Animal 15 (2021) 100013

Contents lists available at ScienceDirect

Animal
The international journal of animal biosciences

Effect of substituting wet corn gluten feed and corn stover for alfalfa hay
in total mixed ration silage on lactation performance in dairy cows
G.N. Zhang 1,2, Y. Li 1, C. Zhao, X.P. Fang, Y.G. Zhang ⁎
College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China

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

Article history: Wet corn gluten feed (WCGF) is a high moisture feed containing rapidly digestible, non-forage fiber and protein.
Received 11 September 2019 The objective of this study was to investigate the effect of substituting WCGF and corn stover for alfalfa hay in
Received in revised form 22 July 2020 total mixed ration (TMR) silage on lactation performance and nitrogen balance in dairy cows. Nine multiparous
Accepted 28 July 2020
Holstein dairy cows (BW = 532 ± 28.9 kg and day in milk = 136 ± 5.6 d; mean ± SD) were used in a replicated
Available online 6 February 2021
3 × 3 Latin square design with 21-d periods (14 d of diet adaption and 7 d of sample collection). Groups were
Keywords:
balanced for parity, day in milk, and milk production and consumed one of three treatment diets during each pe-
By-products riod. The treatment diets were fed as TMR and contained similar concentrate mixtures and corn silage but differ-
Milk composition ent proportions of roughage and WCGF. The three treatments were: (1) 0% WCGF, 0% corn stover, and 22.1%
Nitrogen balance alfalfa hay (0% WCGF); (2) 6.9% WCGF, 3.4% corn stover, and 11.8% alfalfa hay (7% WCGF); and (3) 13.3%
Nutrient digestibility WCGF, 4.9% corn stover, and 3.9% alfalfa hay (13.3% WCGF). Compared to the 0% WCGF diet, the cows fed the
Ruminal parameter 7% and 13.3% WCGF diets had a higher milk yield and concentration of milk fat, protein, lactose, and total solids.
Effective degradability of DM was higher in the cows fed the 7% and 13.3% WCGF diets than it was with the 0%
WCGF diet. Cows fed the 13.3% WCGF had a higher CP effective degradability and a lower rumen undegraded pro-
tein than cows fed the 0% WCGF diet. The concentration of ruminal volatile fatty acids and ammonia-N was
higher in cows fed the 7% and 13.3% WCGF diets than cows fed the 0% WCGF diet. The fecal N was lower in
cows fed the 7% and 13.3% WCGF diets than it was in cows fed the 0% WCGF diet. Milk N secretion and milk N
as a percent of N intake were higher in cows fed the 13.3% WCGF diet than cows fed the 0% and 7% WCGF
diets. In conclusion, it appears that feeding a TMR silage containing WCGF and corn stover in combination, replac-
ing a portion of alfalfa hay, may improve lactation performance and nitrogen utilization for lactating dairy cows.
© 2021 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
Wet corn gluten feed is a high moisture feed containing rapidly di-
gestible, non-forage fiber and protein. The use of corn stover as rough-
age by ruminants is limited by its poor digestibility. Total mixed ration
ensiling has become an effective method of preservation of high-mois-
ture by-products in animal feed. This experiment showed that feeding
wet corn gluten feed and corn stover in combination can efficiently re-
place a portion of alfalfa hay in the dairy cows rations.
Introduction
Alfalfa hay represents a large part of the total cost of the diet. Identi-
fication of less expensive alternative feeds, such as corn stover and wet
⁎ Corresponding author.
E-mail address: zhangyonggen@sina.com (Y.G. Zhang).
1
These authors contributed equally to this study.
2
Present address: College of Animal Science and Technology, Northeast Agricultural
University, Harbin, 150030, China.
corn gluten feed (WCGF), to decrease feed costs is an urgent need. The
WCGF, a feed of rapidly digestible, non-forage fiber and protein, is co-
produced in wet corn milling (Pan et al., 2014) and has been applied
as a popular feedstuff for ruminant animals. Seven billion tons of corn
stover are produced as an economical feedstuff annually (Sánchez and
Cardona, 2008).
However, the high moisture concentration of WCGF causes storage
issues (Pan et al., 2014). Traditionally, WCGF is dried to approximately
90% DM for dry corn gluten feed (DCGF); however, the drying process
can waste energy, cause heat damage, and produce air pollution. Ensiled
WCGF alone is often challenging to ferment adequately due to its high
moisture concentration and lower sugar substrate levels (Pan et al.,
2014). A practice in Japan is to mix wet by-products with dry feeds to
prepare low-moisture total mixed ration (TMR) silage, which has
become an effective method of preservation of high-moisture by-prod-
ucts (Nishino et al., 2010). Ensiling as TMR has some advantages, such as
improving aerobic stability and digestibility, labor saving during the
preparation, and transportation convenience (Nishino et al., 2010).
High-moisture WCGF can be mixed with other feeds as TMR silage for
dairy cows. However, the small particle size of a non-forage source of

https://doi.org/10.1016/j.animal.2020.100013
1751-7311/© 2021 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/).
G.N. Zhang, Y. Li, C. Zhao et al. Animal 15 (2021) 100013

fiber does not stimulate rumination activity as efficiently as dietary for- Table 1
age (Mertens, 1997). Allen and Grant (2000) reported that the physi- Ingredients and chemical composition1 of dairy cow experimental diets.

cally effective NDF of CGF was 11% of NDF based on rumination time Items1 Treatment1 SEM P-value
and ruminal pH value. Hao et al. (2017) reported that DCGF and Chinese 0% WCGF 7% WCGF 13.3% WCGF
wild rye grass could efficiently replace a portion of alfalfa hay in the ra-
Ingredient, % of DM
tion of lactating dairy cows. Therefore, it is essential to consider the
Alfalfa hay 22.1 11.8 3.9
combination of WCGF and long fiber feeds for use in the ration of lactat- Wet corn gluten feed 0 6.9 13.3
ing cows. It is economical to use corn stover as a feedstuff due to its high Corn stover 0 3.4 4.9
production and reduced cost. However, the use of corn stover as rough- Corn silage 27.8 27.8 27.8
age by ruminants is limited by its poor digestibility due to the fiber Ground corn 24.6 24.6 24.6
Soybean meal 9.8 9.8 9.8
structure of corn stover. It was reported that combined lactic acid bacte- Cottonseed meal 4.9 4.9 4.9
ria with fibrolytic enzyme (EN) could increase in vitro DM digestibility DDGS2 7.4 7.4 7.4
and in vitro NDF digestibility of TMR silages containing rape straw com- extruded soybean 1.0 1.0 1.0
pared with control TMR silages by decreasing DM loss and making a Premix3 2.5 2.5 2.5
loose structure of cell wall (Liu et al., 2016). So, replacing a portion of al- Chemical composition
falfa hay with corn stover and WCGF may conserve other feeds in a TMR DM,% of FM 47.2a 44.9b 45.1b 0.12 <0.0001
silage for dairy cows and not only reduce the feed cost but also effi- OM,% of DM 93.2 92.9 92.9 0.45 0.87
CP, % of DM 19.4 19.3 19.8 0.16 0.09
ciently utilize the high moisture concentration of WCGF and poor di-
NDF, % of DM 34.4 35.1 33.9 0.52 0.33
gestibility of corn stover. However, there is little information about ADF, % of DM 21.9a 20.5b 18.6c 0.19 <0.0001
the effect of feeding TMR silage containing WCGF and corn stover in EE,% of DM 3.39 3.29 3.44 0.07 0.33
combination, replacing a portion of alfalfa hay to dairy cows. NFC,4 % of DM 35.9 34.6 35.0 0.53 0.27
This study aimed to investigate the effect of substituting WCGF and Starch, % of DM 20.6 22.4 22.6 0.81 0.24
NEL,5 Mcal/kg of DM 1.67 1.64 1.66 0.01 0.07
corn stover for alfalfa hay in a TMR silage on fermentation quality, lacta-
tion performance, and nitrogen balance in lactating dairy cows. Fermentation profile
pH 4.68a 4.64ab 4.62b 0.02 0.05
Lactic acid, % of DM 3.51b 3.56ab 3.63a 0.02 0.03
Material and methods Acetic acid, % of DM 0.60 0.59 0.59 0.02 0.99
Ammonia-N, % of Total N 5.67b 5.63b 6.23a 0.10 0.01
Animals and experimental design
FM = fresh material; OM = organic matter; EE = ether extract; NFC = non-fibrous car-
bohydrate; NEL = net energy lactation.
Animal use and care for this study were approved by the Animal a–c
Means within a row with different superscripts differ (P < 0.05).
1
Care Advisory Committee, Northeast Agricultural University (Harbin, 0% WCGF = 0% of DM wet corn gluten feed, 0% corn stover, and 22.1% alfalfa hay; 7%
China), and all the experimental procedures for this research were ap- WCGF = 6.9% of DM wet corn gluten feed, 3.4% corn stover, and 11.8% alfalfa hay; 13.3%
WCGF = 13.3% of DM wet corn gluten feed, 4.9% corn stover, and 3.9% alfalfa hay. The
proved by the university’s guidelines for animal research. The feeding same as below.
experiment was conducted at the Songhuajiang Dairy Farm (Harbin 2
DDGS = Distillers Dried Grains with Solubles.
city, Heilongjiang province, China). Nine multiparous Holstein dairy 3
Premix was composed of vitamin A, vitamin D3, vitamin E, manganese sulfate, zinc
cows (BW = 532 ± 28.9 kg and day in milk = 136 ± 5.6 d; mean ± sulfate, magnesium oxide, limestone, and sodium chloride. Chemical composition of pre-
mix contained (DM basis) 99.17% ash, 14.25% Ca, 5.40% P, 4.93% Mg, 0.05% K, 10.64% Na,
SD) were used in a replicated 3 × 3 Latin square design with 21-d pe-
2.95% Cl, 0.37% S, 12 mg/kg Co, 500 mg/kg Cu, 4 858 mg/kg Fe, 25 mg/kg I, 800 mg/kg
riods (14 d of diet adaption and 7 d of sample collection). Groups Mn, 10 mg/kg Se, 1 800 mg/kg Zn, 180 000 IU/kg vitamin A, 55 000 IU/kg vitamin D, and
were balanced for parity, day in milk, and milk production while they 1 500 IU/kg vitamin E.
4
consumed one of three treatment diets during each period. The treat- NFC = 100 − % NDF − % CP − % ether extract − % ash.
5
ment diets were fed as a TMR and contained similar concentrate mix- Calculated based on Ministry of Agriculture of P.R. China (MOA, 2004).

tures and corn silage but different proportions of roughage and WCGF.
The constituents of the three treatments were (1) 0% WCGF, 0% corn
stover, and 22.1% alfalfa hay (0% WCGF); (2) 6.9% WCGF, 3.4% corn sto-
ver, and 1.8% alfalfa hay (7% WCGF); and (3) 13.3% WCGF, 4.9% corn sto-
ver, and 3.9% alfalfa hay (13.3% WCGF). Cows were housed in individual
stall barns with free access to drinking water, were fed twice daily (0600
and 1630 h) at 105% ad libitum intake, and were milked twice daily at
0600 and 1830 h.
Preparation of total mixed ration silage
The ingredient and chemical composition is shown in Table 1. Three
diets were formulated to be isonitrogenous with a forage-to-concen-
trate ratio of 50:50 (DM basis) and meet the animals’ requirements
for nutrition based on the Cornell–Penn–Miner Dairy model version
3.08.01 (Boston et al., 2000). Three TMR silages were prepared, and
WCGF was obtained from the Cargill Biochemical Co., Ltd., Songyuan,
China. The corn stover was chopped to a length of 2 cm using a manual
forage chopper. Alfalfa hay was chopped to a length of 3–4 cm using a
manual forage chopper. These cutting lengths did not cause any sorting.
Lactic acid bacteria and fibrolytic ENs were used as additives for ensiling
the TMR. A combination of Lactobacillus plantarum and Lactobacillus
buchneri (LAB; applied at a ratio of 1:1) was isolated from whole-
plant corn silage. The combined LAB was added at the level of 1011
cfu/g of fresh material (FM), which satisfies a population of the initial
lactic acid bacteria of more than 105 cfu/g/FM, considered a crucial fac-
tor in ensuring successful silage fermentation (Weinberg and Muck,
1996). Fibrolytic ENs were purchased from a company (XS Biotechnol-
ogy Co., Ltd., Beijing, China) and had been extracted from Trichoderma
reesei. The activity of EN was measured as follows: cellulase at 10 000
U/g. Additives were dissolved in water and applied using a sprayer by
spraying uniformly onto the mixture. Total mixed ration silages were
packaged with polyethylene stretch film, which was stored outdoors
at 16–30 °C for all 60 days of fermentation. The chemical composition
of forages and WCGF is shown in Table 2.
Sample collection, measurements, and analyses
Diet chemical composition and nutrient intake
All three diets offered and refusals were weighed and collected on
days 15, 16, and 17 of each period to determine the DM intake (DMI).
The DM concentration of the diet was determined by drying samples
at 65 °C for 48 h in a forced-draft oven (DGX-9243B-1, Fuma Laboratory
Co., Ltd., Shanghai, China). The dried sample was ground through a 1-
mm screen in a micro plant-grinding machine (FZ102, Taisite Instru-
ment Co., Ltd, Tianjin, China) and analyzed for DM, CP, ash, and starch
according to the procedures 934.01, 976.05, 942.05, and 982.30, respec-
tively, of the Association of Official Analytical Chemists (AOAC) (AOAC,

2
G.N. Zhang, Y. Li, C. Zhao et al.
Table 2
Chemical composition of alfalfa hay, corn stover, and wet corn gluten feed used in the
three dairy cow diets (mean ± SD).
Item Alfalfa hay Corn stover Wet corn gluten feed
DM, % of FM 89.4 ± 0.49 92.7 ± 0.90 62.9 ± 0.46
OM, % of DM 88.4 ± 0.51 92.5 ± 0.35 92.9 ± 0.35
CP, % of DM 18.8 ± 0.06 9.70 ± 0.20 23.9 ± 0.40
NDF, % of DM 39.2 ± 0.31 51.6 ± 0.21 44.7 ± 0.35
ADF, % of DM 30.6 ± 0.35 46.6 ± 0.83 10.6 ± 0.25
EE, % of DM 2.70 ± 0.05 2.84 ± 0.03 6.64 ± 0.17
Starch, % of DM 1.01 ± 0.04 1.60 ± 0.06 11.8 ± 0.08
FM = fresh material; OM = organic matter; EE = ether extract.
1990). The NDF and ADF concentrations were sequentially analyzed
using the Ankom A200 fiber analyzer (Ankom Technology, Macedon,
NY, USA). The NDF was determined according to the method of Van
Soest et al. (1991) using heat-stable alpha-amylase and sodium sulfite,
and ADF was determined according to the procedure 954.01 of the
AOAC (AOAC, 1990). The NDF was corrected for ash. The fresh sample
of all three diets was separated and stored at −20 °C for future analysis
of pH, ammonia-N (NH3–N), and volatile fatty acids (VFAs). A fresh
sample of silage (10 g) was diluted in 90 ml of distilled water and stored
in the refrigerator at 4 °C for 24 h, and then, the extracts were filtered
through two layers of cheesecloth. The pH was immediately measured
using a pH meter (PB-10; Sartorius Co., Göttingen, Germany). Ammo-
nia-N concentration was determined by steam distillation into boric
acid and titration with dilute hydrochloric acid (10 ml). The VFA con-
centration was determined by gas chromatography (GC-8A; Shimadzu
Corp., Kyoto, Japan), as described by Hao et al. (2017). Nutrient intake
was calculated by the difference between specific nutrients in the feed
offered and refused.
Milk and ruminal fluid parameter
All cows were scored for body condition from 1 (thin) to 5 (fat)
(Edmonson et al., 1989) on day 20 of each period by three trained ob-
servers. Milk production was recorded, and milk samples were collected
during six consecutive milkings on days 15, 16, and 17 of each period.
Each day, a 50-ml aliquot of milk was mixed according to the actual pro-
duction by volume that reflected the am and pm milkings, preserved
with potassium dichromate, and stored at 4 °C until analyzed for pro-
tein, fat, lactose, milk urea nitrogen (MUN), and TS and somatic cell
count (SCC) by IR analysis (Laporte and Paquin, 1999) using an auto-
mated near-IR milk analyzer (MilkoScan, Foss Electric, Hillerød, Den-
mark) in the Heilongjiang Dairy Herd Improvement Testing Center
(Harbin, China). On days 19, 20, and 21 of each period, ruminal fluid
samples (approximately 200 ml) were collected using an oral stomach
tube approximately 3 h after the morning feeding (Wang et al., 2014).
We threw away the first 100–200 ml of fluid collected via the stomach
tube to reduce the risk of contamination of rumen fluid samples by sa-
liva. The pH was immediately measured using a pH meter (PB-10; Sar-
torius Co., Göttingen, Germany). The samples were centrifuged at 3 000
×g for 15 min, and the supernatant was separated and stored at −20 °C
for future analysis of NH3–N and VFA. The methods of NH3–N and VFA
assays were the same as the methods used for the analysis of a fresh
sample of all three diets.
Feces and urine analysis
On days 19, 20, and 21 of each period, approximately 500 g of fecal
sample was spot collected from the rectum at 0600 and 1800 h and
composited for individual cows. Fecal samples were dried at 65 °C in a
forced-air oven, ground through a 1-mm screen in a micro plant-grind-
ing machine (FZ102, Taisite Instrument Co., Ltd, Tianjin, China), and an-
alyzed for DM, CP, ash, NDF, and ADF using the same procedures as
described for feed. Indigestible NDF was used as an indirect marker to
calculate total-tract apparent nutrient digestibility. Incubated diet and
3
Animal 15 (2021) 100013
feces in the rumen of three ruminally cannulated multiparous Holstein
cows (average ± standard deviation; parity: 2.67 ± 0.58 lactations,
day in milk: 133 ± 4.16 d, and milk yield: 27.7 ± 1.53 kg/d) were
assessed for 288 h as described by Lee and Hristov (2013). Urine sam-
ples for individual cows were collected during urination with stimula-
tion at 0600 and 1800 h on days 19, 20, and 21 of each period and
then pooled by volume for each cow; 10 ml of each subsample was acid-
ified with 40 ml of H2SO4 (0.036 mol/l) and frozen (−20 °C). Urine sam-
ples were analyzed for urea N using the colorimetric method
(Rahmatullah and Boyde, 1980).
In situ degradation
In situ rumen degradation of DM and CP of the three experimental
diets with three ruminally cannulated Holstein cows was determined
using the method described by Hao et al. (2017). Seven grams of a
TMR sample was weighed and placed into numbered nylon bags (10
× 20 cm, 50-μm pore size; Ankom Technology, Macedon, NY, USA). All
nylon bags were tied to an end of a 40-cm polyester mesh bag, which
was used to hold the bags in the rumen. According to the report of
Schadt et al. (2014), bags were inserted in the rumen immediately be-
fore feeding and removed after 2, 4, 8, 12, 16, 24, 36, and 48 h. After in-
cubation, all bags were removed from the rumen and rinsed under cold
tap water until the water was clear. The bags were then dried at 65 °C
for 48 h and weighed. The residues and original diet samples were
ground and analyzed for DM, CP, and NDF. The constants of the in situ
degradation were estimated by the non-linear model described by
Orskov et al. (1980): p = a + b [1 – exp(−ct)], where p is the rate of dis-
appearance at time t (h), a is the rapidly degradable fraction (the soluble
fraction), b is the slowly degradable fraction, and c is the degradation
rate (c > 0). The effective degradability (dg) was calculated according
to the equation: dg = a + bc/ (c + kp), where a, b, and c are the con-
stants described above. The passage rate outflow from the rumen (kp)
was assumed to be 4.6%/h (Krizsan et al., 2010).
Statistical analysis
Data on milk production and components, nutrient intake and di-
gestibility, feed efficiency, nitrogen utilization, and rumen fermentation
were analyzed using PROC MIXED of SAS (version 9.3, SAS Institute Inc.,
Cary, NC, USA). The following model was used for the analysis as a rep-
licated 3 × 3 Latin square design: Yijkm = μ + Si + C(S)ij + Pk + Tm +
Eijkm, where Yijkm is the dependent variable, μ is the overall mean, Si is
the fixed effect of square i, C(S)ij is the random effect of cow j within
square i, Pk is the fixed effect of period k, Tm is the fixed effect of treat-
ment m, and Eijkm is the residual error. In situ DM and CP degradation
constants (a, b, c, and dg) and ruminally undegraded protein (RUP)
were analyzed using PROC GLM of SAS. Differences between the diets
were detected using Duncan’s adjustment. Statistical significance was
defined at P ≤ 0.05 with highly significant values at P < 0.01; trends
were declared at 0.05 < P ≤ 0.1.
Results
Feed characteristic
As shown in Table 1, the ADF concentration of the 0% WCGF was the
highest (P < 0.0001), followed by 7% WCGF and 13.3% WCGF. The 13.3%
WCGF diet had a lower pH value and higher lactic acid concentration
than that of 0% WCGF diet (P = 0.05 and P = 0.03, respectively). No dif-
ferences were observed between the 7% WCGF and 0% WCGF or 13.3%
WCGF diet for both pH and lactic acid concentration. The 13.3% WCGF
diet had a higher NH3–N/total N than the other two treatment diets
(P = 0.009), and there were no differences between the 0% WCGF and
7% WCGF diets.
G.N. Zhang, Y. Li, C. Zhao et al. Animal 15 (2021) 100013

Feed intake, milk production, and components Table 4

The degradation of DM, CP, and RUP of dairy cow experimental diets.

The results of feed intake, milk production, and components are Item Treatment SEM P-values
shown in Table 3. No differences were found between the treatment 0% WCGF 7% WCGF 13.3% WCGF
diets in DMI (P = 0.31), milk fat concentration (P = 0.95), milk lac-
DM degradation
tose concentration (P = 0.91), milk TS concentration (P = 0.76),
a, % 25.0b 31.4a 33.4a 0.90 0.01
MUN (P = 0.76), SCC (P = 0.59), and body condition score (BCS) b, % 61.0a 51.8b 51.9b 1.85 0.05
(P = 0.91). Cows fed the 13.3% WCGF diet had a higher milk yield c, %/h 4.11 4.90 4.56 0.93 0.84
(P = 0.004) and energy-corrected milk (ECM) (P < 0.0001) compared dg1 51.3b 57.0a 58.1a 1.18 0.05
with the other treatments. The yields of milk fat (P < 0.0001) and lac- CP degradation
a, % 29.0b 36.2a 40.7a 1.00 0.009
tose (P = 0.006) were higher for the cows fed the 13.3% WCGF diet b, % 53.7a 46.5ab 44.0b 1.77 0.05
compared with those of the other diets, and there were no differences c, %/h 4.71 4.94 5.00 0.59 0.94
between the 0% and 7% WCGF diets. Cows fed the 13.3% WCGF diet dg1 55.1b 59.1ab 62.6a 1.11 0.04
had a higher yield of milk protein (P < 0.0001), compared to those RUP,2 % of CP 44.9a 40.9ab 37.4b 1.11 0.04
in the other treatment diets, followed by 7% and 0% WCGF diets. The WCGF = wet corn gluten feed; a = the rapidly degradable fraction; b = the slowly
milk TS yield and milk protein concentration (P = 0.007) were higher degradable fraction; c = the degradation rate; dg = the effective degradability; RUP =
(P = 0.01 and P = 0.007, respectively) in the cows fed the 13.3% rumen undegradable protein; RDP = rumen degradable protein.
a,b
Means within a row with different superscripts differ (P < 0.05).
WCGF diet compared with those fed the 0% WCGF diet, and there 1
dg = a + bc/(c + kp) (Orskov et al., 1980), assuming a passage rate (kp) of 4.6%/h
were no differences between the 7 and 0% WCGF diets or between (Krizsan et al., 2010).
2
the 7% and 13.3% WCGF diets. Milk efficiency was lower (P = 0.002) RUP = 100 – RDP.
in the cows fed the 0% WCGF diet compared with those fed other
diets, and there were no differences between the 7% and 13.3% Rumen fermentation
WCGF diets.
The rumen fermentation characteristics of the three treatment diets
are presented in Table 5. Cows fed the 0% WCGF diet had lower concen-
In situ rumen digestion trations of NH3–N (P = 0.01) and total VFA (P < 0.0001) than cows fed
the other two treatment diets, and there were no differences between
The data on in situ rumen digestion of DM and CP of the three treat- the 7% and 13.3% WCGF diets. There were no significant differences in
ment diets are shown in Table 4. For the DM degradation, cows fed the rumen pH value (P = 0.84), molar proportion of acetate (P = 0.57), pro-
0% WCGF diet had a lower a value (P = 0.01) and dg of DM degradation pionate (P = 0.75), and butyrate (P = 0.27) and the ratio of acetate to
(P = 0.05) and a higher b value (P = 0.05) than the other two treatment propionate between the three treatment diets (P = 0.77).
diets, and there were no differences between the 7% and 13.3% WCGF
diets. For the CP degradation, cows fed the 0% WCGF diet had lower a Nutrient intake and digestibility
value (P = 0.009) and RUP (P = 0.04) of CP degradation than with
the 13.3% WCGF diet, and there were no differences between the 7% The total amount of nutrient intake and apparent total-tract nutrient
WCGF and 0 or 13.3% WCGF diets. Cows fed the 13.3% WCGF diet had digestibility of the three treatment diets are shown in Table 6. Cows fed
a higher dg (P = 0.04) for CP degradation than that of 0% WCGF diet, the 0% WCGF diet had higher ADF intake compared with the other treat-
and there were no differences between the 7% WCGF and 0% or 13.3% ment diets (P < 0.0001), followed by 7% and 13.3% WCGF diets. No sig-
WCGF diets. nificant differences between the three treatment diets were observed in
organic matter (OM), CP, and NDF intake (P = 0.25, 0.37, and 0.43, re-
spectively). Total-tract apparent digestibility of DM, OM, CP, NDF, and
ADF was lower for the cows fed the 0% WCGF diet compared with
Table 3
those fed the other treatment diets (P = 0.0002, 0.02, <0.0001, 0.01,
Lactation performance in dairy cows fed the experimental diets.
and 0.006, respectively), and there were no differences between the
Item Treatment SEM P-values 7% and 13.3% WCGF diets.
0% WCGF 7% WCGF 13.3% WCGF

DMI, kg/d 16.6 16.0 15.9 0.38 0.31 Nitrogen balance

Yield, kg/d
Milk 26.3c 27.1b 28.6a 0.31 0.004 Nitrogen utilization of the three treatment diets is presented in
ECM1 28.4b 29.2b 30.9a 0.33 <0.0001
Table 7. No significant differences were observed between the three
Fat 0.99b 1.02b 1.08a 0.01 <0.0001
Protein 0.78c 0.83b 0.89a 0.01 <0.0001
Lactose 1.35b 1.39b 1.46a 0.02 0.006
Total solids 3.17b 3.34ab 3.49a 0.07 0.01 Table 5
Milk composition, % Ruminal pH, NH2-N, and VFA in dairy cows fed the experimental diets.
Fat 3.79 3.78 3.78 0.02 0.95
Protein 2.98b 3.06ab 3.12a 0.03 0.007 Item Treatment SEM P-values
Lactose 5.14 5.15 5.12 0.09 0.91
0% WCGF 7% WCGF 13.3% WCGF
Total solids 12.1 12.3 12.2 0.25 0.76
MUN, mg/dL 15.1 14.6 14.7 0.58 0.79 pH 6.27 6.21 6.19 0.09 0.84
Milk efficiency2 1.71b 1.84a 1.95a 0.05 0.003 NH2-N, mg/dl 15.2b 17.0a 17.2a 0.48 0.01
SCC, ×103 24.2 26.4 26.9 2.00 0.59 Total VFA, mmol/l 76.9b 85.5a 87.6a 1.08 <0.0001
Body condition score 2.93 2.93 2.94 0.05 0.91 Molar proportion, mmol/100 mmol
Acetate 67.6 68.4 68.3 0.70 0.57
WCGF = wet corn gluten feed; DMI = DM intake; MUN = milk urea nitrogen; SCC =
Propionate 21.7 21.5 21.2 0.42 0.75
somatic cell count; ECM = energy-corrected milk.
a–c Butyrate 10.7 10.1 10.5 0.54 0.27
Means within a row with different superscripts differ (P < 0.05).
1 Acetate: propionate 3.14 3.19 3.23 0.09 0.77
ECM (kg) = 0.3246 × milk yield (kg) + 13.86 × fat yield (kg) + 7.04 × protein yield
(kg) (Orth, 1992). WCGF = wet corn gluten feed; VFA = volatile fatty acids.
2 a,b
Milk efficiency = ECM/DMI. Means within a row with different superscripts differ (P < 0.05).

4
G.N. Zhang, Y. Li, C. Zhao et al. Animal 15 (2021) 100013

Table 6 dairy cows (Huhtanen et al., 2002). Mohammadzadeh et al. (2012) ob-
Nutrient intake and total-tract apparent digestibility in dairy cows fed the experimental served that a greater concentration of organic acid produced by lactic
diets.
acid bacteria limited DMI. The ADF intake of the cows fed the 13.3%
Item Treatment SEM P-value WCGF diet was lower than that of the cows fed the 0% and 7% WCGF
0% WCGF 7% WCGF 13.3% WCGF diets due to the lower ADF concentration of the 13.3% WCGF diet. Gen-
erally, cows fed diets containing low-quality corn stover may have
Intake, kg/d
OM 15.5 14.8 14.8 0.35 0.25 lowered nutrient digestibility. In this study, cows fed the 7% and 13.3%
CP 3.23 3.08 3.15 0.07 0.37 WCGF diets improved nutrition digestibility due to high digestibility of
NDF 5.73 5.70 5.50 0.13 0.43 CP and fiber in WCGF (Mullins et al., 2010). The NDF in CGF was highly
ADF 3.53a 3.14b 2.84c 0.07 <0.0001 degradable (Firkins et al., 1991; VanBaale et al., 2001). At the same time,
Digestibility, %
DM 68.1b 72.2a 72.5a 0.73 0.001
the addition of lactic acid bacteria and fibrolytic ENs improved the di-
OM 59.3b 62.3a 63.2a 0.98 0.02 gestibility of NDF and ADF by loosening the structure of the cell wall
CP 70.2b 73.6a 73.9a 0.44 <0.0001 (Liu et al., 2016). Thus, digestibility of OM was increased in cows fed
NDF 42.3b 45.6a 47.2a 1.06 0.01 the 7% and 13.3% WCGF diets. Hao et al. (2017) also observed that
ADF 27.9b 31.4a 34.8a 1.15 0.01
cows fed the 7% and 11% DCGF diets demonstrated an increase of digest-
WCGF = wet corn gluten feed; OM = organic matter. ibility of OM.
a–c
Means within a row with different superscripts differ (P < 0.05). Moreover, Wang et al. (2014) demonstrated that a higher total con-
centration of ruminal VFA was indicative of higher nutrient digestibility.
Higher ruminal total VFA concentration in the cows fed the 7% and
treatment diets in N intake, retained N concentration, and proportion of 13.3% WCGF diets was indicative of higher nutrient digestibility, sug-
N intake (P = 0.37, 0.19, and 0.15, respectively). Cows fed the 13.3% gesting an increase in the energy supply to support milk production.
WCGF diet had higher milk N secretion (P < 0.0001) compared with Therefore, the use of 7% and 13.3% WCGF diets can improve nutrient di-
the other treatment diets, followed by the 7% and 0% WCGF diets. The gestibility while maintaining high milk production in cows. Higher di-
fecal N excretion and proportion of N intake were higher for cows fed gestibility can improve the efficiency of energy utilization and milk
the 0% WCGF diet than for those fed the other two treatment diets yield in dairy cows (Weiss et al., 2009). A similar increase in the yields
(P = 0.03 and 0.006, respectively), and there were no differences be- of milk and ECM has been reported due to an improvement in feed effi-
tween the 7% and 13.3% WCGF diets. Cows fed the 0% WCGF diet had ciency when WCGF replaced dietary forages (Mullins et al., 2010). Ace-
a lower milk N expressed as a proportion of N intake, and lower urinary tate is the main lipogenic precursor in the mammary gland during
N, expressed as a proportion of N intake compared with that in the other lactation (Smith, 1983). No significant differences were observed in
two treatment diets (P = 0.001, 0.002, and <0.0001, respectively), and the milk fat content due to unchanged ruminal acetate molar proportion
there were no differences between the 7% and 13.3% WCGF diets. (Table 5) between the three treatment diets. In the present study, the
yields of milk protein, fat, and lactose were higher when the cows
were fed WCGF due to an increase in the milk yield. These results are
Discussion
consistent with the result of Mullins et al. (2010), who observed that in-
creasing dietary WCGF linearly increased milk production and yield of
In this study, there was no effect on DMI of the cows fed three diets
milk components. Milk protein secretion in dairy cows is closely associ-
despite the addition of corn stover, which has poor digestibility and pal-
ated with the supply of metabolizable protein, which consists of the in-
atability. This result is consistent with the data of Wang et al. (2014),
testinally absorbable dietary protein, intestinally absorbable microbial
who observed that inclusion of 30% corn stover to replace alfalfa hay
protein, and intestinally absorbable endogenous true protein (NRC,
and Chinese wild rye hay did not affect DMI in dairy cows. Low-quality
2001). An increase in milk protein is related to supplementation with
forage diets with lower energy density may result in the consumption of
a certain proportion of DCGF, which was more digestible, resulting in
more DM to compensate for the need for additional energy (Wang et al.,
a higher energy intake than other treatments, which might promote mi-
2014). Certain studies have reported that the addition of WCGF with
crobial CP synthesis (Allen and Grant, 2000; Hao et al., 2017). Cows fed
higher rumen passage rates leads to decreased rumen fill per unit of
the 13.3% WCGF diet showed increased milk protein concentration,
feed intake, stimulating greater DMI (Mullins et al., 2010; Sullivan et
which is consistent with the data of Hao et al. (2017), who observed
al., 2012). However, the cows fed the 7% and 13.3% WCGF diets did
that feeding cows the 11% DCGF diet resulted in an increase in milk pro-
not demonstrate an increase in DMI due to a higher concentration of or-
tein concentration.
ganic acid and NH3–N in the TMR silage. It was reported that the percent
The DM degradation in cows fed the 7% and 13.3% WCGF diets was
of acetic acid in the total acid was negatively correlated with silage in-
higher, resulting from a higher a value (although a lower b value) com-
take (Wilkins et al., 1971). In addition, the NH3–N concentration in
pared with 0% WCGF. Montgomery et al. (2004) found that higher deg-
grass silage was reported to be related inversely to DMI in lactating
radation of WCGF resulted in higher a and c values. Hao et al. (2017)
also observed that cows fed the 11% DCGF diet had higher DM degrada-
tion, resulting from higher a and c values (although lower b). Cows fed
Table 7
Nitrogen balance in dairy cows fed the experimental diets.
the 13.3% WCGF diet had higher CP degradation (Table 3), resulting
from a higher a value (although a lower b value) compared with that
Item Treatment SEM P-values in the 0% WCGF diet. A higher a value was mostly attributable to the
0% WCGF 7% WCGF 13.3% WCGF higher concentration of rapid and higher CP degradation of WCGF
N intake, g/d 516.7 492.6 503.5 11.8 0.37 (Montgomery et al., 2004). Thus, the RUP percentage of CP was lower
Milk N secretion, g/d 122.7c 129.8b 140.0a 1.88 <0.0001 for cows fed the 13.3% WCGF diet compared with that of the 0% WCGF
% of N intake 23.8b 26.5a 28.0a 0.71 0.001 diet. Ruminal NH3–N concentration is positively correlated with the
Fecal N excretion, g/d 201.1a 179.1b 174.0b 6.95 0.03
rumen degradable protein (RDP) intakes (Reynolds and Kristensen,
% of N intake 38.8a 36.3b 34.5b 0.87 0.006
Urinary N excretion, g/d 116.2b 123.4a 127.5a 2.00 0.002 2008). The NH3–N concentration was higher for cows fed the 7% and
% of N intake 16.7b 23.1a 23.5a 0.63 <0.0001 13.3% WCGF diets than that for the 0% WCGF diet because of higher in
Retained N, g/d 76.6 60.4 61.9 7.16 0.19 situ CP degradation. The protein of CGF is rapidly degraded in the
% of N intake 14.8 12.1 12.1 1.18 0.15 rumen, thus increasing the ruminal NH3–N concentration (Hao et al.,
a–c
Means within a row with different superscripts differ (P < 0.05). 2017). Total VFA concentration in the rumen is increased with the

5
G.N. Zhang, Y. Li, C. Zhao et al.
addition of CGF (Hao et al., 2017). Biricik et al. (2007) observed that
total VFA increased when corn silage was replaced with DCGF in dairy
cows. Although the concentration of VFA increased in cows fed the 7%
and 13.3% WCGF diets, no significant differences in the pH values
were observed between the three treatment diets. Several studies indi-
cated that ruminal pH was not increased with the addition of CGF,
which caused an increase in alkaline ammonia ions (Krehbiel et al.,
1995; Hao et al., 2017).
The conversion of ingested dietary N into milk N, referred to as milk
N efficiency, is rather low in dairy cows; much of the dietary N intake is
lost in feces and urine, thus polluting the environment (Mcallister and
Text, 2011). In the present experiment, cows given the 13.3% WCGF
diet had a higher milk N secretion and milk N expressed in percent of
N intake due to higher milk yield. Fecal N consisted mainly of undi-
gested feed N and endogenous N (McDonald et al., 2002). Greater CP
degradation of the 7% and 13.3% WCGF diets results in less fecal N excre-
tion and concentration than those in the 0% WCGF diet. Mutsvangwa et
al. (2016) found that cows fed the low-RDP diet had a greater fecal N ex-
cretion compared with those fed the high-RDP diet. However, urinary N
concentrations in cows fed the 7% and 13.3% WCGF diets were higher
than in the 0% WCGF diet due to higher CP degradation and reduced
amino acid (AA) balance (Hao et al., 2017). Excessive concentrations
of NH3–N in the rumen resulted in high levels of N excreted mainly in
the urine (Whelan et al., 2011). Colmenero and Broderick (2006) ob-
served that the excretion of fecal N was lower than that of urinary N
when there was any increase in N intake in diets higher than 605 g/d
(16.5% of DM dietary CP). In this study, the excretion of fecal N was
higher than that of urinary N due to a lower dietary CP intake (492.6–
516.7 g/d). Moreover, N retention may depend on the amount of digest-
ible proteins and AA composition of the protein and on the energy
available for the protein accretion in the body (Valkeners et al., 2008).
There are two limitations in this study. First, the packing bag of TMR
silage needs to be opened manually or mechanically, which leads to
time-consuming and laborious feeding of TMR silage. Second, TMR si-
lage is wrapped and compacted into pieces, resulting in poor fluidity
of feed and inconvenient distribution and feeding.
Conclusions
Cows fed the 7% and 13.3% WCGF diets demonstrated a higher yield
of milk and milk components. Compared with 0% WCGF, cows fed the
7% and 13.3% WCGF diets had higher nutrient digestibility and milk pro-
tein concentration. Milk N secretion and milk N as a percent of N intake
were higher for cows fed the 13.3% WCGF diet compared to cows fed the
other diets. A higher RDP concentration in the 13.3% WCGF diet de-
creased fecal N excretion. Thus, it appears that feeding TMR silage con-
taining WCGF and corn stover in combination, replacing a portion of
alfalfa hay, improves lactation performance and nitrogen utilization
for lactating dairy cows.
Ethics approval
All procedures of animal handling were approved by the Animal
Care Advisory Committee, Northeast Agricultural University (Harbin,
China).
Data and model availability statement
None of the data was deposited in an official repository.
Author ORCIDs
Y. G. Zhang (Corresponding author): 0000-0001-5974-4651.
6
Animal 15 (2021) 100013
Author contributions
G.N.Z. and Y.L. contributed equally to this work, and they are both
co-first authors. G.N.Z., Y.L., C.Z., and X.P.F. participated in the design of
this study and performed most of the experiments. G.N.Z. performed
the statistical analysis and drafted the main manuscript. Y.G.Z. super-
vised the work. Y.G.Z. revised the final version of the manuscript. All au-
thors have read and approved the final version of the manuscript.
Declaration of interest
The authors declare no conflicts of interest.
Acknowledgements
None.
Financial support statement
This study was financially supported by the China Agriculture Re-
search System (CARS-36), the China Postdoctoral Science Foundation
for the 65th batch (2019M651252), the Postdoctoral Foundation in
Heilongjiang Province (LBH-Z17035), and the Young Talents "Project
of Northeast Agricultural University" (18QC35).
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