ME4 (Mcal/kg) 2.82 2.85 Romania). Rumen contents were squeezed through 4
NEG4 (Mcal/kg) 1.25 1.25 layers of cheesecloth and the fluid was stored at −20°C
DM 90 92
CP 19.61 19.73 pending analysis. For stabilization, 10 mL of the rumen
NDF 23.0 24.5 fluid was acidified with 3 mL of 25% metaphosphoric
Ether extract 3.96 3.87 acid and stored (−20°C) until analysis for VFA by gas
Ash 11.2 11.4
Ca4 0.75 0.78 chromatography.
P4 0.42 0.47 Blood samples were collected 3 h after the morning
1
Control = diet without alfalfa hay; 12.5% AH = diet with 12.5% meal on d 21, 35, and 74 of the study period. Blood was
alfalfa hay; 25% AH = diet with 25% alfalfa hay. withdrawn from the jugular vein into evacuated tubes
2
Prilled saturated FFA (Milk Specialties, Dundee, IL). containing K2 EDTA (Becton Dickinson Vacutainer
3
Contained per kilogram of supplement: 250,000 IU of vitamin A, Systems, Franklin Lakes, NJ) and immediately placed
50,000 IU of vitamin D, 1,500 IU of vitamin E, 2.25 g of Mn, 120 g of
Ca, 7.7 g of Zn, 20 g of P, 20.5 g of Mg, 186 g of Na, 1.25 g of Fe, 3 g on ice. The tubes were then centrifuged at 2,000 × g
of S, 14 mg of Co, 1.25 g of Cu, 56 mg of I, and 10 mg of Se. (4°C) for 20 min within 2 h of sampling. The plasma
4
Calculated from NRC (2001). was separated and stored at −20°C for subsequent
analysis.

Behavioral data were monitored by direct observa- Chemical and Biochemical Analyses
tions of all calves as the total time (min) devoted to each
monitored behavior for a duration of 4 d, at 2 wk before Total mixed rations were sampled weekly at the
weaning (d 37 and 44 of the experiment) and 2 wk after time of feeding and frozen in plastic bags. Samples
weaning (d 58 and 56 of the experiment). Calves were were thawed and composited into 1 sample per month.
observed 1 h after the solid feed was offered and 2 h The composited samples were mixed thoroughly and
immediately after the morning milk feeding during the ground to pass a 1-mm screen in a Wiley mill (Ogaw
preweaning week. During the postweaning week, calves Seiki Co. Ltd., Tokyo, Japan) before chemical analyses.
were observed for 1 h before and 2 h after the solid Feed and refusal samples were analyzed for their DM
feed was offered. Thus, the total time for observing the content according to the procedures of AOAC (1990).
behavior of each calf was equal to 12 h (6 h before and Crude protein (method 988.05; AOAC, 1990) and NDF
6 h after weaning). Calves were observed every minute, without the use of sodium sulfite and with α-amylase
and the following behaviors were recorded: standing, included (Van Soest et al. 1991) were also analyzed.
lying, eating, ruminating, and non-nutritive oral behav- Rumen samples were analyzed for VFA by gas chro-
iors (when the animal licked any surface, tongue rolled, matography (model CP-9002, Chrompack, Middelburg,
or consumed wood shavings). the Netherlands) with a 50-m (0.32 mm i.d.) silica-
Feces were scored daily for physical shape and consis- fused column (CP-Wax Chrompack Capillary Column,
tency according to the procedure described in Heinrichs Varian, Palo Alto, CA). Helium was used as the carrier
et al. (2003): 1 = normal; 2 = soft to loose; 3 = loose gas and the oven initial and final temperatures were 55
to watery; 4 = watery, mucous, slightly bloody; and 5 and 195°C, respectively. The detector and injector tem-
= watery, mucous, and bloody. peratures were set at 250°C. Crotonic acid (1:7, vol/
On d 35 and 71 of the study period, rumen fluid sam- vol) was used as the internal standard. Plasma BHBA
ples were obtained by using a stomach tube 4 h after was determined by autoanalyzer using a laboratory
the morning feeding, and rumen pH was measured im- kit (Randox Laboratories Ltd, Ardmore, UK). Plasma
mediately (HI 8318, Hanna Instruments, Cluj-Napoca, concentration of glucose was measured using a serum

Journal of Dairy Science Vol. 98 No. 10, 2015

7134 NEMATI ET AL.

spectrophotometer (UNICCO, 2100, Zistchemi Co., were not affected by AH level, PS, or their interaction
Tehran, Iran). (P > 0.05; Table 3). No significant 2-way interactions
(week × level and week × PS of AH) were found for
Statistical Analyses gain-to-feed ratio, indicating that the effect of level and
PS of AH on gain-to-feed ratio was similar throughout
Data of DMI, ADG, and gain-to-feed ratio were sub- the experiment. No significant 3-way interactions (week
jected to ANOVA for a complete randomized design × level × PS of AH) were found for DMI, ADG, and
with repeated measurements using PROC MIXED in gain-to-feed ratio (Table 3).
SAS (version 9.2; SAS Institute, Cary, NC) with the
individual calf as the experimental unit. The model Rumen Fermentation Parameters
included level, PS of AH, the interaction between level and Blood Metabolites
and PS of AH, and the interactions of level × week,
PS × week, and level × PS × week for DMI, ADG, Regardless of the level of AH, ruminal pH was greater
and gain-to-feed ratio data. An autoregressive (order for MPS than for FPS on d 35 and 70 of the study (P
1) covariance structure was chosen based on the Akaike < 0.01; Table 4). No interaction was observed between
and Bayesian information criteria. Statistical analyses forage PS and the level of AH on ruminal pH (Table 4).
were conducted over 3 periods: preweaning (wk 1 to Ruminal pH was not affected by AH level, PS, or
7), postweaning (wk 8 to 10), and overall (wk 1 to 10) their interaction (Table 4). Regardless of the AH level,
periods. Rumen fermentation, behaviors, fecal score, acetate concentrations were greater on d 70 of the
and structural growth variables were analyzed using study in calves fed MPS than in those fed FPS (P <
a similar model but without the effect of time. For 0.05; Table 4). Regardless of PS, propionate concentra-
structural growth variables, the initial measurements tion tended to be greater at 12.5% inclusion of AH
were included as a covariate for the statistical analysis than at 25% (P = 0.08; Table 4). The concentration
of body measurements. When differences for dietary of butyrate was not affected by AH level, PS, or their
treatments or interactions were detected, means sepa- interaction. Furthermore, regardless of the AH level,
ration was conducted using a Tukey’s adjustment for the acetate-to-propionate ratio was greater (P < 0.05)
the probability. Significance was declared at P ≤ 0.05 on d 35 and 70 of the study in calves fed MPS than in
and trends were considered when 0.05 < P < 0.10. those fed FPS (Table 4). Blood glucose concentration
was not affected by AH level, PS, or their interaction
(Figure 4). However, regardless of the AH level, blood
RESULTS
BHBA concentrations were greater on d 70 of the study
All the calves were healthy with no clinical symp- period in calves fed MPS than in those fed FPS (P <
toms of systemic disease or mortality throughout the 0.05; Figure 5).
experiment.
Animal Behavior and Structural Growth
Calf Performance Regardless of AH level, calves receiving the MPS
treatment devoted greater time to eating starter feed
Medium PS promoted total DMI (TDMI) when AH
and ruminating than those receiving FPS (P < 0.01;
was supplemented at 25% of dietary DM compared
Table 5). Moreover, calves on the MPS diet spent less
with the 12.5% level (Figure 1; Table 3). Total DMI
time performing non-nutritive oral behaviors compared
was not affected by AH PS during the preweaning pe-
with those on the FPS diet (Table 5).
riod, although TDMI was greater for calves fed MPS
Body length, withers height, heart girth, hip width,
than for those fed FPS during the postweaning and
and hip height were not affected by AH level, PS, or
overall periods (P < 0.05; Table 3). Final BW (d 70)
their interaction (Table 6). However, body girth at the
increased (P < 0.01) from 75.18 to 84.1 kg when PS
end of experiment increased with increasing PS from
changed from fine (1 mm) to medium (3 mm; Figure
fine to medium, regardless of the AH level (P < 0.05;
2; Table 3). Mean ADG and gain-to-feed ratio of the
Table 6).
calves did not differ across the treatments before wean-
ing. Regardless of the AH level, ADG was greater
DISCUSSION
in calves fed MPS than in those fed FPS during the
postweaning and overall periods (P < 0.01; Figure 3). Calf Performance
No interaction was found in this study between AH
level and PS regarding their effects on TDMI, BW, It has been widely recognized that feeding forage in-
or ADG. In general, gain-to-feed ratio and fecal score creases postweaning TDMI (Castells et al., 2012; Terré
Journal of Dairy Science Vol. 98 No. 10, 2015
 ALFALFA LEVEL AND PARTICLE SIZE IN CALVES 7135

Figure 1. Weekly mean (±SEM) total DMI (kg) in calves fed different diets. M-FPS = medium level (12.5%) of alfalfa hay (AH) with fine
particle size (PS; ), M-MPS = medium level (12.5%) of AH with medium PS (), H-FPS = high level (25%) of AH with fine PS (), H-MPS
= high level (25%) of AH with medium PS (). For each time point, * denotes significant difference at P < 0.05; ** denotes significant differ-
ence at P < 0.01.

et al., 2013). It seems that forage particle size may affect mm) grass hay (0.68 vs. 0.63 ± 0.02 kg of gain/kg of
postweaning DMI in young calves. Providing AH with DMI).
a fine PS at medium and high supplementation levels The results obtained for fecal scores are in agreement
may not have provided sufficient mechanical stimuli to with those reported by Coverdale et al. (2004) who did
increase the physical capacity and muscular develop- not find differences in the fecal scores of calves fed dif-
ment of the reticulorumen (Tamate et al., 1962), or ferent inclusion levels of bromegrass hay compared with
rumen motility (Nocek, 1997; Owens et al., 1998), re- their control group.
sulting in similar values of DMI, BW, ADG, and gain-
to-feed ratio compared with non-forage-supplemented Rumen Fermentation Parameters
calves. In the present study, the greater DMI with MPS and Blood Metabolites
diets may be explained by the increased capacity and
development of the reticulorumen (Khan et al., 2011; In the current study, ruminal pH ranged from 5.05
Montoro et al., 2013). This is in line with the results to 5.72 in calves supplemented with higher levels of
of a recent study (Montoro et al., 2013), which showed forage after weaning (5.22 to 5.72) compared with be-
that the greater PS of grass hay with a DM inclusion fore weaning (5.05 to 5.51), which is consistent with
level of 10% improved starter feed intake by dairy calves the findings of Mirzaei et al. (2015). Anderson et al.
after weaning. The improved rumen environment and (1987) also reported that pH in the rumen of dairy
enhanced muscular development of the rumen may, in calves did not exceed 6 during the first 10 wk of life.
turn, have contributed to the increased intake of solid Krause et al. (2002) reported that mean ruminal pH
feed during the postweaning period (Coverdale et al., was positively correlated with the time spent on rumi-
2004; Khan et al., 2011; Castells et al., 2012). Mon- nating and chewing. Our results concerning forage PS
toro et al. (2013) reported that calves fed 10% coarsely are in agreement with those of other authors (Krause
chopped (3 to 4 cm) grass hay tended to have a greater et al., 2002; Teimouri Yansari et al., 2004), confirming
gain-to-feed ratio than those fed 0% finely ground (2 that forage PS plays a major role in maintaining opti-

Journal of Dairy Science Vol. 98 No. 10, 2015

7136 NEMATI ET AL.

Table 3. Effects of forage level (12.5 vs. 25%) and particle size (fine vs. medium) on BW, DMI, ADG, gain-to-feed ratio, and fecal score in
dairy calves (n = 15 per treatment)

P-value2

Treatment1 Treatment Interaction3

Item M-FPS M-MPS H-FPS H-MPS SEM L PS L × PS 1 2 3

DMI, kg/d
Preweaning 0.80 0.82 0.79 0.83 0.03 0.98 0.46 0.86 0.52 0.72 0.58
Postweaning 1.49 1.69 1.45 2.02 0.08 0.23 0.01 0.11 0.08 0.01 0.15
Overall 1.01 1.08 0.99 1.19 0.05 0.51 0.04 0.35 0.05 0.01 0.13
BW, kg
Initial, d 1 42.25 42.63 43.18 42.7 1.16 0.23 0.43 0.64 — — —
At weaning, d 51 63.10 65.12 63.02 64.99 1.31 0.99 0.15 0.94 — — —
Final, d 70 76.52 80.22 75.18 84.10 2.22 0.52 0.01 0.25 — — —
ADG, kg/d
Preweaning 0.41 0.45 0.41 0.45 0.02 0.99 0.13 0.94 0.35 0.20 0.13
Postweaning 0.64 0.72 0.58 0.89 0.07 0.37 0.01 0.08 0.86 0.01 0.47
Overall 0.48 0.53 0.46 0.58 0.03 0.50 0.01 0.23 0.72 0.01 0.47
Gain-to-feed ratio4
Preweaning 0.47 0.47 0.46 0.49 0.03 0.72 0.61 0.58 0.81 0.65 0.79
Postweaning 0.42 0.42 0.40 0.45 0.04 0.87 0.35 0.51 0.98 0.12 0.86
Overall 0.45 0.46 0.44 0.48 0.03 0.83 0.34 0.34 0.95 0.57 0.91
Fecal score5 2.46 2.64 2.59 2.67 0.15 0.59 0.39 0.74 — — —
1
Treatment: M-FPS = medium level (12.5%) of alfalfa hay (AH) with fine particle size (PS); M-MPS = medium level (12.5%) of AH with me-
dium PS; H-FPS = high level (25%) of AH with fine PS; H-MPS = high level (25%) of AH with medium PS.
2
Statistical comparisons: L = level of alfalfa hay; PS = particle size of alfalfa hay; L × PS = L by PS interaction.
3
Interactions 1, 2, and 3 = level of AH × week; PS of AH × week; level of AH × PS of AH × week.
4
Gain-to-feed ratio was calculated by dividing ADG (g) by average daily DMI.
5
Scoring was as follows: 1 = normal; 2 = soft to loose; 3 = loose to watery; 4 = watery, mucous, slightly bloody; 5 = watery, mucous, and bloody.

Figure 2. Weekly BW (±SEM; kg) in calves fed different diets. M-FPS = medium level (12.5%) of alfalfa hay (AH) with fine particle size
(PS; ), M-MPS = medium level (12.5%) of AH with medium PS (), H-FPS = high level (25%) of AH with fine PS (), H-MPS = high level
(25%) of AH with medium PS ().

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 ALFALFA LEVEL AND PARTICLE SIZE IN CALVES 7137
Table 4. Effects of forage level (12.5 vs. 25%) and particle size (fine vs. medium) on rumen fermentation parameters in dairy calves (n = 15
per treatment)

Treatment1 P-value2

Item M-FPS M-MPS H-FPS H-MPS SEM L PS L × PS

Rumen pH
d 35 5.05 5.45 5.13 5.51 0.04 0.12 0.01 0.83
d 70 5.22 5.48 5.23 5.72 0.07 0.13 0.01 0.15
Total VFA, mmol/L
d 35 101.10 94.65 91.72 98.19 5.88 0.63 0.99 0.29
d 70 110.1 105.7 96.1 103.8 4.54 0.11 0.72 0.21
Individual VFA, mol/100 mol
Acetate
d 35 52.11 50.89 46.23 54.66 3.84 0.78 0.36 0.23
d 70 49.95 53.99 46.06 56.24 2.46 0.74 0.01 0.24
Propionate
d 35 35.68 32.27 33.41 31.49 1.95 0.45 0.20 0.71
d 70 39.66 37.04 34.81 34.92 1.86 0.08 0.51 0.48
Butyrate
d 35 13.29 11.48 12.08 12.03 0.88 0.72 0.32 0.34
d 70 20.5 14.68 15.25 12.68 2.43 0.16 0.11 0.52
Acetate:propionate
d 35 1.46 1.57 1.37 1.74 0.06 0.54 0.01 0.08
d 70 1.26 1.48 1.33 1.62 0.09 0.30 0.02 0.69
1
Treatment: M-FPS = medium level (12.5%) of alfalfa hay (AH) with fine particle size (PS); M-MPS = medium level (12.5%) of AH with me-
dium PS; H-FPS = high level (25%) of AH with fine PS; H-MPS = high level (25%) of AH with medium PS.
2
Statistical comparisons: L = level of alfalfa hay; PS = particle size of alfalfa hay; L × PS = L by PS interaction.

Figure 3. Weekly ADG (±SEM; kg) in calves fed different diets. M-FPS = medium level (12.5%) of alfalfa hay (AH) with fine particle size
(PS; ), M-MPS = medium level (12.5%) of AH with medium PS (), H-FPS = high level (25%) of AH with fine PS (), H-MPS = high level
(25%) of AH with medium PS (). For each time point, * = significant difference at P < 0.05; ** = significant difference at P < 0.01.

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7138 NEMATI ET AL.

Figure 4. Blood glucose concentrations (±SEM; mg/dL) in calves fed different diets. M-FPS = medium level (12.5%) of alfalfa hay (AH)
with fine particle size (PS; ), M-MPS = medium level (12.5%) of AH with medium PS (), H-FPS = high level (25%) of AH with fine PS cane
(), H-MPS = high level (25%) of AH with medium PS ().

mal rumen pH and that increasing the PS of AH can al. (2012) reported that cows fed long AH had higher
improve ruminal pH, which may be the result of greater ruminal pH compared with those fed finely chopped
time spent ruminating in dairy calves. Nasrollahi et AH diets (6.46 vs. 6.26). However, this observation is

Figure 5. Blood BHBA concentrations (±SEM; mmol/L) in calves fed different diets. M-FPS = medium level (12.5%) of alfalfa hay (AH)
with fine particle size (PS; ), M-MPS = medium level (12.5%) of AH with medium PS (), H-FPS = high level (25%) of AH with fine PS (),
H-MPS = high level (25%) of AH with medium PS (). For each time point, * = significant difference at P < 0.05.

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 ALFALFA LEVEL AND PARTICLE SIZE IN CALVES 7139
Table 5. Effects of forage level (12.5 vs. 25%) and particle size (fine vs. medium) on total time devoted to performing different behaviors during
8 h of observation in dairy calves (n = 15 per treatment)

Treatment1 P-value2

Item M-FPS M-MPS H-FPS H-MPS SEM L PS L × PS

Standing (min) 186.8 177.9 180.3 169.5 7.45 0.48 0.28 0.70
Lying (min) 217.5 213.9 225.3 212.6 6.59 0.58 0.25 0.45
Eating (min) 24.15 30.93 21.13 35.52 1.43 0.72 0.01 0.06
Ruminating (min) 21.17 33.91 20.37 39.38 2.1 0.24 0.01 0.11
Non-nutritive oral behaviors (min) 30.42 23.20 32.95 22.98 2.5 0.65 0.01 0.59
1
Treatment: M-FPS = medium level (12.5%) of alfalfa hay (AH) with fine particle size (PS); M-MPS = medium level (12.5%) of AH with me-
dium PS; H-FPS = high level (25%) of AH with fine PS; H-MPS = high level (25%) of AH with medium PS.
2
Statistical comparisons: L = level of alfalfa hay; PS = particle size of alfalfa hay; L × PS = L by PS interaction.

inconsistent with the report of Mirzaei et al. (2015), ings of Khan et al. (2011). The reduced blood glucose
who found no significant differences in the ruminal pH concentration with age observed in calves regardless of
of calves fed AH of different PS. treatment may be attributed to a physiological shift in
The total VFA concentrations in the current study the primary energy source from glucose to VFA when
were not affected by AH level, PS, or their interac- the rumen in young calves becomes functional (Ham-
tion, which is in agreement with the results reported mon et al., 2002). In agreement with previous studies
by Mirzaei et al. (2015). However, previous studies did (Greenwood et al., 1997; Coverdale et al., 2004; Naeem
not observe any differences in total rumen VFA concen- et al., 2012), increasing blood BHBA concentration as
trations between calves offered different forage levels calves aged was mainly due to a shift in the sources of
and those not receiving any forage (Thomas and Hinks, physiological fuel during the transition from liquid to
1982; Suárez et al., 2007). In the current study, insuf- solid diets (Khan et al., 2011).
ficient particle size resulted in reduced ruminal pH and In the current study, the greater concentrations of
acetate-to-propionate ratio. The reduction in rumen blood BHBA (an indicator of metabolic function of the
pH impairs the growth of cellulolytic bacteria, result- rumen wall) in calves fed MPS compared with FPS
ing in declining acetate-to-propionate ratio (Grant et suggested that ruminal development was normal and
al., 1990). that rumen walls were equally efficient in converting
Blood glucose concentration was also observed to de- butyrate to BHBA (Khan et al., 2011).
crease with calf age, which is consistent with the find-

Table 6. Effects of forage level (12.5 vs. 25%) and particle size (fine vs. medium) on structural growth in dairy calves (n = 15 per treatment)

Treatment1 P-value2

Item M-FPS M-MPS H-FPS H-MPS SEM L PS L × PS

Body length
Weaning 52.39 54.10 52.93 53.43 0.8 0.90 0.19 0.44
Final 57.06 57.33 57.24 58.43 0.8 0.46 0.41 0.58
Body girth
Weaning 108.8 111.0 109.8 113.8 1.78 0.41 0.08 0.47
Final 116.1 119.5 117.8 123.8 2.00 0.14 0.02 0.51
Withers height
Weaning 83.52 82.74 83.44 83.80 0.71 0.49 0.77 0.42
Final 85.39 84.96 85.41 85.46 0.44 0.81 0.85 0.47
Heart girth
Weaning 94.27 95.77 94.37 95.30 0.92 0.85 0.22 0.63
Final 100.7 99.9 98.9 101.6 1.57 0.96 0.41 0.13
Hip width
Weaning 19.57 20.02 19.73 20.31 0.51 0.85 0.23 0.49
Final 23.30 23.15 23.49 23.92 0.32 0.12 0.61 0.38
Hip height
Weaning 89.40 88.94 89.41 88.85 0.56 0.95 0.56 0.11
Final 91.57 90.67 90.44 91.39 0.84 0.69 0.84 0.60
1
Treatment: M-FPS = medium level (12.5%) of alfalfa hay (AH) with fine particle size (PS); M-MPS = medium level (12.5%) of AH with me-
dium PS; H-FPS = high level (25%) of AH with fine PS; H-MPS = high level (25%) of AH with medium PS.
2
Statistical comparisons: L = level of alfalfa hay; PS = particle size of alfalfa hay; L × PS = L by PS interaction.

Journal of Dairy Science Vol. 98 No. 10, 2015

7140 NEMATI ET AL.

Animal Behavior and Structural Growth Animal Science, College of Agriculture, Malayer Uni-
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