ANIMAL-100179; No of Pages 8

Animal xxx (xxxx) xxx

Contents lists available at ScienceDirect

Animal
The international journal of animal biosciences

Effects of supplemental fat sources and forage feeding levels on growth

performance, nutrient digestibility, ruminal fermentation, and nitrogen
utilization in dairy calves
A. Karimi a, Y.A. Alijoo a,⁎, M. Kazemi-Bonchenari b, M. Mirzaei b, H. Sadri c,d
a
Department of Animal Science, Faculty of Agriculture, Urmia University, 5756151818 Urmia, Iran
b
Department of Animal Science, Faculty of Agriculture and Natural Resources, Arak University, 38156-8-8349 Arak, Iran
c
Department of Clinical Science, Faculty of Veterinary Medicine, University of Tabriz, 516616471 Tabriz, Iran
d
Institute of Animal Science, Physiology and Hygiene Unit, University of Bonn, 53115 Bonn, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Knowledge regarding the potential interactions between supplemental fat source and fiber level in starter diet of
Received 1 July 2020 dairy calves is lacking. The aim of the present study was to investigate the effects of supplemental saturated fat
Received in revised form 10 January 2021 [palm fat (PLF) containing 86% palmitic acid (C16:0)] vs. unsaturated fat [soybean oil (SBO) containing 51%
Accepted 11 January 2021
linoleic acid (C18:2)] and forage level on feed intake, growth performance, ruminal fermentation, nutrient digest-
Available online xxxx
ibility, and metabolic traits in dairy calves. Forty newborn Holstein female calves (BW = 39.7 ± 1.8 kg) were
Keywords:
assigned to 1 of 4 treatment groups (each consisting of 10 animals) in a 2 × 2 factorial arrangement of fat source
Alfalfa [soybean oil vs. palm fat; 3% of starter based on DM basis] and alfalfa hay level (0 vs. 15%, on DM basis): SBO or PLF
Fatty acid with (AH) or without (NAH) alfalfa hay. Calves had ad libitum access to water and starters throughout the study
Fiber and a constant amount of milk was offered among experimental calves during the pre-weaning period. All calves
Nitrogen efficiency were weaned on day 63 of age and remained in the study until day 73 of age. The results showed that the lowest
Physiology and the highest starter intake and average daily gain during pre-weaning period was observed when calves re-
ceived SBO-AH and PLF-AH, respectively. Accordingly, the lowest wither and hip heights at weaning time (day
63) and final wither height (day 73) were observed in SBO-AH group across treatments. Calves received PLF-
AH had the highest weaning and final BW compared to other groups. Feed efficiency tended to be higher in
PLF groups compared with SBO calves. Calves fed SBO-AH had the lowest digestibility of organic matter and neu-
tral detergent fiber and also total short chain fatty acid concentrations in rumen compared with other groups. The
SBO calves had lower urinary allantoin, urinary purine derivatives, and microbial protein synthesis than PLF
calves; however, urinary nitrogen increased with SBO supplementation. In summary, the supplementation of
SBO rich in C18:2 and AH during the pre-weaning period resulted in negative responses on growth performance,
digestibility, and ruminal fermentation profile. Therefore, the inclusion SBO rich in C18:2 along with forage in the
starter is not recommendable for young dairy calves.
© 2021 The Authors. Published by Elsevier Inc. on behalf of The Animal Consortium. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Implications Introduction

Fat supplementation can compensate the low energy intake in dairy
calves. Forage inclusion in the calf starter supply lower energy level
compared to concentrate feed fraction in dairy calves. The present
study evaluated the interaction of fat source soybean oil (as unsaturated
fatty acid source) vs. palm fat (as saturated fatty acid source) with for-
age level in starter. Results show that forage can be incorporated in
starter when palm fat was supplemented in diet, but concurrent feeding
of forage and soybean oil in starter negatively influence animal growth
performance, digestibility, and ruminal fermentation in young calves.
⁎ Corresponding author.
E-mail address: y.alijoo@urmia.ac.ir (Y.A. Alijoo).
Supplementation of dairy calves' diets with different source of fatty
acids (FA) could be a strategy to increase energy density of the diet
(Hill et al., 2011; Ghasemi et al., 2017) and may compensate energy de-
mand in the animals with limited solid feed intake (Kazemi-Bonchenari
et al., 2016). Although some studies have reported improvement in per-
formance of dairy calves supplemented with a blend of FA (Hill et al.,
2011; Ghasemi et al., 2017) or with omega-3 FA supplied through fish
oil (Ballou and DePeters, 2008), whereas others have observed even
lower performance in dairy calves supplemented with soybean oil in
starter (Hill et al., 2015; Ghorbani et al., 2020). Decreased starter intake
(Araujo et al., 2014), decreased nutrient digestibility such as fiber or
protein (Hill et al., 2015), and altered rumen microbial activity

https://doi.org/10.1016/j.animal.2021.100179
1751-7311/© 2021 The Authors. Published by Elsevier Inc. on behalf of The Animal Consortium. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: A. Karimi, Y.A. Alijoo, M. Kazemi-Bonchenari, et al., Effects of supplemental fat sources and forage feeding levels on
growth performance, nutrient digest..., Animal, https://doi.org/10.1016/j.animal.2021.100179
A. Karimi, Y.A. Alijoo, M. Kazemi-Bonchenari et al. Animal xxx (xxxx) xxx

(Palmquist and Jenkins, 1980; Fiorentini et al., 2013) have been sug- basis). Hence, treatments were (1) starter with SBO but without AH
gested as the reasons for unfavorable effects of fat supplementation in supplementation (SBO-NAH), (2) starter with SBO and 15% AH
dairy calves' starters. The sources of FA and environmental temperature supplementation (SBO-AH), (3) starter with PLF but without AH sup-
(Ghasemi et al., 2017), incorporating in milk replacer (Ballou and plementation (PLF-NAH), and (4) starter with PLF and 15% AH supple-
DePeters, 2008) compared to feeding in the starter (Hill et al., 2015), mentation (PLF-AH).
FA delivery methods in starter (Berends et al., 2018; Ghorbani et al., Calves were separated from their dams shortly after birth, housed in
2020), and FA profile of supplemental source (Quigley et al., 2019; individual pens, and fed 5 l of colostrum within the first 12 h of life (2.5 l
Kandi et al., 2020) may affect the pre-ruminant animals' response to of colostrum within 2 h of life and 2.5 l in a second feeding). The calves
specific FA supplementation. Supplemental FA are of particular interest received 4 l of whole milk/d from d 3 to 10, 7 l/d from d 11 to 53, and 3 l/
to increase dietary energy density, whereas higher level of dietary FA d from d 54 to 63 in galvanized tin buckets (twice daily at 09:00 and
could be toxic to rumen microbes (Palmquist and Jenkins, 1980; 18:00 h). The average composition of offered milk was 3.16 ± 0.09%
Fiorentini et al., 2013) and may decline nutrient digestibility and impair fat, 3.05 ± 0.06% CP, 4.84 ± 0.06% lactose, 11.9% total solids, and 1.5 ×
ruminal fermentation. Previous works on dairy cows and cross bred 105 cells/ml somatic cell count. All the calves were weaned on d 63
heifers have indicated that unsaturated FA (UFA) rich in linoleic acid and remained in the study until d 73 of age. Experimental diets were
(C18:2) and linolenic acid (C18:3) have more detrimental effects on ru- formulated to meet the National Research Council (NRC) (2001) nutri-
minal microbes and fermentation pattern than saturated FA (Palmquist ent requirements. The SBO source (Naz Industrial Vegetable Oil Co.,
and Jenkins, 1980; Fiorentini et al., 2013). This is specifically more im- Isfahan, Iran) with the following fatty acids compositions: C16:0 =
portant in the case of pre-ruminants with undeveloped rumen and 12.1%, C18:0 = 5.2%, C18:1 = 21.8%, C18:2 = 51.2%, C18:3 = 8.1%,
not well-established microbial community (Fiorentini et al., 2013; and other fatty acids = 1.6% and the PLF source (rumen protected, En-
Cersosimo et al., 2019). ergizer RP-10, IFFCO, Johor, Malaysia) with the following fatty acids
Similar to FA content in starter, controversial reports along with compositions: C12:0 = 2.3%, C14:0 = 4.2%, C16:0 = 86.0%, C18:0 =
much interesting exist regarding the evaluating the effects of dietary 2.0%, C18:1 = 4.1%, and other fatty acids = 1.4% were used. Ingredients
starter fiber content on rumen development and animal performance and chemical composition of the experimental calf starters are
(Beiranvand et al., 2014; Mirzaei et al., 2017). Contrast to FA supple-
mentation which increases energy density per unit of starter feed, for-
age inclusion reduces this parameter due to lower energy content of
forage than concentrate fraction (Beiranvand et al., 2014). Therefore, it Table 1
Ingredients and chemical composition of experimental starters fed to dairy calves.
could be postulated that lower energy content of starters when forage
is incorporated may be compensated with fat supplementation. Supple- Item Treatments1
mental fat, in contrast, may have some toxic effects to rumen microor- SBO PLF
ganisms, adhere to the feed particles and create a physical barrier
NAH AH NAH AH
that make an obstacle for microbial activity and fiber digestibility
(Palmquist and Jenkins, 1980). However, these negative effects may Ingredient (% of DM)
Alfalfa hay, chopped 0 15 0 15
be minimized by supplementing ruminal inert fat sources. To the best
Barley grain 15 15 15 15
of our knowledge, there is no study regarding the interaction of differ- Corn grain 43.5 30.5 43.5 30.5
ent supplemental fat source and forage feeding level in dairy calves. Soybean meal 33 31 33 31
We hypothesized that supplementing an inert fat source which is less Soybean oil 3 3 0 0
accessible to ruminal microbes may avoid the negative ruminal fermen- Palm fat 0 0 3 3
Vitamin and mineral premix2 2 2 2 2
tation aspects, and from other view, can compensate the lesser energy Calcium carbonate 1.5 1.5 1.5 1.5
content supplied through forage incorporation in the starters. Sodium bicarbonate 1.0 1.0 1.0 1.0
The first aim of the present study was to evaluate the effects of soy- Magnesium oxide 0.5 0.5 0.5 0.5
bean oil (SBO), rich in C18:2 as UFA and more accessible for ruminal mi- Salt 0.5 0.5 0.5 0.5
Chemical composition (% of DM, unless otherwise
crobes, compared with palm fat (PLF), rich in palmitic acid (C16:0) as
indicated)
saturated FA and less accessible for ruminal microbes on growth perfor- DM3 89 89 89 89
mance and ruminal fermentation in dairy calves. The second objective CP3 22.5 22.5 22.5 22.5
of the present study was to determine the interaction effect of fat Metabolizable energy,4 MJ/kg 12.54 12.49 12.54 12.49
sources (SBO vs. PLF) and alfalfa hay inclusion levels (0 vs. 15%, DM Ether extract3 5.88 5.84 5.85 5.86
NDF3 15.4 23.65 15.4 23.65
basis) in the starter on growth performance, digestibility, ruminal fer- Ca3 0.9 0.9 0.9 0.9
mentation, urinary purine derivatives (PD), and urinary nitrogen excre- P3 0.4 0.4 0.4 0.4
tion in Holstein dairy calves. FA5 composition, g/100 g of total FA
C12:0 0.67 0.52 1.65 1.60
C14:0 0.85 0.71 2.95 2.73
Material and methods
C16:0 13.55 12.10 49.5 48.2
C16:1 0.52 0.50 0.60 0.53
All the animal procedures and experimental methods applied were C18:0 3.99 3.72 2.25 2.18
approved by the Animal Care and Use Committee of Urmia University C18:1 20.41 20.78 11.55 11.72
(IACUC Protocol #IR2018011) outlined by the Iranian Council of Animal C18:2 51.12 52.10 26.50 27.43
C18:3 7.05 7.89 3.21 3.96
Care (1995).
Others 1.84 1.68 1.79 1.65
1
Treatments were (1) soybean oil supplementation in starter with no alfalfa hay (SBO-
Calves, housing, and diets
NAH); (2) soybean oil supplementation in starter with alfalfa hay (SBO-AH); (3) palm fat
supplementation in starter with no alfalfa hay (PLF-NAH); (4) palm fat supplementation
The present study was carried out at a commercial dairy farm (Avin- in starter with alfalfa hay (PLF-AH).
2
Dasht Dairy Co.), Qazvin, Iran. A total of forty female Holstein dairy Contained per kilogram of supplement: 500 000 IU of vitamin A, 100 000 IU of vitamin
calves (about 3 days old, 39.2 ± 1.8 kg of BW; n = 10 calves per treat- D, 500 IU of vitamin E, 7 500 mg of Mn, 100 g of Ca, 8 250 mg of Zn, 30 g of P, 20.5 g of Mg,
20 g of Na, 60 mg of Co, 2 375 mg of Cu, 56 mg of I, and 40 mg of Se.
ment) were randomly assigned to experimental diets in a 2 × 2 factorial 3
Values were chemically analyzed in laboratory.
arrangement with the factors of fat supplement type [SBO vs. PLF; 3% of 4
Calculated from NRC (2001).
starter based on DM basis] and forage addition level (0 or 15%, on DM 5
FA = fatty acid.

2
A. Karimi, Y.A. Alijoo, M. Kazemi-Bonchenari et al.
presented in Table 1. Calves were fed starters in meal-form with average
geometric mean particle size of 0.73 ± 0.1 mm. Alfalfa hay was chopped
(geometric mean particle size of 2.8 ± 0.14 mm) and then well mixed
with starter feed in forage-supplemented groups before feeding to
dairy calves. Starter was fed ad libitum to permit at least 10% orts
(i.e., the portion of the starter not consumed over a 24-h period). The
calves had free access to water throughout the experimental period.
Data recording, sample collection, and analysis
The amounts of starters offered (at 0800 h) and refused were re-
corded (at 0730 h) daily throughout the experiment. Measurement of
BW was taken by 10 d intervals during experimental period using an
electronic balance. Average daily gain (ADG) was calculated as the dif-
ference between BW taken every 10 d apart divided by 10. Feed effi-
ciency (kg of BW gain/kg of total DM intake [DMI]) was also
calculated in a 10 d interval (Rastgoo et al., 2020). Total DMI was consid-
ered as liquid feed DMI + starter feed DMI. Samples collected from
feeds and orts were dried in a convection oven (60 °C for 48 h). Subsam-
ples of dried feeds and orts were well mixed and ground in a mill (Ogaw
Seiki CO., Ltd., Tokyo, Japan) to pass a 1-mm screen and were analyzed
for CP, ether extract (Association of Official Analytical Chemists [AOAC],
2002), and NDF without sodium sulfite, but with the inclusion of α-
amylase (Van Soest et al., 1991). The nonfibrous carbohydrate compo-
nent was calculated based on NRC, 2001 equation as follows: 100 –
(CP + NDF + ether extract + ash).
To determine apparent digestibility, fecal samples were obtained
during 4 consecutive days (from d 70 to 73) via rectal palpation by
hand at 6, 12, and 18 h after the morning meal. The collected fecal sam-
ples were dried at 60 °C for 72 h in a forced-air oven, ground to pass a
1-mm screen in a Wiley mill (Ogaw Seiki Co., Ltd., Tokyo, Japan), and
then mixed thoroughly. All samples were analyzed for CP, ether extract,
and NDF based on the procedures described above. Acid-insoluble ash
was used as an internal marker to estimate the apparent total-tract di-
gestibility of organic matter (OM), CP, NDF, and ether extract (Van
Kuelen and Young, 1977).
The growth indices including heart girth, body length, body girth,
wither height, hip height, and hip-width were taken at d 3 (Initial), d
63 (weaning), d 73 (the final day of measurements) of age in the morn-
ing and before feeding.
Rumen fluid (30 ml) was collected on d 35 (pre-weaning) and d
70 (post-weaning) of the experiment using a stomach tube fitted to
a vacuum pump 3–4 h after morning feeding; the first 10 ml was
discarded because of possible saliva contamination, and rumen pH
was measured immediately (HI 8314 membrane pH meter; Hanna In-
struments, Villafranca, Italy). The rumen samples were squeezed
through 4 layers of cheesecloth. A 10-ml aliquot was preserved with
2 ml of 25% metaphosphoric acid and frozen at − 20 °C until analysis
for short chain fatty acids (SCFA). After thawing at room temperature,
the rumen samples were analyzed for SCFA using gas chromatogra-
phy (model CP-9002; Chrompack, Delft, the Netherlands) with a 50
m (0.32 mm ID) silica-fused column (CP-Wax Chrompack Capillary
Column; Varian, Palo Alto, CA) as previously described (Kazemi-
Bonchenari et al., 2016).
A ruminal fluid subsample was thawed at room temperature and
clarified by centrifuging (15 000 ×g for 20 min), then decanted and an-
alyzed for NH3-N concentration using a modified phenol-hypochlorite
(Broderick and Kang, 1980). The spot sampling technique was used to
estimate microbial protein synthesis in the rumen. This technique is
based on the PD excretion obtained via urine as explained in dairy calves
during the post-weaning period (Kazemi-Bonchenari et al., 2020). Urine
volume excretion rate per day was estimated by urinary creatinine ex-
cretion using the following model; BW × 26.8/creatinine concentration
3
Animal xxx (xxxx) xxx
(mg/l) as reported by Dennis et al. (2017) in post-weaned dairy calves.
Spot urine samples were collected on 3 days during the post-weaning
period from each calf during the morning (between 0900 and 1100 h)
and during the afternoon (between 1500 and 1700 h). Samples (ap-
proximately 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 then stored at −20 °C for analysis. The concentra-
tions of creatinine, urea N, uric acid, and allantoin were measured in
thawed urine samples as described extensively in our previous work
(Kazemi-Bonchenari et al., 2017). Estimated daily urine output was
used to calculate daily urinary excretion of allantoin + uric acid as
total daily PD. The ruminal microbial N synthesis was calculated from
daily total dairy PD output using the following equation described by
Chen and Gomes (1992): Microbial nitrogen (g N/d) = X (mmol/d) ×
70/(0.116 × 0.83 × 1 000), 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 and Gomes, 1992).
Statistical analyses
Statistical analyses were conducted for 3 periods according to Sup-
plementary Material S1: pre-weaning (d 3 to 63), post-weaning (d 64
to 73), and the entire period (d 3 to 73) using PROC MIXED of SAS (ver-
sion 9.1; SAS Inst. Inc., Cary, NC). The following model was adopted:
Yijklm = μ + Calfi + FSj + AHk + Pl + (FS × P)il + (AH × P)kl + (FS ×
AH)jk + (FS × AH × P)jkl + β(Xi − X̅) + εijklm, where Yijklm is the de-
pendent variable; μ is the overall mean; FSj is the effect of fat source
(j = soybean oil and palm fat); AHk is the effect of alfalfa hay inclusion
(k = no alfalfa hay supplementation and alfalfa hay supplementation);
Pl is the effect of period; (FS × P)jl is the interaction between fat source
and period; (AH × P)kl is the interaction between alfalfa hay inclusion
and period; (FS × AH)jk is the interaction between fat source and AH in-
clusion; (FS × AH × P)jkl is the tripartite effect of fat source, AH inclusion
and period; β(Xi − X̅) is the covariate variable and εijklm is the overall
error term. The model contained calf within treatment as a random ef-
fect and the first-order autoregressive covariance structure (AR1) was
determined as the most appropriate covariance structure for all re-
peated statements according to the Akaike's information criterion and
Bayesian information criterion. The BW and growth indices in the initial
day of experiment (d 3) were used as a covariate for weaning and final
measurements of related items. Effects were considered to be signifi-
cant when P ≤ 0.05, and it has been considered to have tendency was
considered when 0.05 < P ≤ 0.10. All reported values trough the tables
are least squares means.
Results
Starter intake, performance, and nutrient digestibility
The results showed that the highest and the lowest starter intake
during pre-weaning period were observed in PLF-AH and SBO-AH treat-
ments, respectively (P < 0.05) (Table 2). Total DMI tended to be low in
calves received SBO-AH as compared with other groups (P = 0.07).
Based on the supplemented fat source, individual fatty acids intake
was differed among experimental starter diets with the greatest
amount of C16:0 intake for PLF diet compared to the greatest intake
amount for C18:2 that found for SBO diet. The greatest and the lowest
ADG for pre-weaning and entire periods were observed for PLF-AH and
SBO-AH treatments, respectively (P < 0.05). Accordingly, the greatest
and the lowest BW at weaning time were found for PLF-AH and SBO-
AH treatments, respectively (P < 0.05). The post-weaning period starter
A. Karimi, Y.A. Alijoo, M. Kazemi-Bonchenari et al. Animal xxx (xxxx) xxx

Table 2 The interaction between fat source and forage level in the starter
Starter intake, average daily gain, feed efficiency, and nutrient digestibility in dairy calves was significant in the current study, where the lowest digestibility of
fed different fat sources (soybean oil vs. palm fat) with different dietary forage inclusion
level (0 vs. 15%, DM basis) (n = 10 calves per treatment).
OM (P = 0.02) and NDF (P = 0.05) was found for SBO-AH (Table 2). Di-
gestibility of ether extract was not influenced by the treatments. How-
Item Treatments1 SEM P-value2 ever, digestibility of CP was reduced in calves supplemented with SBO
SBO PLF Fat AH Fat × AH as compared with PLF (P = 0.03).
NAH AH NAH AH
Structural growth
Starter feed
intake, g/day
Pre-weaning 552ab 443b 538ab 635a 56.9 0.09 0.85 0.04 Results showed that the calves fed SBO-AH diet had the lowest
Post-weaning 1 721 1 655 1 885 1 965 112.8 0.03 0.97 0.53 wither height, both in weaning time and final measurement, and the
Entire period 719 616 731 825 75.0 0.13 0.91 0.18 lowest hip height in weaning time (P < 0.05) (Table 3). Body length
Milk intake, g 651 649 650 652 15.8 0.95 0.98 0.99
DM/d
and heart girth were not influenced by fat source, forage feeding level,
Total DMI, (milk 1 203 1 094 1 190 1 277 61.4 0.15 0.85 0.07 or their interaction. Hip height was reduced (P = 0.05) and hip width
DM intake + tended to be reduced (P = 0.07) in SBO calves than in PLF groups in
starter DM the final measurement. The body girth of calves in the final measure-
intake), g/d
ment was increased when animals fed AH compared with that of NAH
Individual fatty
acid intake, g/d (P = 0.04).
C16:0
Pre-weaning 3.6 2.2 12.0 15.7 1.05 0.01 0.29 0.09 Ruminal fermentation, urinary purine derivatives, and urinary nitrogen
Post-weaning 10.9 9.6 43.8 42.1 2.29 0.01 0.52 0.93
Entire period 7.1 5.8 27.9 28.8 1.50 0.01 0.80 0.26
C18:0 The highest ruminal concentrations for NH3-N (P = 0.05) and the
Pre-weaning 1.06 0.69 0.55 0.71 0.08 0.01 0.26 0.10 lowest ruminal concentration of SCFA (P < 0.01) during the pre-
Post-weaning 3.2 2.9 1.9 1.9 0.21 0.01 0.42 0.69 weaning period were found in calves received SBO-AH treatment
Entire period 2.1 1.7 1.2 1.3 0.13 0.01 0.36 0.11 (Table 4). The ruminal pH tended to be greater in the post-weaning pe-
C18:1
Pre-weaning 5.4 3.8 2.9 3.9 0.47 0.01 0.56 0.07
riod for calves offered AH in the starter (P = 0.08). The supplemental
Post-weaning 16.4 16.5 10.2 10.3 1.12 0.01 0.97 0.98 SBO compared with PLF increased ruminal NH3-N, but reduced ruminal
Entire period 10.8 10.1 6.5 7.1 0.70 0.01 0.79 0.21 SCFA (P = 0.02) and butyrate (P = 0.05) concentrations during the
C18:2 post-weaning period.
Pre-weaning 13.6 9.7 6.4 8.9 1.16 0.01 0.55 0.07
In comparison with PLF, SBO reduced urinary excretion of allantoin,
Post-weaning 41.2 41.4 23.4 23.9 2.76 0.01 0.89 0.95
Entire period 27.2 25.5 14.7 16.4 1.73 0.01 0.32 0.19 PD, and microbial protein synthesis, but increased urinary nitrogen,
C18:3
Pre-weaning 1.8 1.4 0.78 1.2 0.16 0.01 0.76 0.06
Table 3
Post-weaning 5.6 6.2 2.8 3.4 0.39 0.01 0.14 0.97
Growth parameters in dairy calves fed different fat sources (soybean oil vs. palm fat) with
Entire period 3.7 3.8 1.7 2.3 0.24 0.01 0.41 0.19
different dietary forage inclusion level (0 vs. 15%, DM basis) (n = 10 calves per treatment).
Average daily
gain, g/day Item Treatments1 SEM P-value2
Pre-weaning 583ab 541b 599ab 671a 34.0 0.02 0.59 0.05
Post-weaning 712 765 767 987 84.5 0.05 0.06 0.30 SBO PLF Fat AH Fat × AH
Entire period 601ab 573b 623ab 716a 33.6 0.01 0.28 0.03 NAH AH NAH AH
BW, kg
Initial 39.3 38.7 39.5 39.2 0.78 0.61 0.53 0.81 Heart girth, cm
Weaning 74.3ab 71.2b 75.5ab 79.6a 1.68 <0.01 0.77 0.03 Initial (d 3) 80.4 80.6 81.7 81.3 0.97 0.27 0.89 0.73
Final 81.4 b
78.8b 83.1 b
87.9a 1.82 0.01 0.52 0.04 Weaning (d 63) 102.2 99.7 100.2 100.5 1.23 0.61 0.32 0.26
Feed efficiency3 Final (d 73) 108.6 106.4 105.6 108.5 1.50 0.79 0.88 0.11
Pre-weaning 0.491 0.498 0.504 0.537 0.03 0.06 0.74 0.51 Body length, cm
Post-weaning 0.413 0.459 0.409 0.502 0.04 0.76 0.17 0.72 Initial (d 3) 49.6 48.5 49.7 49.0 0.72 0.63 0.22 0.82
Entire period 0.456 0.463 0.470 0.519 0.04 0.08 0.54 0.49 Weaning (d 63) 62.8 60.1 61.1 63.6 1.59 0.57 0.99 0.10
Nutrient Final (d 73) 63.8 63.4 64.6 65.4 1.60 0.35 0.91 0.70
digestibility, Body girth, cm
g/kg Initial (d 3) 80.4 79.0 80.4 81.0 0.87 0.24 0.62 0.26
Organic matter 780a 738b 804a 809a 9.6 0.01 0.12 0.02 Weaning (d 63) 115 115 113 112 1.73 0.19 0.66 0.88
CP 747 730 782 773 16.7 0.03 0.43 0.81 Final (d 73) 116 120 118 122 1.98 0.40 0.04 0.89
NDF 640ab 588b 659ab 686a 22.0 0.02 0.27 0.05 Wither height, cm
Ether extract 820 811 817 840 12.4 0.30 0.56 0.19 Initial (d 3) 82.1 80.9 81.6 82.0 0.67 0.63 0.53 0.32
Weaning (d 63) 98.6a 93.5b 95.4ab 97.7a 1.03 0.61 0.19 <0.01
a,b
Means within a row with different superscript letters are different (P < 0.05). Final (d 73) 102.8a 98.3b 100.2a 101.0a 0.89 0.92 0.07 <0.01
1
Treatments were (1) soybean oil supplementation in starter with no alfalfa hay (SBO- Hip height, cm
NAH); (2) soybean oil supplementation in starter with alfalfa hay (SBO-AH); (3) palm fat Initial (d 3) 80.9 80.1 80.6 80.2 0.79 0.89 0.40 0.77
supplementation in starter with no alfalfa hay (PLF-NAH); (4) palm fat supplementation Weaning (d 63) 95.6a 92.4b 94.2a 95.5a 0.82 0.30 0.22 0.01
in starter with alfalfa hay (PLF-AH). Final (d 73) 97.5 97.2 98.5 99.1 0.67 0.05 0.79 0.68
2
Statistical comparisons: fat; different fat source in starter (soybean oil vs. palm fat); Hip width, cm
AH = alfalfa hay inclusion level (0 vs. 15%, DM basis); Fat × AH = interaction between Initial (d 3) 15.2 14.6 14.8 15.0 0.28 0.87 0.34 0.20
fat source × alfalfa hay inclusion level. Weaning (d 63) 20.8 19.9 20.7 20.3 0.59 0.73 0.26 0.71
3
kg of BW gain/kg of total DM intake. Final (d 73) 21.9 21.3 23.2 22.2 0.63 0.07 0.19 0.74
a,b
Means within a row with different superscript letters are different (P < 0.05).
1
Treatments were (1) soybean oil supplementation in starter with no alfalfa hay (SBO-
intake was reduced in calves supplemented with SBO as compared with NAH); (2) soybean oil supplementation in starter with alfalfa hay (SBO-AH); (3) palm fat
PLF (P = 0.03). The greatest BW in the final measurement was observed supplementation in starter with no alfalfa hay (PLF-NAH); (4) palm fat supplementation
in starter with alfalfa hay (PLF-AH).
for PLF-AH (P = 0.04). The feed efficiency tended to be lower in calves 2
Statistical comparisons: fat; different fat source in starter (soybean oil vs. palm fat);
supplemented with SBO compared with PLF during pre-weaning period AH = alfalfa hay inclusion level (0 vs. 15%, DM basis); Fat × AH = interaction between
(P = 0.06) and entire period of the study (P = 0.08). fat source × alfalfa hay inclusion level.

4
A. Karimi, Y.A. Alijoo, M. Kazemi-Bonchenari et al. Animal xxx (xxxx) xxx

Table 4
Ruminal fermentation parameters, urinary purine derivatives and nitrogen excretion, and microbial protein synthesis in dairy calves fed different fat sources (soybean oil vs. palm fat) with
different dietary forage inclusion level (0 vs. 15%, DM basis) (n = 10 calves per treatment).

Item Treatments1 SEM P-value2

SBO PLF Fat AH Fat × AH

NAH AH NAH AH

Ruminal pH
d 35 5.65 5.74 5.79 5.84 0.07 0.10 0.37 0.75
d 70 5.94 6.18 5.92 6.01 0.09 0.25 0.08 0.34
Ruminal NH3-N, mg/dl
d 35 8.98b 10.63a 8.40b 7.65b 0.59 0.01 0.46 0.05
d 70 13.42 14.35 12.45 12.08 0.49 0.02 0.57 0.19
Total short-chain fatty acids, mM/l
d 35 84.4ab 71.1b 87.8ab 90.7a 1.74 0.01 0.03 <0.01
d 70 106 101 117 116 1.96 0.02 0.20 0.41
Acetate (A), mmol/100 mol
d 35 50.9 50.1 47.9 48.7 1.09 0.12 0.70 0.96
d 70 49.2 49.9 48.2 47.2 0.78 0.11 0.89 0.36
Propionate (P), mmol/100 mol
d 35 29.9 30.9 32.9 31.7 1.15 0.22 0.78 0.43
d 70 29.4 30.7 30.1 31.0 1.22 0.54 0.60 0.79
A: P
d 35 1.68 1.67 1.50 1.56 0.08 0.11 0.75 0.65
d 70 1.70 1.68 1.61 1.58 0.09 0.27 0.75 0.98
Butyrate, mmol/100 mol
d 35 13.9 13.6 14.4 14.5 0.69 0.27 0.79 0.30
d 70 14.9 13.2 15.6 16.1 0.82 0.05 0.41 0.20
Branched-chain volatile fatty acids,3 mmol/100 mol
d 35 5.28 4.53 4.80 5.09 0.43 0.56 0.59 0.14
d 70 6.49 6.18 6.09 5.68 0.48 0.45 0.61 0.88
Urinary purine derivatives and nitrogen excretion
Allantoin, mmol/d 14.3 12.8 17.5 16.8 1.19 0.02 0.38 0.69
Uric acid, mmol/d 0.93 1.01 0.98 1.05 0.05 0.39 0.19 0.88
PD,4 mmol/d 15.2 13.8 18.4 17.9 1.20 0.01 0.42 0.73
Microbial protein yield, g/d 81.4 73.9 98.8 95.9 6.44 0.01 0.42 0.73
Urinary nitrogen, g/d 20.6 21.5 17.3 19.4 1.04 0.02 0.15 0.59
Urinary nitrogen, % of total N intake 37.1 39.4 29.2 31.3 1.93 <0.01 0.28 0.94
a,b
Means within a row with different superscript letters are different (P < 0.05).
1
Treatments were (1) soybean oil supplementation in starter with no alfalfa hay (SBO-NAH); (2) soybean oil supplementation in starter with alfalfa hay (SBO-AH); (3) palm fat sup-
plementation in starter with no alfalfa hay (PLF-NAH); (4) palm fat supplementation in starter with alfalfa hay (PLF-AH).
2
Statistical comparisons: fat; different fat source in starter (soybean oil vs. palm fat); AH = alfalfa hay inclusion level (0 vs. 15%, DM basis); Fat × AH = interaction between fat source ×
alfalfa hay inclusion level.
3
Branched-chain volatile fatty acids (BCVFA) are the molar proportion of valerate + isovalretae.
4
Purine derivatives (allantoin + uric acid).

regardless of forage supplementation level (P < 0.05). No effect of AH
and its interaction with fat source was found regarding the urinary PD
and microbial protein synthesis during the post-weaning period.
Discussion
Interaction effect of fat source and forage level
Supplementation of different fat sources such as SBO that is rich in
C18:2 (Ghorbani et al., 2020) or forage incorporation (Beiranvand
et al., 2014; Mirzaei et al., 2017) has both shown the potential to influ-
ence starter intake in dairy calves. In the current study, starter intake
was influenced by the interaction of fat source and forage feeding. Inter-
estingly, both the lowest and greatest starter intakes during pre-
weaning period were observed when forage was included in the diet
(443 and 635 g/d for SBO-AH and PLF-AH, respectively), pointing that
starter intake was more closely associated with fat source rather than
forage level. It has been suggested that the feed intake reduction by
fat supplements may be due in part to the decreased palatability and re-
duced nutrient digestibility (Ghorbani et al., 2020). Other potential
mechanisms, such as ‘greasiness’ negative effect of fat on DMI
(Drackley et al., 1994), or oxidation of FA in the liver and controlling ap-
petite (Harvantine and Allen, 2005), as suggested in dairy cows, might
also be involved in dairy calves. Furthermore, the UFA from calcium
salts of FA may also decrease feed intake through decreasing the meal
size or increasing inter-meal interval (Harvantine and Allen, 2005). In
view of limited cellulolytic activity in young calves during pre-
weaning period, supplemental UFA supplied through linseed oil rich
in C18:3 (Ikwuegbu and Sutton, 1982) or soybean oil rich in C18:2
(Ghorbani et al., 2020) could further negatively influence ruminal mi-
crobes' activity and consequently fiber digestibility. Physical coating of
the fiber by fat or modified fiber-digesting bacteria population due to
toxic effects of fat may be responsible in part for the lower fiber digest-
ibility found in SBO-AH group (Soliva et al., 2004). In agreement with
previous works (Hill et al., 2015; Ghorbani et al., 2020), our results sug-
gest that SBO supplementation was associated with reduced digestibil-
ity of OM and NDF in dairy calves, which was more exacerbated when
forage was included in the starter. It is notable here that using calcium
salts of long chain FA that are protected through rumen metabolism
may have actually increase some nutrient digestibility in dairy cows
(Weld and Armentano, 2007). The increased nutrient digestibility due
to the supplementation of calcium salts in dairy cows was not related
to concurrent decreases in DMI, indicating another mechanism besides
changes in DMI is responsible for this increment (Weld and Armentano,
2007). The aforementioned mechanism is need to be more evaluated in
dairy calves by supplementing the starters with protected FA sources.
Supplemental PLF neither reduced starter intake, nor influenced nutri-
ent digestibility in the current study. Ghasemi et al. (2017) reported a
greater starter intake in dairy calves fed starters supplemented with
SBO and PLF as compared with control diets, but with no changes in

5
A. Karimi, Y.A. Alijoo, M. Kazemi-Bonchenari et al.
digestibility of nutrients. However, the latter authors studied the effects
of supplemental fat in a cold stress condition with constant fiber content
among the starters. Dietary supplementation with 4% hydrogenated
palm oil in steers reared in intensive system was associated with only
modified FA profile and some meat quality traits with no negative ef-
fects on feed intake and growth performance (Castro et al., 2016).
In the current study, the lowest (541 g/d) and the greatest (671 g/d)
ADG during pre-weaning period were, respectively, found in SBO-AH
and PLF-AH calves, which coincided with a similar trend in BW, wither
height, and hip height at weaning. These changes along with the lower
digestibility of OM and NDF in SBO-AH calves might reflect a more effi-
cient tissue accretion in PLF-AH calves than in SBO-AH calves.
Beside the lower nutrient digestibility, the lower concentrations of
SCFA seem to be responsible for the lower growth performance ob-
served in calves fed SBO-AH diet. The lower starter intake as well as
the lower OM digestibility in SBO-AH group may reduce substrate for
ruminal microbial fermentation, and hence SCFA was reduced in the
rumen. In ruminants, main energy requirement (approximately 70%)
is supplied through SCFA produced in the rumen (Bergman, 1990);
thus, the lower SCFA in the calves fed SBO-AH would be expectedly ac-
companied by lower supply of energy for growth.
In the current study, the greatest ruminal NH3-N concentrations
during the pre-weaning period were found in SBO-AH treatment. It
has been shown that most cellulolytic bacteria require ammonia
for growth (Griswold et al., 2003); hence, the lower digestibility of
fiber found in SBO-AH calves could be indicator of limited fiber-
digesting bacteria activity, leading to accumulation of ruminal
NH3-N in this group.
Effect of fatty acid source supplementation
The starter intake, ADG, feed efficiency, hip height, and hip width
were or tended to be lower in at the end of experiment in SBO calves
as compared with PLF calves. These data show that the reduced feed in-
take in animals supplemented with SBO compared with PLF continued
even after weaning without having an interaction with forage feeding
level.
In the current study, an interaction between fat source and forage
level was only observed in the pre-weaning period but not in the
post-weaning period, which may suggest that microbial community is
more extensively established after weaning as compared with pre-
weaning period (Cersosimo et al., 2019); thus, less problem regarding
the negative effect of supplemental UFA in starters along with a high di-
etary forage level would be expected during the post-weaning period.
Besides the lower feed intake, the lower digestibility of CP may have
also contributed in the lower ADG and feed efficiency observed in of
SBO calves compared with PLF calves.
The lower concentrations of SCFA in SBO calves reflects the lower ru-
minal fermentation rate of the starters supplemented with SBO as com-
pared with that of PLF. Chalupa et al. (1986) suggested that UFA such as
SBO are more likely to alter microbial fermentation and therefore are
more available to exert toxic effects on ruminal microorganisms. Satu-
rated fatty acids such as PLF may be less toxic to ruminal microorgan-
isms, as they react more readily with metal ions and form insoluble
salts within the rumen (Palmquist and Jenkins, 1980). Palm oil has
been shown to have the least negative effect on ruminal fermentation
in the rumen of goats compared with olive and sunflower oils that
was postulated to be related to the differences in FA profile among the
tested oils (Nur Atikah et al., 2018). Regarding the reduced ruminal bu-
tyrate concentrations with SBO, Maia et al. (2010) stated that butyrate
producing bacteria activity was reduced with linseed oil as a UFA sup-
plement. Similarly, Ikwuegbu and Sutton (1982) found that linseed oil
rich in C18:3 led to a decreased proportion of butyrate among rumen
SCFA. Butyrate has been shown to have pivotal role in rumen
6
Animal xxx (xxxx) xxx
development (Beiranvand et al., 2014); thus, lower butyrate concentra-
tions in ruminal fluid may indicate less potential to proliferations of cells
in the rumen of SBO calves compared with PLF calves. Lower ruminal
NH3-N concentrations in calves supplemented with PLF than SBO in
the post-weaning period indicates the lower ammonia capture by rumi-
nal microbes in SBO diets. In agreement with our results, Nur Atikah
et al. (2018) reported that the lowest ruminal ammonia concentration
was found in goats supplemented with palm oil as compared with
those supplemented with olive oil or sunflower oil, the latter authors
proposed an improvement of N utilization efficiency with palm oil,
likely mediated by reduced protozoa count which was also coincided
with improved digestibility of fiber (Nur Atikah et al., 2018). However,
the detrimental effects of UFA supplied through linseed oil containing
high amount of C18:3 on ruminal fermentation and nutrient digestibil-
ity have been previously shown (Ikwuegbu and Sutton, 1982). Besides
effects on nutrient digestibility, appetite control, and ruminal microbial
modifications, flaxseed oil has also shown inflammatory effect in dairy
calves during pre-weaning period as compared with palm oil (Tsai
et al., 2017); thus, negative effects of SBO on calf growth performance
both in pre- and post-weaning periods in the current study mediated,
at least in part, by its inflammatory effect is also likely.
The higher urinary nitrogen concentrations (21.0 vs. 18.3 g/d for SBO
and PLF diets, respectively) and lower total PD and microbial protein
synthesis (77.6 vs. 97.3 g/d for SBO and PLF diets, respectively) in
SBO-supplemented diets may point that PLF supplementation was
more efficient in N utilization rather than SBO diets. It has already
been shown that the amount of feed intake has a critical role on urinary
PD excretion in ruminant (Singh et al., 2007). Furthermore, the amount
of digested OM in the rumen is the main factor affecting urinary PD ex-
cretion and thus microbial protein synthesis in ruminants by providing
energy demands for microbes (Clark et al., 1992). The current results in-
dicate that both low starter intake and low digestibility of nutrients
found for SBO may be associated with lower microbial protein synthesis
and lower efficiency of N utilization; all of which can negatively influ-
ence growth performance of dairy calves. Thus, it is likely that greater
amino acids were supplied through PLF diets compared with SBO
diets, due to greater microbial protein synthesis and CP digestibility. Ir-
respective of the mechanisms involved in altered digestibility of nutri-
ents or ruminal fermentation pattern, our results show beneficial
effect of feeding starters supplemented with PLF in terms of nitrogen
metabolism as compared with SBO-supplemented starters.
Effect of forage feeding level
Forage level did not influence feed intake, ADG, and feed efficiency
during the pre-weaning period, but tended to improve ADG in the
post-weaning period. Although some previous works have reported
greater ADG in forage fed calves compared with control diets; however,
gut fill factor should also be considered when forage is included in the
starter of dairy calves (Beiranvand et al., 2014). This is specifically rele-
vant to the current study because we observed no differences in terms
of feed intake and feed efficiency between NAH and AH starters and
only ADG was influenced with forage. It is notable that geometric
mean particle size of forage in the current study which might influence
the responses was similar to the values reported in previous calf studies
(Beiranvand et al., 2014; Mirzaei et al., 2017). The reasons for the lack of
a forage response in the examined variables in the current study are not
known, but are likely related to the level of fat supplement used. Most of
the previous studies that have reported positive effects of forage on
starter intakes of dairy calves have used a relatively low level of fat sup-
plement (Beiranvand et al., 2014; Mirzaei et al., 2017) as compared to
that (5.8%, DM basis) tested in the present study. Thus, further studies
that include evaluation of forage inclusion concurrently with low and
A. Karimi, Y.A. Alijoo, M. Kazemi-Bonchenari et al.
high levels of fat supplements are needed to better explore the interac-
tion between forage and fat supplements in the starters of dairy calves.
Conclusion
The inclusion of fat supplements (3% of starter DM) containing palm
FA (about 86% of C16:0) and alfalfa hay (15% of starter DM) in the starter
can result in better animal performance in calves fed whole milk during
the milk feeding period. However, the use of PUFA from soybean oil
(about 51% of C18:2) and alfalfa hay (15% of starter DM) affected nega-
tively both the animal performance and digestibility of nutrients in
young calves. Moreover, the N utilization efficiency was negatively in-
fluenced by soybean oil through reducing the urinary PD excretion
and increasing the urinary nitrogen excretion. Under the conditions of
the present study and variables evaluated, the use of a rumen-
protected fat supplement as the major source of C16:0 in the starter
can be used with alfalfa hay to improve growth performance, digestibil-
ity, and nitrogen metabolism in young dairy calves. The effect of individ-
ual fatty acids intakes from different fat supplements in dairy calves
should be evaluated further.
Supplementary materials
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.animal.2021.100179.
Ethics approval
All the animal procedures were approved by the Animal Care and
Use Committee of Urmia University (IACUC Protocol #IR2018011)
outlined by the Iranian Council of Animal Care (1995).
Data and model availability statement
None of the data were deposited in an official repository.
Author ORCIDs
Y. A. Alijoo, https://orcid.org/0000-0001-7012-2407.
M. Kazemi-Bonchenari, https://orcid.org/0000-0002-4051-1097.
M. Mirzaei, https://orcid.org/0000-0002-1445-2986.
Author contributions
A. Karimi, Conceptualization, Data curation, Investigation, Resources,
Writing – original draft.
Y. A. Alijoo, Conceptualization, Formal analysis, Funding acquisition,
Project administration, Resources, Software, Supervision, Validation, Vi-
sualization, Wring – original draft.
M. Kazemi-Bonchenari, Conceptualization, Formal analysis, Investi-
gation, Methodology, Project administration, Supervision, Validation,
Writing – review & editing.
M. Mirzaei, Conceptualization, Data curation, Investigation, Method-
ology, Software, Validation, Writing – original draft.
H. Sadri, Conceptualization, Formal analysis, Investigation, Valida-
tion, Writing – review & editing.
Declaration of interest
The authors declare that they have no conflict of interest.
Acknowledgements
Great appreciations to Mr. Mohammad-Ali Daghighi (Avin-Dasht
Dairy Co.), Mr. Khamesi and Mr. Sedghi for their assistance throughout
7
Animal xxx (xxxx) xxx
the experiment. Thanks to Mrs. Afshar, Dr. Omidi-Mirzaei, Mr. Molaei
and Mr. Arabi for helping this work throughout the experiment.
Financial support statement
The data in this study was developed as a part of the first author PhD
thesis (Grant No. 94/269-1004) that was financially supported by dep-
uty of research and technology in Urmia University.
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