Effects of whey powder on fermentation quality, nutritive value, and digestibility of alfalfa silage

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Journal of Animal and Feed Sciences, 30, 1, 2022, 65–72 https://doi.org/10.22358/jafs/146345/2022
The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Jabłonna
Effects of whey powder on fermentation quality, nutritive value,
and digestibility of alfalfa silage
S. Özüretmen1,*, H. Özelçam1 and H.H. İpçak2
1 Ege University, Faculty of Agriculture, Department of Animal Science, İzmir, 35100, Turkey
2 Dicle University, Faculty of Agriculture, Department of Animal Science, Diyarbakır, 21280, Turkey
KEY WORDS: alfalfa, digestibility, net energy,
silage, whey powder
Introduction
Alfalfa (Medicago sativa L.) is a perennial le-
gume with a high yield which is also rich in vita-
mins and minerals. It can be used as animal feed in
the form of green herbage, hay, or silage. Because
grazing on fresh alfalfa can cause tympani, it is
very limited (Jonker and Yu, 2016). Although the
dried form is generally preferred as animal feed, the
leaves are easily broken and lost during this process
when drying to the appropriate storage moisture of
12%, also the botanical fraction balance (leaf:stem
ratio) shifts toward the cellulosic structure. This
decreases protein content, digestibility, and quality
of the forage, reduces the alfalfa nutritional value,
and increases the cost of feed. On the other hand,
it could be possible to obtain successful silage by
using additives that stimulate fermentation, such as
water-soluble carbohydrates (Keener, 2019).
Whey is a byproduct resulting from the pro-
cesses of converting milk into cheese. It has high
water content (>90%) and its major constituent
is lactose (77% of the total solids). Whey pow-
der (WP) is the dried form of it. It contains high
content of lactose (69–76%), low content of wa-
ter (<7%) and is rich in protein (β-lactoglobulin
and α-lactalbumin), vitamins (B2, B5 and C) and
minerals (EWPA, 2016). Each year, more than
200 mln t of whey is generated globally, and this value
is increasing by ~2% each year (Mariotti et al., 2020).
ABSTRACT. The aim of the study was to investigate the effects of whey powder
(WP) on the fermentation quality, nutritive value, and digestibility of ensiled
alfalfa (Medicago sativa L.). Alfalfa treated with different doses of WP (0, 2, and
4% fresh matter silage) was ensiled in plastic drums for 60 days. The results
of the study revealed that the physicochemical composition and fermentation
quality of the alfalfa silage improved and that mold growth was inhibited in
the groups treated with 2 and 4% WP in comparison to that in the control.
Production of CO2 (day 7) was much lower in silages treated with 2 and 4% WP
(3.77 and 1.85 g/kg dry matter (DM), respectively) than in the control group
(21.36 g/kg DM). In addition, in vivo dry matter digestibility (DMD) was much
higher in the group treated with 4% WP (76.45%) than in the control one (55.82%).
In this treatment group, all apparent digestibility of coefcients in vivo from crude
nutrient contents and cell wall fractions signicantly increased and hence raised
the net lactation energy value from 1.18 to 1.31 Mcal/kg DM. However, although
the in vitro DMD was higher in the silages treated with WP than in the control one
and the dose was signicant, there was no strong correlation between in vivo
and in vitro values. According to our results, WP could provide an advantage
for the conservation of alfalfa silage. In addition, WP could be evaluated as
a sustainable silage additive.
Received: 21 September 2021
Revised: 15 December 2021
Accepted: 1 February 2022
* Corresponding author:
e-mail: sema.ozuretmen@cpturkiye.com

66 Effects of whey powder on alfalfa silage quality
However, a large part of whey is processed to dried
form to be used in the food and feed industry (Królc-
zyk et al., 2016; El-Tanboly et al, 2017). In February
2022 the price of WP used as animal feed ranged be-
tween 1.154–1.340 €/t in Europe and USA (Anony-
mous, 2022). So that, WP could be more aordable
than other silage additives like inoculants or organic
acids, if its use becomes more widespread. It is well
known that WP used economically in milk replaces
is healthy for young animals. This by-product has
been evaluated for increasing calf growth and can be
used as both a major protein and an energy source
for replacing milk (Huuskonen, 2017). It can be
also used in concentrate feed replacing starch due to
high lactose content (El-Shewy, 2016). Some stud-
ies have revealed that whey has the potential as a
silage additive (Castaño and Villa, 2017; Keener,
2019). But the studies on dried form of whey as si-
lage additive are limited. However, WP can also have
a stimulating eect on alfalfa with low water-solu-
ble carbohydrates during fermentation because of
its high lactose content and can be more economical
than molasses. So, the aim of the present study was to
investigate the eects of WP at dierent doses (0, 2,
and 4%) on the fermentation quality, nutritive value,
and digestibility of ensiled alfalfa.
Material and methods
Alfalafa and whey powder
Alfalfa was harvested in the early bloom of the
fth cutting and was chopped to ~1.5–2.0 cm and
allowed to wilt until the dry matter (DM) content was
~29% before ensiling (Table 1). Whey protein was
purchased from Maybi (Malkara-Tekirdağ,Turkey)
and was obtained from sweet, fresh, pasteurized
whey. The whey had been spray-dried without using
any preservatives or additives and demineralized.
Whey powder containing less protein and more
lactose was preferred in the present study to be better
carbohydrate source for alfalfa silage fermentation.
Silage preparation
Alfalfa treated with dierent doses (0, 2, 4%
fresh matter silage) of WP was ensiled in 120-l plas-
tic drums. All groups were ensiled again in 2 kg jars
for aerobic stability. The ensiling was determined
according to silage-making techniques (Kılıç,
1986). Three replicates were used for each group
in a completely randomised design and 60 days of
storage. Four analyses from each replicate drum was
performed to identify chemical and microbiological
properties, and in vitro DM digestibility.
Chemical analyses
Samples were dried at 65 °C for 48 h and were
ground in a grinder with 1 mm sieve. All samples
were analyzed using the following methods: crude
nutrients (DM, crude protein (CP), ether extract
(EE), and crude bre (CF)) using the Weende analy-
sis (Menke and Huss, 1975), cell-wall components
(neutral detergent bre (NDF), acid detergent bre
(ADF), acid detergent lignin (ADL)) using the bre
bag system as modied by Goering and Van Soest
(1970), organic acids (acetic acid (AA), butyric acid
(BA), lactic acid (LA)) using the distillation method
adapted from Lepper (Naumann and Bassler, 1993),
water-soluble carbohydrates using a spectropho-
tometer and an anthrone-thiourea method (Anony-
mus, 1986), ammonia nitrogen (NH3-N) rates using
the microdistillation method (Anonymus, 1986),
and aerobic stability using the method of Ashbell
et al. (1991). The nitrogen-free extract (NfE) was
calculated as %NfE = 100% – (CP% + CF% + ash%
+ fat%) and the nonbrous carbohydrate (NFC)
was calculated as NFC = 100 − (NDF% + CP% +
fat% + ash%) (all nutrients are in dry matter) (NRC,
2001). Hemicellulose (HEM) content was calculat-
ed from NDF – ADF, and cellulose (CEL) content
from ADF − ADL. Before ensiling, the fresh mate-
rial buer capacity was detected using the method
of Playne and McDonald (1966).
All pH values of the samples were measured us-
ing a desktop pH meter (Hanna HI2211-02; Chen-
nai, Tamil Nadu, India). The physical characteristics
of all silages were determined using three dierent
parameters of colour, odour, and structure.
Table 1. Chemical composition of alfalfa before ensiling, % dry matter
(DM)
Alfalfa chemical composition
DM* 28.94 NFC 17.55
OM 84.62 NDF 46.92
CA 15.38 ADF 37.91
AIA 4.64 ADL 7.26
CP 17.45 HEM 9.02
EE 2.70 CEL 30.65
CF 29.65 WSC 4.70
NfE 34.83 BC 73.78*
Whey powder chemical composition
DM 95.0 EE 0.24
CA 6.50 CP 14.20
Lactose 75.60 pH 6.83
* value of fresh matter silage; OM – organic matter, CA – crude ash,
AIA – acid insoluble ash, CP – crude protein, EE – ether extract,
CF – crude bre, NfE – nitrogen-free extract, NFC – nonbrous
carbohydrate, NDF – neutral detergent bre, ADF – acid detergent
bre, ADL – acid detergent lignin, HEM – hemicellulose, CEL –
cellulose, WSC – water-soluble carbohydrate, BC – buffer capacity

S. Özüretmen et al. 67
The Flieg score:
Flieg score = 220 + (2 × DM% − 15) − 40 × pH,
was calculated according to silage dry matter (DM)
content and pH value (Kılıç, 1986; DLG, 1987).
The relative feed value (RFV) was calculated
according to Rohweder et al. (1978) from the equa-
tion:
RFV = dry matter intake (DMI) × digestible dry
matter (DDM) / 1.29,
where: DMI, % of body weight (BW) = 120 / (neutral
detergent bre (NDF), % of dry matter (DM)); and
DDM, % of DM = 88.9 – 0.779 × (acid detergent
bre (ADF), % of DM).
Microbial analyses
The colonies of mold-yeast were counted using
Tournas et al. (1998) and counts were expressed as
colony-forming units per g (CFU/g). Malt extract
agar was used for the enumeration of mold/yeast
and the Petri plates were incubated at 25 °C for
3–5 days under aerobic conditions.
In vitro digestibility
In vitro DM digestibility (IVDMD) of the
silages was determined using the enzyme technique,
which is based on incubating forages with pepsin
(2000 FIP-U/g) and cellulase enzyme (Onozuka R
10 from Trichoderma viride; Merck, Darmstadt,
Germany). The incubation time and temperature
of each enzyme were 24 h at 38 °C, respectively.
After incubation, the forages were washed, dried at
105 °C for 24 h, and incinerated in a mue furnace
at 550 °C for 4 h (De Boever et al., 1986).
In vivo digestibility
All procedures concerning animal usage were
approved by the Ethics Committee of Ege University,
Izmir, Turkey (No: 2016-001).
Nine Kıvırcık rams, 1–1.5 years old with simi-
lar physical characteristics and 45.0 ± 3.0 kg body
weight were used in the study. They were fed twice/
day at 8:30 and 16:30 and had ad libitum access to
drinking water and lick blocks during the trial. The
study was conducted using a completely randomised
design (3 rams per each treatment).
To evaluate in vivo digestibility, animals were
maintained in individual pens for 12 days (7 days
of adjustment period + 5 days of sampling). Feed
intake was calculated as 1.2 fold animal mainte-
nance requirements and feed (3.5 kg of fresh matter
silage). The manure was collected in the morning
before feeding during the sampling period. The ma-
nure picked up from each group was weighed and
100–150 g of it was kept into a jar by adding
3–5 drops of chloroform. There were three jars of
the manure from one animal. Each jar with manure
was analysed four times. All samples were kept in
the refrigerator at −20 °C until analysis. The appar-
ent digestibility of coecient (ADC) of the groups
was calculated according to GfE (1991):
ADC (%) = (feed intake − throw out with manure)
/ feed intake) × 100.
Total digestible nutrients (TDN) were calcu-
lated according to NRC (2001) and Küçük (2019):
TDN (%) = ((CP (%) × ADC of CP) + (CF (%) ×
ADC of CF) + (NfE (%) × ADC of NfE) + (EE (%)
× ADC of EE × 2.25),
where: CP – crude protein, ADC – apparent digest-
ibility of coecient, CF – crude bre, NfE – nitro-
gen free extract, EE – ether extract.
Net energy for lactation (NEL) was calculated
according to NRC (2001):
NEL (Mcal/kg) = 0.0245 × TDN (%) − 0.12,
where: TDN – total digestible nutrients.
Statistical analyses
All data were conducted to one-way analysis
of ANOVA by employing the procedure SPSS ver-
sion 22.0 (IBM Corp., Armonk, NY, USA) package
software (SPSS, 2013). A completely randomised
design was used according to the following model:
yij = µ + αi + εij,
where: yij – dependent variable, µ - overall mean, αi
– xed eect of treatment (i = 1 to 3), εij – random
error.
Duncan multiple comparison test was used to
compare the dierences among the mean values
(P < 0.05).
Results
Chemical composition
In comparison with the control group (Table 2),
the addition of WP to alfalfa silage led to an increase
in the content of DM of 4.42% in the samples with
4% WP and of 1.63% in the samples with 2% WP
(P < 0.05). No eect of WP addition on OM, CP, and
EE (P > 0.05) was observed.
The addition of WP also increased the NfE con-
tent in the silage in comparison to control samples
(P < 0.05), but there was no dose-depending eect
(P > 0.05). On the other hand, NFC values in si-
lages ranged from 17.06 to 21.92%, and the dier-
ence between the groups was important (P < 0.05).
The addition of WP also aected RFV values in the
silages. According to the data, in the groups treated

68 Effects of whey powder on alfalfa silage quality
with WP higher RFV values than in the control one
were noted, and this eect increased with the WP
addition (P < 0.05).
The addition of 4% WP was more eective on
NDF fraction – the lowest NDF value was found in
this group (P < 0.05). Similarly, only treatment with
4% WP had a signicant eect on the ADF fraction
in the silage and lowered its value (P < 0.05). How-
ever, the both doses of WP had no eect on ADL,
HEM, and CEL contents in the silages (P > 0.05).
Fermentation quality, aerobic stability
and microbiology
The data presented in Table 3 shows that WP de-
creased the pH value and improved the fermentation
quality of studied silages. As the dose increased, the
silage’s acid composition improved, and the pH of
the groups decreased, with the lowest value found
in the 4% WP group. On the other hand, WP also
increased the Flieg score by improving the physical
properties (structure, smell, and colour). We have
also observed that the rams fed silage treated with
4% WP consumed it readily, which can demonstrate
that WP improves the physical quality and enhances
the palatability of the silage.
The silages treated with WP had higher LA and
AA but lower BA in comparison with the control
group (P < 0.05); however, no dierence was ob-
served among the WP treatment groups (P > 0.05).
Although this improvement in silage acids increased
with the dose, the dierence among the treatment
groups was not statistically signicant. There was
no signicant eect on NH3-N in the silages treat-
ed with 2% WP; however, the NH3-N in the silage
treated with 4% WP decreased in comparison to that
in the control group.
At day 7 it was observed that the addition of
WP reduced the CO2 content in the silages and that
the WP dose had a crucial eect (P < 0.05). Accord-
ing to the data, the lowest level of CO2 was found
in the 4% WP group, and aerobic deterioration was
the highest in the control group. Hence, the highest
improvement of aerobic stability was determined in
the group with 4% WP. In addition, it was observed
that mold growth was prevented in the groups treat-
ed with WP.
In vitro and in vivo digestibilities
As shown in Table 4, the addition of WP to si-
lages inuenced IVDMD (P < 0.05). In all groups
values between 62.25 and 67.87% were noted; in the
control group IVDMD was the lowest whereas in
the 4% WP group – the highest. However, there was
no strong correlation between in vitro and in vivo
values for DMD.
Treatment with 4% WP had a signicant eect
on the digestibility of all nutrients in alfalfa silage
Table 2. Effect of whey protein (WP) on crude nutrients, nonbrous
carbohydrate (NFC) and relative feed values (RFV), and cell-wall
fractions in alfalfa silage, % dry matter (DM)
Indices WP treatement, % P-value
0 (n = 3) 2 (n = 3) 4 (n =3)
DM 29.85 ± 0.18c 31.48 ± 0.18b 34.27 ± 0.13a<0.001
OM 81.34 ± 0.22 81.98 ± 0.20 81.25 ± 0.31 0.090
CP 19.16 ± 0.32 18.88 ± 0.16 18.72 ± 0.12 0.320
EE 3.48 ± 0.08 3.22 ± 0.13 3.25 ± 0.10 0.182
CF 25.89 ± 0.45c 24.19 ± 0.01b 22.62 ± 0.43a 0.002
NfE 32.79 ± 0.39b 35.67 ± 0.30a 36.87 ± 0.57a<0.001
NFC 17.06 ± 0.46c 19.22 ± 0.29b 21.92 ± 0.26a<0.001
RFV 144.64 ± 4.36b148.35 ± 1.73b167.12 ± 5.09a 0.005
NDF 41.69 ± 0.35b 40.88 ± 0.22b 37.27 ± 0.24a 0.001
ADF 30.56 ± 0.72b 30.73 ± 0.50b 28.33 ± 0.38a 0.004
ADL 7.30 ± 0.16 6.99 ± 0.26 6.78 ± 0.14 0.124
HEM 10.41 ± 0.54 9.37 ± 0.53 8.96 ± 0.35 0.109
CEL 23.88 ± 1.27 23.07 ± 0.36 21.61 ± 0.41 0.153
OM – organic matter, CP – crude protein, EE – ether extract,
CF – crude bre, NfE – nitrogen-free extract, NDF – neutral deter-
gent bre, ADF – acid detergent bre, ADL – acid detergent lignin,
HEM – hemicellulose, CEL – cellulose; a–c means within the row with
different superscripts are signicantly different at P < 0.05
Table 3. Effect of whey protein (WP) on fermentation characteristics, aerobic stability, and the microbial population in alfalfa silage
Indices WP treatment, % P-value
0 (n = 3) 2 (n = 3) 4 (n = 3)
pH 5.94 ± 0.16b 5.11 ± 0.21a 5.09 ± 0.04a<0.001
Flieg score 27 (low) 60 (good) 70 (good) -
Lactic acid, % DM 4.77 ± 0.29b 8.03 ± 0.68a 8.11 ± 0.32a<0.001
Acetic acid, % DM 2.40 ± 0.12b 3.11 ± 0.16a 3.47 ± 0.14a<0.001
Butyric acid, % DM 1.98 ± 0.10b 0.82 ± 0.20a 0.51 ± 0.08a<0.001
NH3-N, g/kg DM 3.57 ± 0.14b 3.57 ± 0.01b 2.75 ± 0.06a<0.001
CO2, g/kg DM (day 7) 21.36 ± 1.00c 3.77 ± 0.52b 1.85 ± 0.22a<0.001
Total mold/yeast, log CFU/g 1.36 ± 0.73 N/A N/A -
DM – dry matter, CFU – colony forming unit, N/A – not available; a–c means within the row with different superscripts are signicantly different at
P < 0.05

S. Özüretmen et al. 69
(P < 0.05). The greatest apparent digestibility coef-
cient (ADC) values were found in the silages with
4% WP (P < 0.05). No eect on ADC digestibility
in silage samples with 2% WP was noted (P > 0.05).
However, it was observed that WP addition inu-
enced carbohydrate digestibility. In silages treat-
ed with WP higher ADC of CF and NfE contents
were noted, and this eect increased with the dose
(P < 0.05). Treatment with WP improved the digest-
ibility of silages, and dose greatly aected ADC of
NDF and ADF. The greatest ADC of NDF was in
the 4% WP silage (P < 0.05). In addition, both WP
doses had a signicant eect on ADF digestibility
(P < 0.05). Accordingly, the lowest ADC of ADF
was observed in the control silage, followed by that
in the 2 and 4% treatment groups (P < 0.05). The
digestibility of crude nutrients was consistent with
ADC and the parameters increased in the group
treated with WP 4%.
Values of TDN were higher in silages treated
with WP than in control one (P < 0.05). In addition,
there was a signicant dierence among groups
concerning NEL value (P < 0.05). Accordingly, in
the silage treated with 4% WP the highest NEL value
was observed; whereas, in the control group it was
the lowest (P < 0.05).
Discussion
Chemical composition. It was found that DM
and NFC contents of the silages increased along
with the increasing dose of WP. It could be associ-
ated with the high DM content (95%) in WP, and
so the ability of WP to improve silage fermentation.
On the other hand, NFC could play a critical role as
a fermentation stimulant in the rumen because of the
carbohydrate. It was shown that NFC as a carbohy-
drate source balancing with nitrogen (N) as a protein
source improved rumen microorganisms and N e-
ciency in the rumen (Ma et al., 2015). In the present
study, slight increase of NFC in the groups treated
with WP (with high lactose content) had a positive
eect on alfalfa silage digestibility in the rumen be-
cause it stimulated N eciency and improved ru-
men microorganisms.
Whey powder treatment improved silages by
decreasing their NDF and ADF contents. These re-
sults are in line with the results of previous studies
on the eect of WP on the cellulose content in the
silage (Dash et al., 1974; Fallah, 2019). According
to chemical analyses data, it could be stated that WP
used as a fermentation stimulant had a signicant
eect on the carbohydrate structure of the ensiling
material by increasing the rate of NFC versus de-
creasing the rates of NDF and ADF.
Fermentation quality, aerobic stability and
microbiology. The pH value is an important indica-
tor reecting silage fermentation toward dominant
microorganisms either desired or not desired. In the
present study WP improved the silage fermentation
but not as enough to lower the pH to < 5. Bijelić
et al. (2015) have reported that DM has a direct ef-
fect on pH. Also, Kung (2010) stated that legume si-
lages have a pH value of more than 4.6 to 4.8 due to
low DM value of fresh material (< 30%) that causes
Table 4. Effect of whey powder (WP) on in vitro dry matter digestibility (IVDMD), in vivo digestibility, total digestible nutrients (TDN) and net energy
lactation (NEL) in alfalfa silage, in dry matter (DM)
Indices WP treatment, % P-value
0 (n =3) 2 (n = 3) 4 (n = 3)
IVDMD, % 62.25 ± 1.10c64.91 ± 0.49b67.87 ± 0.65a 0.010
In vivo, %
Crude nutrients
DM 55.82 ± 2.29b59.83 ± 2.09b76.45 ± 1.51a<0.001
OM 62.18 ± 1.71b66.02 ± 1.66b77.32 ± 1.50a<0.001
CP 65.70 ± 1.74b70.37 ± 1.44b77.87 ± 1.93a<0.001
EE 68.53 ± 2.08b69.38 ± 1.64b81.10 ± 1.28a<0.001
CF 63.87 ± 1.63c70.37 ± 1.44b77.87 ± 1.93a<0.001
NfE 58.21 ± 2.51c66.28 ± 1.50b79.46 ± 1.21a<0.001
Cell wall fractions
NDF 68.23 ± 1.69b66.00 ± 1.44b77.12 ± 1.65a<0.001
ADF 59.38 ± 2.73c61.86 ± 1.85b72.78 ± 1.72a<0.001
TDN, % 53.19 ± 0.63c58.37 ± 0.33b66.79 ± 0.93a<0.001
NEL, Mcal/kg 1.18 ± 0.01c 1.31 ± 0.02b 1.52 ± 0.02a<0.001
OM – organic matter, CP – crude protein, EE – ether extract, CF – crude bre, NfE – nitrogen-free extract, NDF – neutral detergent bre,
ADF – acid detergent bre; a–c means within the row with different superscripts are signicantly different at P < 0.05

70 Effects of whey powder on alfalfa silage quality
clostridial fermentation. Clostridia is undesirable
bacteria in silages as it causes protein degradation,
DM loss, and production of toxins. However, butyric
acid is a marker for growing clostridia and it was not
identied in the silages treated with WP in this study.
The second reason for such low pH values is the high
buering capacity of legumes which might be a barri-
er. On the other hand, pH values of the silages treated
with WP were compatible with some previous studies
(Şengül and Aydın, 2019; Kang et al., 2021). Nev-
ertheless, the addition of 2 and 4% WP was enough
to improve other fermentation characteristics, such as
aerobic stability and the microbial population in the
alfalfa silages, without changing the pH.
Lactic acid is the primary acid responsible for
the decrease in silage pH, and the ratio of LA should
constitute 65–70% of the total acids in silages that
have undergone successful fermentation. Acetic acid
is the second most commonly found volatile acid in
silages, and BA should not be >0.5% in well-pre-
served silages because it induces ketosis in lactating
cows. High ratios of BA in silage also indicate that
feed proteins are broken down and the silage begins
to spoil (McDonald et al., 2010; Kung et al., 2018).
When the prole of the volatile acids in the silage
groups was examined, we observed that the silages
treated with WP had higher LA and AA but lower
BA than the control. Hence, it is possible that WP
could be a good source for improving end products
of legume silages. It was found that the AA ratio was
slightly higher in the WP groups than in the control
one. This moderate increment could be benecial
for improving aerobic stability. Even so, raising
the LA ratio and reducing the BA ratio supported
the fermentation to occure at the desired level and
that the Lactobacillus species were dominant in the
fermentation environment in the present study. Fur-
thermore, AA showed a protective property for si-
lages after opening and improved aerobic stability by
reducing the CO2 ratio (Kung et al., 2018). Danner
et al. (2003) have shown that AA plays a critical role
in inhibiting the organisms that create spoilage and
successfully inhibits these organisms. These ndings
are conrmed in our study where the mold growth
was not observed in silages treated with WP. Hence,
the improvement in aerobic stability and prevention
of mold growth in the treatment groups was associ-
ated with the increase in the AA ratio in these silages.
Our results indicated that adding 4% WP eectively
improved the fermentation quality of the silage.
In vitro and in vivo digestibilities. It was ob-
served that in comparison with the in vivo digest-
ibility, the in vitro results for DMD were higher.
Barchiesi-Ferrari et al. (2011) have shown that cel-
lulase concentration, incubation time, and washing
type have a signicant eect on enzymatic DMD.
During incubation, enzymatic activity or enzyme
type might have aected and altered the results of
the study. Similarly, it has been reported that as the
incubation time increases when using the enzymatic
method, while other methods, such as in situ, may
decrease it (Olowu and Yaman Fırıncıoğlu, 2019).
Additionally, this inconsistency between in vitro
and in vivo results could be related to the unpre-
dicted eects of feed additives such as WP on ru-
men health. Promoting to grow rumen microbiota in
a living organism might be an unpredicted eect of
4% WP addition.
Previous studies have generally shown that WP
addition to silage improves the fermentation process
(Castaño and Villa, 2017; Fallah, 2019). In some
studies, it was also observed that addition of WP
signicantly increases the digestibility of the alfalfa
silage, which is believed to be a result of its high
lactose content (Dash et al., 1974; Keener, 2019). Be-
cause WP has a stimulating eect as a carbohydrate
on fermentation during ensiling, it is presumed that
the silages treated with it are of high quality. In the
present study, we observed that the WP groups had
higher in vivo digestibility resulting from the suc-
cessful fermentation process. Hence, the increase in
DMD in the treated silages was associated with the
increased DM content resulting from the addition of
WP. This increment in DM rate supported in vitro and
in vivo DMD in the group treated with 4% WP. On
the other hand, silage treated with 4% WP had the
NEL value closest to that of alfalfa hay (NRC, 2001).
Conclusions
Chemical composition and fermentation quality
of alfalfa silage was improved by the treatment with
whey powder (WP). The additive provided a crucial
improvement in aerobic stability, and prevent the
mold growth. Also, 4% WP addition signicantly
increased in vivo nutrient digestibility. Whereas the
addition of both 2 and 4% WP aected the digest-
ibility of crude bre and acid detergent bre. Ac-
cordingly, it is recommend that silage additives such
as WP could be successfully used with legumes as
alfalfa, that are dicult to ensilage, and in order to
avoid losses in silage digestibility. Also, using whey
in ruminant nutrition could prevent environmental
pollution caused by industrial wastes and support
investments for ecological recycling facilities in
cheese factories pulverizing whey to powder.

S. Özüretmen et al. 71
Acknowledgements
This study was supported by TÜBİTAK 1002
- Short Term R&D Funding Programme (No: 116
O 110) and was included part of Sema ÖZÜRET-
MEN’s PhD thesis. Those who participated were as
follows: SÖ: original draft, conceptualization, inves-
tigation, data collection, formal analysis, and writing;
HÖ: original draft, conceptualization, project admin-
istration, supervision, validation, and editing; HHİ:
in vitro digestibility and the correlation between
in vitro and in vivo parameters and contributed to
writing this article. The authors also thank TÜBİTAK
for nancial support.
Conict of interest
The authors declare that there is no conict of
interst.
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