veterinary

sciences

Article
Peculiarities in the Amino Acid Composition of Sow Colostrum
and Milk, and Their Potential Relevance to Piglet Development
Renjie Yao 1, *, An Cools 1 , Anneleen Matthijs 2 , Peter P. De Deyn 3 , Dominiek Maes 2 and Geert P. J. Janssens 1

1 Department of Veterinary and Biosciences, Faculty of Veterinary Medicine, Ghent University,

9820 Merelbeke, Belgium
2 Department of Internal Medicine, Reproduction and Population Medicine, Faculty of Veterinary Medicine,
Ghent University, 9820 Merelbeke, Belgium
3 Laboratory of Neurochemistry and Behaviour, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences,
Faculty of Medicine and Health Sciences, University of Antwerp, 2610 Antwerp, Belgium
* Correspondence: renjie.yao@ugent.be

Simple Summary: Sow’s milk, serving as the primary nutrient source, contributes to the morpholog-
ical and functional maturation of piglets’ gastrointestinal tract. The abrupt diet transition at weaning
has required piglets to familiarize a solid diet earlier through supplementation with creep feed apart
from milk during the suckling period. We assume that the composition of sow milk is the gold
standard and could inspire the optimization of creep feed for suckling piglets. Therefore, in this
study, we investigated how conserved and variable the amino acid profile of sow colostrum and
milk is, compared with other literature and species. The amino acid profile of sow milk is conserved
compared to actual content throughout the lactation period and is rich in glycine, valine, and proline
compared to other species. Similarities with characteristic differences were observed within these
comparisons, which may reflect the nutritional requirements for preweaning piglets.

Abstract: The composition of mother’s milk is considered the ideal diet for neonates. This study in-
vestigated how conserved or variable the amino acid profile of sow colostrum and milk is throughout
lactation, compared with other studies in sows and other species. Twenty-five sows (parity one to
seven) from one farm with gestation lengths of 114 to 116 d were sampled on d 0, 3, and 10 after
Citation: Yao, R.; Cools, A.; Matthijs, parturition. The total amino acid profile of the samples was analyzed through ion-exchange chro-
A.; Deyn, P.P.D.; Maes, D.; Janssens, matography, and the results were displayed as the percentage of total amino acid and compared with
G.P.J. Peculiarities in the Amino Acid
literature data. Most of the amino acid concentrations in sow milk decreased significantly (p < 0.05)
Composition of Sow Colostrum and
throughout the lactation period, while the amino acid profile generally showed a conserved pattern,
Milk, and Their Potential Relevance
especially from d 3 to d 10, and was rather similar across different studies. Glutamine + glutamate
to Piglet Development. Vet. Sci. 2023,
was the most abundant amino acid in milk at all sampling moments, accounting for 14–17% of
10, 298. https://doi.org/10.3390/
vetsci10040298 total amino acids. The proportions of proline, valine, and glycine in sow milk nearly accounted for
11%, 7%, and 6% respectively, and were higher compared to human, cow, and goat milk, while the
Academic Editor: Marios Mataragas
methionine proportion was less than the other three. Compared to the large variations often reported
Received: 8 March 2023 in macronutrient concentrations, the amino acid profile of sow milk in the present study, as well as in
Revised: 7 April 2023 others, seems well conserved across the lactation period. Similarities with characteristic differences
Accepted: 12 April 2023 were also observed between sow milk and piglet body composition, which might reflect the nutrition
Published: 17 April 2023 requirements of preweaning piglets. This study warrants further research exploring the link between
the whole amino acid profile and the particular amino acids for suckling piglets and could facilitate
insight for optimizing creep feed.

Copyright: © 2023 by the authors.

Keywords: amino acids; colostrum; sow milk; creep feed
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
1. Introduction
creativecommons.org/licenses/by/ Amino acids from sow milk play a key role in offspring development. Apart from their
4.0/). traditional role as “building blocks” for protein synthesis, amino acids also have important

Vet. Sci. 2023, 10, 298. https://doi.org/10.3390/vetsci10040298 https://www.mdpi.com/journal/vetsci

Vet. Sci. 2023, 10, 298 2 of 12

functional roles in metabolism [1]. They are involved in features such as regulating gene ex-
pression and cell signaling [2–4]. They participate in the maintenance, growth, and immunity
of organisms. For example, proline is dominantly present in collagen and is also involved
in the activation of the mammalian target of rapamycin (mTOR) cell signaling for initiating
protein synthesis [5–7]. Amino acids can be used as alternative sources of other important
metabolites, such as glucose, fatty acids, and nitric oxide. Specifically, branched-chain amino
acids (BCAA) can be catabolized in the mammary gland to branched-chain α-keto acids
(BCKA), which can be further decarboxylated into a series of metabolites during lactation
which enter the citric acid cycle for producing energy in the mammary gland [8–11]. These
examples demonstrate that amino acids are not only necessary as structural components of
proteins, they can also support the gut development of neonatal piglets.
Amino acids are traditionally classified as essential amino acids (EAA) and nonessen-
tial amino acids (NEAA). The EAAs are those that the organisms cannot synthesize de
novo and, therefore, they must be supplied by the diet [12]. This definition may lead to an
underestimation of the functions and importance of some NEAA. Therefore, some NEAA
are reconsidered as conditionally essential amino acids that the organisms normally can
synthesize in adequate amounts, but which must be obtained from the diet on particular
occasions [13]. For instance, the NEAA glutamine and glutamate, the two most abundant
amino acids in sow milk, are synthesized by the mammary tissue starting from BCAA and
subsequently secreted into the milk. This means that the uptake of BCAA by the sow’s
mammary gland is higher than their output in milk [14,15]. This conversion of amino acids
in the mammary gland during lactation is reasonably considered a natural process to opti-
mize the diet of the offspring. These observations suggest that these abundant amino acids
in milk may have a high requirement in the newborn and that supplementation of these
NEAA may be beneficial for the intestinal health and growth of suckling piglets [16–20].
From an evolutionary perspective, the amino acid profile in milk should be used as the gold
standard for understanding the nutritional requirements of neonates [21]. Many amino
acids display remarkable metabolic versatility, and it can be hypothesized that natural
evolution has optimized the amino acid composition of sow colostrum and milk to support
neonatal piglet growth.
The present study investigated the amino acid profile of sow colostrum and milk.
Knowledge about the amino acid profile and comparison with the body composition
of suckling piglets and other species’ amino acid profile of milk may contribute to the
optimization of feeding suckling piglets.

2. Materials and Methods

2.1. Animals and Housing
In the present experiment, 25 sows (breed: Topigs 20; with average parity of 3.65 ± 1.65
(min 1–max 7)) were selected for colostrum and milk sampling. The sows had a gestation
length of 114 to 116 days and were housed in a commercial farm in Belgium. The day of
farrowing was defined as d 0 of lactation. During the whole lactation period, all sampled
sows were managed in the same way according to the regular procedures of the commercial
farm. The average litter size of these sows was 15.5, with, on average, 13.3 live-born piglets.
The sows were offered the same lactation diet and the feeding scheme was identical for all
sows. All diets applied for sows in this commercial farm met the nutrient requirements for
this pig category and feeding stage. They were housed in a climate-controlled farrowing
unit with a 14 h light and 10 h dark cycle. Each sow had access to an individual drinking
nipple and water was available unrestrictedly with a nipple flow rate of 1.5–2 L/min. The
health condition of the sows on this farm was supervised continuously.

2.2. Milk Samples and Chemical Analysis

The colostrum and milk (around 25 mL per sow) were collected from all teats along
one side of the mammary glands within 10 h after the birth of the first piglet (d 0) and at d
3 and d 10 of lactation. At each sampling point, the milk samples were collected 10 min
Vet. Sci. 2023, 10, 298 3 of 12

after intramuscular injection of 2 mL oxytocin (10 IU/mL) in the necks of sows. Samples
from the different teats of each sow were pooled immediately upon collection and stored at
−20 ◦ C until further analysis.
The total amino acid composition of all of the samples was determined after hydrolysis
of the sample. The samples were first dried with a Savant speed-vac system, after which
they were further dried in a desiccator with potassium hydroxide platelets and phosphorus
pentoxide. Then, 6 N HCl containing 1% phenol and 6% thioglycolic acid was added to
the dried samples. Subsequently, the hydrolysis was carried out under a vacuum and inert
conditions with nitrogen gas to prevent oxidative degradation of the amino acids during the
hydrolysis. The samples were heated at 110 ◦ C for 24 h and then dried under a vacuum.
The samples were washed several times with a solution of water, ethanol, and triethylamine
(2:2:1 v/v) to remove any acid residue. Lithium citrate buffer was added to the dry hydrolysis
product and dilutions were made for amino acid analysis with a Biotronik LC 6001 Amino
Acid Analyzer (Biotronik, Maintal, Germany). The optical density was measured by the
ninhydrin method. As a result of sample hydrolyses, no distinction between glutamine and
glutamate and between asparagine and aspartate was possible. Therefore, these amino acids
are always reported as glutamine + glutamate and asparagine + aspartate.

2.3. Data Selection

Data on amino acid profiles in whole piglet body composition and cow, goat, and human
milk were collected from published studies [22–31]. Cow and goat milk nearly account for
nearly 85% of the global production of milk and dairy products for human consumption, and
humans share similar metabolic and digestive processes with pigs [32–34]. Therefore, we
have made a comparison among these species. We searched in PubMed and Web of Science
using the keywords “amino acid composition”, “sow milk”, “breast milk” and “piglets body
composition” in September 2021. Our selection criteria for the literature search were: (1) The
types of amino acids which were analyzed should include all those we had analyzed, (2) The
milk samples in each study should be hydrolyzed before the analysis so that the results
could show the total composition of amino acid in milk, and (3) except for sow milk if
any treatments or diets were included, we only select the control group as common practice.
Typical information on sows and the methods applied in the present study and six publications
is listed in Table 1. The piglets used in the cited literature studies to analyze the amino acid
profile of the entire body composition were slaughtered at weaning and had not received any
creep feed during the lactation period (Mahan and Shields, 1998).

Table 1. An overview of potential factors influencing sow milk composition in different studies.

Sampling Date
Sow Breed Number of Sows Parity (Range) Method
(Day of Lactation)
This report Topigs 20 25 3.65 (1–7) D3 IEC a
Large White ×
Beyer et al. (2007) German 24 2.41 (1–4) D3 IEC
Landrace
Pre-column with
Wu et al. (1994) F1 hybrid 10 3.3 (-) D3
RP-HPLC b
Mudd et al. (2016) Yorkshire 14 - D7 IEC
Yorkshire × Pre-column with
Rezaei et al. (2022) 30 2.5 (2–3) D3
Landrace RP-HPLC b
Danish Large
Csapo et al. (1998) White + Duroc + 30 - D5 IEC
Landrace
Pre-column with
Kim et al. (2004) PIC Cambrough-22 10 - D7, 14, 21
RP-HPLC b
a IEC: ion-exchange chromatography. b RP-HPLC: reversed-phase high-performance liquid chromatographic.
Vet. Sci. 2023, 10, 298 4 of 12

2.4. Statistical Analysis

The data obtained in this study were expressed as mean ± standard deviation. The
level of each amino acid was expressed as the proportion of the whole amino acid concen-
trations (on a weight basis) both in the current study and the selected publications. The
comparison of amino acid content and profile in sow milk between different dates during
lactation was conducted in SPSS. 28.0 using a mixed model ANOVA of and post hoc Tukey
contrasts to determine the differences. Differences were considered significant at p < 0.05.

3. Results
3.1. Amino Acid Concentrations in Sow Milk at d 0 (Colostrum), d 3, and d 10 after Parturition
The amino acid content of hydrolyzed sow milk (colostrum) is shown in Table 2. Dur-
ing lactation, the total amino acid content decreased more than 50%, from 946 mmol/L in
colostrum (d 0) to 413 mmol/L in milk at d 3, while there was only about a 15% reduction
from d 3 to d 10. From d 0 to d 3, each of the individual amino acid concentrations showed
significant decreases (p < 0.05), while most amino acid concentrations (Thr, Ser, Glu, Gly, Val,
Met, Tyr, Phe, His, and Pro) remained stable (p > 0.05) from d 3 to d 10. Glutamine + glutamate
was the most abundant amino acid, accounting for 137 mmol/L in colostrum, followed by
proline, leucine, asparagine + aspartate, and lysine.

Table 2. Actual amino acid content of sow milk at d 0 (colostrum), 3, and 10 of lactation (mmol/L,
n = 25).

Date of Lactation
Amino Acids p Value
D0 D3 D 10
Asp 78.9 ± 14.2 a 35.8 ± 4.4 b 30.1 ± 3.4 c <0.001
Thr 57.1 ± 12.4 a 19.9 ± 3.4 b 16.6 ± 2.3 b <0.001
Ser 62.1 ± 12.5 a 24.7 ± 3.9 b 21.2 ± 2.9 b <0.001
Glu 137.0 ± 23.7 a 68.4 ± 7.9 b 61.6 ± 6.6 b <0.001
Gly 56.0 ± 11.2 a 23.5 ± 2.9 b 20.9 ± 2.9 b <0.001
Ala 59.8 ± 11.9 a 24.6 ± 3.2 b 20.5 ± 2.6 c <0.001
Val 72.5 ± 13.7 a 26.2 ± 3.7 b 21.9 ± 3.5 b <0.001
Met 11.0 ± 2.6 a 5.5 ± 9.9 b 4.9 ± 0.8 b <0.001
Ileu 38.3 ± 7.2 a 18.5 ± 2.7 b 16.1 ± 2.1 c <0.001
Leu 90.2 ± 17.8 a 37.5 ± 4.9 b 32.0 ± 4.1 c <0.001
Tyr 31.3 ± 6.5 a 11.8 ± 2.0 b 10.1 ± 1.5 b <0.001
Phe 34.5 ± 6.8 a 13.8 ± 1.9 b 12.0 ± 1.6 b <0.001
Lys 62.4 ± 11.2 a 28.6± 3.1 b 24.5 ± 2.7 c <0.001
His 21.1 ± 4.4 a 9.4 ± 1.3 b 8.2 ± 1.0 b <0.001
Arg 36.7 ± 6.9 a 15.6 ± 2.2 b 13.3 ± 1.9 c <0.001
Pro 97.3 ± 16.4 a 49.3 ± 6.7 b 44.7 ± 5.9 b <0.001
EAA * 423.7 ± 79.3 a 174.8 ± 22.1 b 149.5 ± 18.3 c <0.001
NEAA * 522.2 ± 93.7 a 237.9 ± 28.8 b 209.1 ± 22.3 c <0.001
Total AA 945.9 ± 172.3 a 412.8 ± 50.2 b 358.5 ± 40.0 c <0.001
Values were expressed as mean ± standard deviation, n = 25. Values followed by different superscript a–c within a
row were significantly different (p < 0.05). * EAA: essential amino acid; NEAA: nonessential amino acid. Asp and
Glu represent Asn + Asp and Glu + Gln, the following tables and figures are the same.

3.2. Amino Acid Profile of Sow Milk at d 0 (Colostrum), d 3, and d 10 after Parturition
Table 3 represents the amino acid profiles of sow colostrum at d 0 and sow milk at d
3 and d 10 of the lactation period. Since the data was expressed as the proportion of total
amino acids as a weight basis, the conservation of amino acid profiles among different dates
became apparent. Glutamine + glutamate was the most abundant amino acid in sow milk
across the sample dates, occupying about 14–17% of the total amino acid content, followed
by proline and leucine. Although all actual amino acid concentrations in sow milk decreased
significantly throughout the first day of lactation, the proportions of glutamine + glutamate,
proline, lysine, and methionine to the total amino acid rose significantly from d 0 to d 3,
 Vet. Sci. 2023, 10, 298 5 of 12

while those of threonine, serine, leucine, and tyrosine decreased (p < 0.05). From d 3 to d 10,
most of the AA remained stable (p > 0.05) as a proportion of total AA except for asparagine +
aspartate, glutamine + glutamate, alanine, and valine in the course of the ranking based on
the six highest amino acid proportions remained similar along the course of lactation.

Table 3. Amino acid profile of sow milk at d 0, 3, and 10 of lactation (% of total amino acid, n = 25).

Date of Lactation
Amino Acids p Value
D0 D3 D 10
Asp 8.6 ± 0.2 c 8.9 ± 0.2 a 8.7 ± 0.2 b <0.001
Thr 5.6 ± 0.5 a 4.4 ± 0.4 b 4.3 ± 0.3 b <0.001
Ser 5.4 ± 0.2 a 4.8 ± 0.3 b 4.8 ± 0.4 b <0.001
Glu 16.2 ± 0.5 c 18.5 ± 0.2 b 19.2 ± 0.8 a <0.001
Gly 3.5 ± 0.3 a 3.3 ± 0.3 b 3.4 ± 0.5 ab 0.005
Ala 4.4 ± 0.3 a 3.1 ± 0.30 c 3.9 ± 0.3 b <0.001
Val 7.0 ± 0.4 a 5.8 ± 0.4 b 5.6 ± 0.4 c <0.001
Met 1.4 ± 0.2 b 1.6 ± 0.1 a 1.6 ± 0.2 a <0.001
Ileu 4.2 ± 0.2 b 4.6 ± 0.3 a 4.6 ± 0.2 a <0.001
Leu 9.8 ± 0.5 a 9.3 ± 0.4 b 9.1 ± 0.5 b <0.001
Tyr 4.7 ± 0.2 a 4.3 ± 0.3 b 4.3 ± 0.3 b <0.001
Phe 4.7 ± 0.2 a 4.3 ± 0.2 b 4.3 ± 0.2 b <0.001
Lys 7.5 ± 0.3 b 7.9 ± 0.3 a 7.8 ± 0.2 a <0.001
His 2.7 ± 0.1 2.7 ± 0.2 2.8 ± 0.2 0.301
Arg 5.3 ± 0.2 a 5.1 ± 0.3 ab 5.0 ± 0.2 c 0.010
Vet. Sci. 2023, 10, x FOR PEER REVIEW 6 of 13
Pro 9.3 ± 0.6 c 10.6 ± 0.8 b 11.1 ± 0.72 a <0.001
Values were expressed as mean ± standard deviation. Values followed by different superscript a–c within a row
were significantly different (p < 0.05).
3.3. Amino Acid Profile of Sow Milk across Studies
3.3. Amino Acid Profile of Sow Milk across Studies
The amino acid profiles of sow milk throughout lactation showed a similar pattern
(FigureThe
1),amino acid profiles
even across studies of sowvarious
with milk throughout
factors shownlactation showed
in Table a similar+ pattern
1. Glutamine gluta-
(Figure
mate was1),
theeven
mostacross studies
abundant withacid
amino various factors
in these shown
studies, in Table 1.for
accounting Glutamine + gluta-
around 17–23%.
mate was to
According thedifferent
most abundant
studies, amino acid in these
the proportions studies, accounting
of glutamine + glutamate forand
around 17–23%.
asparagine
According to different studies, the proportions of glutamine + glutamate
+ aspartate were more variable than other amino acids. There were some minor numerical and asparagine
+ aspartateinwere
differences more variable
the ranking based onthan other
amino amino
acid acids. There
abundance betweenwere some In
studies. minor numer-
all studies,
ical differences in the ranking based on amino acid abundance between
the proportions of glutamine + glutamate, proline, leucine, lysine, and asparagine +aspar-studies. In all
studies, the proportions of glutamine
tate, however, all ranked in the top five. + glutamate, proline, leucine, lysine, and asparagine
+aspartate, however, all ranked in the top five.

Figure 1. Amino acid profile of sow milk across studies (% of total amino acids, the black spot were
Figure 1. Amino acid profile of sow milk across studies (% of total amino acids, the black spot were
the data collected from the studies shown in Table 1) (Wu et al., 1994, Csapó et al., 1996, Kim et al.,
the data collected from the studies shown in Table 1) (Wu et al., 1994, Csapó et al., 1996, Kim et al.,
2004,Beyer
2004, Beyeretetal.,
al.,2007,
2007,Mudd
Muddetetal.,
al.,2016,
2016,Rezaei
Rezaeietetal.,
al.,2022).
2022).

3.4. Amino Acid Profile of Milk across Animal Species

Figure 2 shows the amino acid profiles of sow, cow, goat, and human milk. Among
these species, most amino acid proportions were relatively similar. Except for human
milk, the proportions of glutamine + glutamate, proline, leucine, valine, lysine, and aspar-
agine + aspartate were still the top six most abundant amino acids, accounting for around
 Figure 1. Amino acid profile of sow milk across studies (% of total amino acids, the black spot were
the data collected from the studies shown in Table 1) (Wu et al., 1994, Csapó et al., 1996, Kim et al.,
2004, Beyer et al., 2007, Mudd et al., 2016, Rezaei et al., 2022).
Vet. Sci. 2023, 10, 298 6 of 12

3.4. Amino Acid Profile of Milk across Animal Species

Figure 2 shows the amino acid profiles of sow, cow, goat, and human milk. Among
3.4. Amino Acid Profile of Milk across Animal Species
these species, most amino acid proportions were relatively similar. Except for human
Figure
milk, the 2 showsof
proportions the amino acid
glutamine profiles ofproline,
+ glutamate, sow, cow, goat, valine,
leucine, and humanlysine,milk. Among
and aspar-
these species, most amino acid proportions were relatively similar. Except for
agine + aspartate were still the top six most abundant amino acids, accounting for around human milk,
the proportions of glutamine + glutamate, proline, leucine, valine, lysine,
18%, 10%, 9%, 7%, 6%, and 8%, respectively. Glutamine + glutamate was the most abun- and asparagine
+ aspartate
dant were stillwas
and methionine thethe
topscarcest
six most abundant
amino amino
acid in milk acids, accounting
of all four species.for
Sow around 18%,
milk had
relatively more proline, glycine, valine, and threonine, but less or the least glutamineand
10%, 9%, 7%, 6%, and 8%, respectively. Glutamine + glutamate was the most abundant +
methionine
glutamate was thelysine
(16.09%), scarcest aminoand
(6.81%), acidmethionine
in milk of all four species.
(1.30%) comparedSowto milk
thehad relatively
other three
more proline,
species. Goat and glycine, valine,
cow milk hadand threonine,
higher but less
proportions of or the least than
glutamine glutamine
sow and+ glutamate
human
(16.09%), lysine (6.81%), and methionine (1.30%) compared to the other three species. Goat
milk, and goat milk had higher lysine than the other species. Arginine was the fifth most
and cow milk had higher proportions of glutamine than sow and human milk, and goat
abundant amino acid in human milk, and human milk had the highest arginine propor-
milk had higher lysine than the other species. Arginine was the fifth most abundant amino
tion.
acid in human milk, and human milk had the highest arginine proportion.

Vet. Sci. 2023, 10, x FOR PEER REVIEW 7 of 13

Figure 2. Amino acid profile of milk across animal species (% of total amino acids) (Ceballos et al.,
Figure 2. Amino acid profile of milk across animal species (% of total amino acids) (Ceballos et al.,
2009,
2009, Garcia-Rodenas
Garcia-Rodenas etet al.,
al., 2016,
2016, GuGu
etetal.,
al.,2020).
2020).

3.5. Comparing Amino Acid Profiles of Sow Milk with Piglet Body Composition
3.5. Comparing Amino Acid Profiles of Sow Milk with Piglet Body Composition
The amino acid pattern in the whole body of piglets near weaning age (8.5 kg body-
The amino acid pattern in the whole body of piglets near weaning age (8.5 kg body-
weight) showed a close resemblance with the average amino acid pattern found in sow
weight) showed a close resemblance with the average amino acid pattern found in sow
milk in the present study (Figure 3). Glutamine + glutamate, proline, and leucine were
milk in the present study (Figure 3). Glutamine + glutamate, proline, and leucine were
most abundant while methionine was still the scarcest in the amino acid profile in sow milk
most abundant while methionine was still the scarcest in the amino acid profile in sow
and piglet body. Two marked differences were found: proline was higher in sow milk than
milk and piglet body. Two marked differences were found: proline was higher in sow milk
in piglets, whereas arginine was lower.
than in piglets, whereas arginine was lower.

Figure 3. Amino acid profile of sow milk in this study and whole piglet body in 8.5 kg body weight
(% of total amino acid).
Figure 3. Amino acid profile of sow milk in this study and whole piglet body in 8.5 kg body weight
4. Discussion
(% of total amino acid).
The amino acid profiles of milk composition included in these studies presented
4. Discussionand also noticeable variations. However, there is a large range of internal
similarities
and external factors that may theoretically influence milk composition (Table 1) though
The amino acid profiles of milk composition included in these studies presented sim-
most studies have focused on the macronutrient concentrations of sow milk. Different
ilarities and also noticeable variations. However, there is a large range of internal and ex-
technologies of amino acid analysis could play a key role in affecting the results since
ternal factors that may theoretically influence milk composition (Table 1) though most
studies have focused on the macronutrient concentrations of sow milk. Different technol-
ogies of amino acid analysis could play a key role in affecting the results since various
levels of sensitivity and the process of sample preparation in different methods could in-
fluence the results directly. In the present study, an automatic amino acid analyzer with
Vet. Sci. 2023, 10, 298 7 of 12

various levels of sensitivity and the process of sample preparation in different methods
could influence the results directly. In the present study, an automatic amino acid analyzer
with an ion-exchange chromatography (IEC) method was applied, while others applied
precolumn derivatization preceding RP-HPLC. Due to the long run time of IEC, the ninhy-
drin is unstable and the other analytes may react with the ninhydrin, whereas this method
is highly reproducible and has good linearity over a broad range [35,36]. Therefore, the
selection of technology could also be the factor causing variance.
As colostrum and milk are the main nutrition sources for neonatal pigs, they can
have a profound impact on neonates in the short and long term through physiological
processes [37]. The data of this study and the comparison with published studies showed
that the amino acid profile (expressed as the proportions of total amino acids) of sow milk
is conserved throughout the lactation period and is only marginally influenced by various
factors and even different breeds. However, the actual concentrations of amino acids in sow
milk decreased significantly during the first days of lactation. As the nutritional component
of maternal milk is considered the “gold standard” for infant feeding, WHO21, [38], infant
formulae are optimized based on milk composition. Therefore, the results of our study
suggest that this conserved amino acid profile of sow milk is suited for the nutritional
requirement of piglet development and may be used as a guide for the design of creep feed
for preweaning piglets.
In previous studies, the modulation of diet structure and the inclusion of feed additives
has been applied to sow diets to optimize the nutritional components (protein, fat, and
lactose) of sow milk. Several studies reported that the changes in dietary fat level and source
could alter both fat and lactose output, and the fatty acid profile of sow milk [39–41]. King
et al. [42] found that an increase in the dietary protein level could improve the total protein
concentration of sow milk, though the proportions of each amino acid were unaffected. Xu
et al. [43] reported that the amino acid concentrations of sow colostrum increased with the
standardized ileal digestible valine:lysine ratio, but the proportions of each amino acid to
total amino acids were stable. These findings combined with the results of the present study
suggest that the macronutrient profile is not as conserved as the amino acid profile in sow
milk, suggesting that there is more flexibility in exploring better nutrition sources for sow
diets while the prospects of applying specific amino acids to sows will likely not result in
changes in the amino acid profile.
In the sow milk samples of our study, the actual amino acid concentrations decreased,
though the amino acid pattern remained stable during lactation, in agreement with data
reported by Aguinaga et al. [44] and Csapo et al. [24]. Even though there were minor
differences among these three studies, the top six most abundant amino acids were the
same and the rankings based on amino acid abundance were similar. That was also the
case in other studies that had analyzed the amino acid profile of sow milk [44,45].
Glutamine plus glutamate were the most abundant amino acids independent of the
lactation stage and study, accounting for 17–23% of the total amino acids. This points
to the high requirement for piglet development. Glutamine has been proposed as an
indispensable amino acid to all tissues, since glutamine, as a protein synthesis precursor,
has many metabolites including glutamate, alanine, aspartate, α-ketoglutarate, and arginine
that are involved in various metabolic processes [46]. Glutamine, as the preferred fuel
for intestinal development and immune cells [13,15], is utilized at approximately 67% by
the small intestine of the pig, and it is the only amino acid in arterial blood absorbed
by the small intestine [47]. A myriad of studies reported the positive effect of glutamine
on piglet-growth performance, intestinal epithelium structure, antioxidant capacity, and
barrier functions [48–51], which are exactly suitable for newborn piglets with immature
digestive functions. Likewise, proline and leucine were also abundant amino acids. Proline,
as the conditionally essential amino acid, is highly abundant in collagen and elastin [52],
and adequate proline is essential for maximal collagen synthesis and growth performance
for animals [53–55]. Apart from these functions, proline also seems to be a precursor for
arginine synthesis in neonates [56]. It is therefore an interesting observation that the higher
Vet. Sci. 2023, 10, 298 8 of 12

proline content in sow milk may be a source for the higher arginine content in the piglet
body. Dietary amino acids are extensively catabolized by the small intestine in the first pass,
and nearly 30–40% of all proline is degraded [57,58]. These reports above also contributed
to the discrepancies in the comparison of the present study. However, it is not clear what
drives the level of these conversions, though it suggests an underestimated role of proline
in suckling piglet nutrition.
Leucine, as a BCAA, is not only the substrate for protein synthesis. It can also stimulate
protein deposition in skeletal muscle by serving as a signal molecule or mTOR signaling
pathway [59,60]. Leucine also performs a main role in the catabolism within the BCAA,
since the complex branched-chain α-keto acid dehydrogenase, which is a key enzyme in
BCAA catabolism, is regulated by leucine intake [61].
Among the different species, the amino acid profiles of milk generally matched in the
numerical comparison, despite minor discrepancies. The typical most abundant amino
acids were similar for these four mammalian species, i.e., glutamine + glutamate, proline,
valine, leucine, lysine, and asparagine, accounting for over 60% of total amino acids. It
should be noted that the level of “Glu” presented in the results is the sum of glutamine
and glutamate, and “Asp” is the sum of asparagine and aspartate due to sample hydrolysis
for amino acid analysis. This may lead to an overestimation of their importance, though
the above arguments do support their importance. It would, however, be interesting for
instance to know the glutamate: glutamine ratios in milk because exactly the interconver-
sion between glutamine and glutamate is under strong feedback regulation of several other
amino acids and nutrients [62–64].
Sow milk had numerically higher proportions of glycine, valine, and proline than
other species. Wang et al. [65] concluded that glycine is a dietary-essential amino acid for
maximal protein accretion in milk-fed piglets and that glycine is also the most abundant free
amino acid in the plasma of pigs [66]. Valine as a BCAA is crucial for regulating the energy
homeostasis, intestinal health, and immunity, which is essential for piglets under intensive
production [67]. The amino acid profiles of cow and goat milk were relatively close, and it
also was reported that the amino acid sequence of cow and goat milk protein showed an
88% homology [68]. Even though the amino acid sequence of cow and goat milk has been
concluded to share a 60% homology with human milk, infant formulae made from these
two milks still required modification of individual amino acids due to the higher proportion
of methionine and phenylalanine in cow and goat milk [69–72]. There was a report that
an infant fed with undiluted goat milk showed signs of severe liver impairment, which
was related to the excessive amounts of methionine and phenylalanine in goat milk [69].
The similar digestive physiology between pigs and humans, and close proportions of
methionine and phenylalanine in their milk, as shown in our study, may point to an
inaccurate attempt at diet design for piglets as well. Interestingly, this general similarity
in amino acid profiles was also observed in nonmammalian body compositions, with the
predominant amino acids in frogs (Xenopus laevis), such as glutamine, glycine, leucine, and
asparagine being generally similar to those in fish species (Engraulis encrasicholus), while
methionine, histidine, and phenylalanine were scarce in both [73]. By contrast, sows are
intensively using methionine as a methyl donor during lactation [74].
Generally, to mimic sow milk, creep feed is often nutrient dense and formulated with
more highly digestible ingredients such as milk byproducts to facilitate the nutrient intake
of immature piglets’ digestive systems [75]. In addition, the level of protein in creep feed
is normally higher than that in sow milk [76]. However, highly digestible creep feed only
improves the performance of piglets during the preweaning period, and coarse grain-based
creep feed benefits postweaning performance more than highly digestible creep feed [77].
Therefore, attention to the amino acid profile of creep feed in close connection with the
weaning diet might be a new impetus for creep feed development.
Finally, the conserved amino acid pattern in milk since d 3 might reflect the nutrition
requirement of piglets in the suckling period. However, whether the overall amino acid
structure or the partial amino acid combination can meet piglets’ needs efficiently remains
Vet. Sci. 2023, 10, 298 9 of 12

a question. Although the nutritional composition of supplementary feed for suckling

piglets can be modified based on data on maternal milk composition, the precise level of
conversion occurring in the first pass in the intestines needs further exploration. It also
remains to be verified whether the conserved amino acid structure is already an ideal state
for alleviating stress from diet transition in the future or the restriction to the development
of preweaning piglets now.
In conclusion, the amino acid pattern of sow milk is relatively stable across lactation
stages and diets. Compared to other species, sow milk is rich in glycine, valine, and proline,
the latter most likely serves as a source for arginine synthesis in the piglets. These findings
provide a basis to improve the amino acid pattern of creep feed or at least identify the
amino acid needs of suckling piglets, even beyond essential amino acids.

Author Contributions: G.P.J.J. and D.M. conceived and designed the experiment. R.Y., A.C., A.M.
and P.P.D.D. performed the experiments. R.Y. wrote the main manuscript text. All authors have read
and agreed to the published version of the manuscript.
Funding: This study received additional funding from Veepeiler Varken—Animal Healthcare Flanders.
Institutional Review Board Statement: The trial was executed in compliance to Directive 2010/63/EU.
All animals included in the trial were housed in accordance with EU legislation on animal husbandry.
Throughout the entire trial period, intramuscular injection of oxytocin prior to sampling was done
and animal health was supervised by a veterinary supervisor and no health problems were observed.
All procedures were common agricultural practices which belonged to “(a), 5, Article 3” (Directivse
2010/63/EU), so Directive could not be applied. The owner of the animal provided permission and
support for this study. The study was reported in accordance with ARRIVE guidelines 2.0.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The datasets used and/or analyzed during the current study are
available from the corresponding author on reasonable request.
Acknowledgments: The authors gratefully acknowledge financial support from China Scholarship
Council for Renjie Yao.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
For the amino acids; the standard tree-letter code has been used; except for “Glu” (Glutamine +
glutamate) and “Asp” (Asparagine + aspartate).

References
1. Fetrow, J.S.; Palumbo, M.J.; Berg, G. Patterns, structures, and amino acid frequencies in structural building blocks, a protein
secondary structure classification scheme. Proteins Struct. Funct. Bioinform. 1997, 27, 249–271. [CrossRef]
2. Jewell, J.L.; Russell, R.C.; Guan, K.-L. Amino acid signalling upstream of mTOR. Nat. Rev. Mol. Cell. Biol. 2013, 14, 133–139.
[CrossRef]
3. Wang, C.; Kang, C.; Xian, Y.; Zhang, M.; Chen, X.; Pei, M.; Zhu, W.; Hang, S. Sensing of L-Arginine by Gut-Expressed Calcium
Sensing Receptor Stimulates Gut Satiety Hormones Cholecystokinin and Glucose-Dependent Insulinotropic Peptide Secretion in
Pig Model. J. Food Sci. 2018, 83, 2394–2401. [CrossRef]
4. Zdraljevic, S.; Fox, B.W.; Strand, C.; Panda, O.; Tenjo, F.J.; Brady, S.C.; Crombie, T.A.; Doench, J.G.; Schroeder, F.C.; Andersen, E.C.;
et al. Natural variation in C. elegans arsenic toxicity is explained by differences in branched chain amino acid metabolism. elife
2019, 8, e40260. [CrossRef]
5. Wu, G.; Bazer, F.W.; Datta, S.; Johnson, G.A.; Li, P.; Satterfield, M.C.; Spencer, T. Proline metabolism in the conceptus: Implications
for fetal growth and development. Amino Acids 2008, 35, 691–702. [CrossRef] [PubMed]
6. Yao, K.; Yin, Y.-L.; Chu, W.; Liu, Z.; Deng, D.; Li, T.; Huang, R.; Zhang, J.; Tan, B.; Wang, W.; et al. Dietary Arginine Supplementation
Increases mTOR Signaling Activity in Skeletal Muscle of Neonatal Pigs. J. Nutr. 2008, 138, 867–872. [CrossRef]
7. Geiger, R.; Rieckmann, J.C.; Wolf, T.; Basso, C.; Feng, Y.; Fuhrer, T.; Kogadeeva, M.; Picotti, P.; Meissner, F.; Mann, M.; et al.
L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity. Cell 2016, 167, 829–842. [CrossRef]
[PubMed]
Vet. Sci. 2023, 10, 298 10 of 12

8. Blachier, F.; Mariotti, F.; Huneau, J.F.; Tomé, D. Effects of amino acid-derived luminal metabolites on the colonic epithelium and
physiopathological consequences. Amino Acids 2007, 33, 547–562. [CrossRef]
9. Morris, S.M., Jr. Arginine metabolism: Boundaries of our knowledge. J. Nutr. 2007, 137, 1602s–1609s. [CrossRef] [PubMed]
10. Montañez, R.; Rodriguez-Caso, C.; Jiménez, F.M.S.; Medina, M.A. In silico analysis of arginine catabolism as a source of nitric
oxide or polyamines in endothelial cells. Amino Acids 2008, 34, 223–229. [CrossRef]
11. Ma, C.; Liu, Y.; Liu, S.; Lévesque, C.L.; Zhao, F.; Yin, J.; Dong, B. Branched chain amino acids alter fatty acid profile in colostrum
of sows fed a high fat diet. J. Anim. Sci. Biotechnol. 2020, 11, 9. [CrossRef] [PubMed]
12. Rose, W.C.; Oesterling, M.J.; Womack, M. Comparative growth on diets containing ten and 19 amino acids, with further
observations upon the role of glutamic and aspartic acids. J. Biol. Chem. 1948, 176, 753–762. [CrossRef] [PubMed]
13. Wu, G. Functional amino acids in growth, reproduction, and health. Adv. Nutr. 2010, 1, 31–37. [CrossRef]
14. Li, P.; Knabe, D.A.; Kim, S.W.; Lynch, C.J.; Hutson, S.M.; Wu, G. Lactating Porcine Mammary Tissue Catabolizes Branched-Chain
Amino Acids for Glutamine and Aspartate Synthesis. J. Nutr. 2006, 139, 1502–1509. [CrossRef] [PubMed]
15. Hou, Y.; Yao, K.; Yin, Y.; Wu, G. Endogenous Synthesis of Amino Acids Limits Growth, Lactation, and Reproduction in Animals.
Adv. Nutr. 2016, 7, 331–342. [CrossRef] [PubMed]
16. Brunton, J.A.; Baldwin, M.P.; Hanna, R.A.; Bertolo, R.F. Proline Supplementation to Parenteral Nutrition Results in Greater Rates
of Protein Synthesis in the Muscle, Skin, and Small Intestine in Neonatal Yucatan Miniature Piglets. J. Nutr. 2012, 142, 1004–1008.
[CrossRef]
17. Cabrera, R.A.; Usry, J.L.; Arrellano, C.; Nogueira, E.T.; Kutschenko, M.; Moeser, A.J.; Odle, J. Effects of creep feeding and
supplemental glutamine or glutamine plus glutamate (Aminogut) on pre- and post-weaning growth performance and intestinal
health of piglets. J. Anim. Sci. Biotechnol. 2013, 4, 29. [CrossRef] [PubMed]
18. Wang, H.; Zhang, C.; Wu, G.; Sun, Y.; Wang, B.; He, B.; Dai, Z.; Wu, Z. Glutamine enhances tight junction protein expression
and modulates corticotropin-releasing factor signaling in the jejunum of weanling piglets. J. Nutr. 2015, 145, 25–31. [CrossRef]
[PubMed]
19. Bertocchi, M.; Bosi, P.; Luise, D.; Motta, V.; Salvarani, C.; Ribani, A.; Bovo, S.; Simongiovanni, A.; Matsunaga, K.; Takimoto, T.;
et al. Dose-response of different dietary leucine levels on growth performance and amino acid metabolism in piglets differing for
aminoadipate-semialdehyde synthase genotypes. Sci. Rep. 2019, 9, 18496. [CrossRef] [PubMed]
20. Zhao, Y.; Albrecht, E.; Sciascia, Q.; Li, Z.; Görs, S.; Schregel, J.; Metges, C.; Maak, S. Effects of Oral Glutamine Supplementation on
Early Postnatal Muscle Morphology in Low and Normal Birth Weight Piglets. Animals 2020, 10, 1976. [CrossRef]
21. Scano, P.; Murgia, A.; Demuru, M.; Consonni, R.; Caboni, P. Metabolite profiles of formula milk compared to breast milk. Food Res.
Int. 2016, 87, 76–82. [CrossRef]
22. Wu, G.; Knabe, D.A.A. Free and protein-bound amino acids in sow’s colostrum and milk. J. Nutr. 1994, 124, 415–424. [CrossRef]
23. Mahan, D.C.; Shields, R.G. Essential and nonessential amino acid composition of pigs from birth to 145 kilograms of body weight,
and comparison to other studies. J. Anim. Sci. 1998, 76, 513–521. [CrossRef]
24. Csapó, J.; Martin, T.G.; Csapó-Kiss, Z.S.; Házas, Z. Protein, fats, vitamin and mineral concentrations in porcine colostrum and
milk from parturition to 60 days. Int. Dairy J. 1996, 6, 881–902. [CrossRef]
25. Kim, S.W.; McPherson, R.L.; Wu, G. Dietary arginine supplementation enhances the growth of milk-fed young pigs. J. Nutr. 2004,
134, 625–630. [CrossRef] [PubMed]
26. Beyer, M.; Jentsch, W.; Kuhla, S.; Wittenburg, H.; Kreienbring, F.; Scholze, H.; Rudolph, P.E.; Metges, C.C. Effects of dietary energy
intake during gestation and lactation on milk yield and composition of first, second and fourth parity sows. Arch Anim. Nutr.
2007, 61, 452–468. [CrossRef] [PubMed]
27. Ceballos, L.; Ramos-Morales, E.; Adarve, G.; Díaz-Castro, J.; Martínez, L.; Sampelayo, M. Composition of goat and cow milk
produced under similar conditions and analyzed by identical methods. J. Food Compos. Anal. 2009, 22, 322–329. [CrossRef]
28. Garcia-Rodenas, C.L.; Affolter, M.; Vinyes-Pares, G.; De Castro, C.A.; Karagounis, L.G.; Zhang, Y.; Wang, P.; Thakkar, S.K. Amino
Acid Composition of Breast Milk from Urban Chinese Mothers. Nutrients 2016, 8, 606. [CrossRef]
29. Mudd, A.T.; Alexander, L.S.; Johnson, S.K.; Getty, C.M.; Malysheva, O.V.; A Caudill, M.; Dilger, R.N. Perinatal Dietary Choline
Deficiency in Sows Influences Concentrations of Choline Metabolites, Fatty Acids, and Amino Acids in Milk throughout Lactation.
J. Nutr. 2016, 146, 2216–2223. [CrossRef]
30. Gu, F.; Wang, D.; Yang, D.; Liu, J.; Ren, D. Short communication: Effects of dietary N-carbamoylglutamate supplementation on the
milk amino acid profile and mozzarella cheese quality in mid-lactating dairy cows. J. Dairy Sci. 2020, 103, 4935–4940. [CrossRef]
31. Rezaei, R.; Gabriel, A.S.; Wu, G. Dietary supplementation with monosodium glutamate enhances milk production by lactating
sows and the growth of suckling piglets. Amino Acids 2022, 54, 1055–1068. [CrossRef]
32. Patterson, J.K.; Lei, X.G.; Miller, D.D. The pig as an experimental model for elucidating the mechanisms governing dietary
influence on mineral absorption. Exp. Biol. Med. 2008, 233, 651–664. [CrossRef]
33. Gerosa, S.; Skoet, J. Milk Availability: Trends in Production and Demand and Medium-Term Outlook; Sematic Scholar: Seattle, WA,
USA, 2012.
34. Gonzalez, L.M.; Moeser, A.J.; Blikslager, A.T. Porcine models of digestive disease: The future of large animal translational research.
Transl. Res. J. Lab. Clin. Med. 2015, 166, 12–27. [CrossRef] [PubMed]
Vet. Sci. 2023, 10, 298 11 of 12

35. Le Boucher, J.; Charret, C.; Coudray-Lucas, C.; Giboudeau, J.; Cynober, L. Amino acid determination in biological fluids by
automated ion-exchange chromatography: Performance of Hitachi L-8500A. Clin. Chem. 1997, 43, 1421–1428. [CrossRef]
[PubMed]
36. Pappa-Louisi, A.; Nikitas, P.; Agrafiotou, P.; Papageorgiou, A. Optimization of separation and detection of 6-aminoquinolyl
derivatives of amino acids by using reversed-phase liquid chromatography with on line UV, fluorescence and electrochemical
detection. Anal. Chim. Acta 2007, 593, 92–97. [CrossRef]
37. Thompson, A.L. Developmental origins of obesity: Early feeding environments, infant growth, and the intestinal microbiome.
Am. J. Hum. Biol. 2012, 24, 350–360. [CrossRef]
38. World Health Organization. WHO guidelines approved by the guidelines review committee. In WHO Recommendations on
Postnatal Care of the Mother and Newborn; World Health Organization: Geneva, Switzerland, 2013.
39. Zhao, P.Y.; Zhang, Z.F.; Lan, R.X.; Liu, W.C.; Kim, I.H. Effect of lysophospholipids in diets differing in fat contents on growth
performance, nutrient digestibility, milk composition and litter performance of lactating sows. Animal 2017, 11, 984–990.
[CrossRef]
40. Jin, C.; Fang, Z.; Lin, Y.; Che, L.; Wu, C.; Xu, S.; Feng, B.; Li, J.; Wu, D. Influence of dietary fat source on sow and litter performance,
colostrum and milk fatty acid profile in late gestation and lactation. Anim. Sci. J. 2017, 88, 1768–1778. [CrossRef]
41. Flummer, C.; Theil, P.K. Effect of β-hydroxy β-methyl butyrate supplementation of sows in late gestation and lactation on sow
production of colostrum and milk and piglet performance. J. Anim. Sci. 2012, 90 (Suppl. 4), 372–374. [CrossRef]
42. King, R.H.; Rayner, C.J.; Kerr, M. A note on the amino acid composition of sow’s milk. Anim. Prod. 2016, 57, 500–502. [CrossRef]
43. Xu, Y.; Zeng, Z.; Xu, X.; Tian, Q.; Ma, X.; Long, S.; Piao, M.; Cheng, Z.; Piao, X. Effects of the standardized ileal digestible valine:
Lysine ratio on performance, milk composition and plasma indices of lactating sows. Anim. Sci. J. 2017, 88, 1082–1092. [CrossRef]
44. Aguinaga, M.A.; Gómez-Carballar, F.; Nieto, R.; Aguilera, J.F. Utilization of milk amino acids by the suckling Iberian piglet. J.
Anim. Physiol. Anim. Nutr. 2011, 95, 771–780. [CrossRef]
45. Elliott, R.F.; Noot, G.W.V.; Gilbreath, R.L.; Fisher, H. Effect of dietary protein level on composition changes in sow colostrum and
milk. J. Anim. Sci. 1971, 32, 1128–1137. [CrossRef]
46. Blachier, F.; Guihot-Joubrel, G.; Vaugelade, P.; Le Boucher, J.; Bernard, F.; Duée, P.; Cynober, L. Portal hyperglutamatemia after
dietary supplementation with monosodium glutamate in pigs. Digestion 1999, 60, 349–357. [CrossRef]
47. Wu, G.; Bazer, F.W.; Johnson, G.A.; Knabe, D.A.; Burghardt, R.; Spencer, T.; Li, X.L.; Wang, J. Triennial Growth Symposium:
Important roles for L-glutamine in swine nutrition and production. J. Anim. Sci. 2011, 89, 2017–2030. [CrossRef]
48. Amorim, A.B.; Berto, D.A.; Saleh MA, D.; Miassi, G.M.; Ducatti, C. Dietary glutamine, glutamic acid and nucleotides increase the
carbon turnover (δ 13C) on the intestinal mucosa of weaned piglets. Animal 2017, 11, 1472–1481. [CrossRef]
49. He, Y.; Fan, X.; Liu, N.; Song, Q.; Kou, J.; Shi, Y.; Luo, X.; Dai, Z.; Yang, Y.; Wu, Z.; et al. l-Glutamine Represses the Unfolded
Protein Response in the Small Intestine of Weanling Piglets. J. Nutr. 2019, 149, 1904–1910. [CrossRef] [PubMed]
50. da Silva, I.V.; Soares, B.P.; Pimpão, C.; Pinto, R.M.A.; Costa, T.; Freire, J.P.B.; Corrent, E.; Chalvon-Demersay, T.; Prates, J.A.M.;
Lopes, P.A.; et al. Glutamine and cystine-enriched diets modulate aquaporins gene expression in the small intestine of piglets.
PLoS ONE 2021, 16, e0245739. [CrossRef] [PubMed]
51. Vázquez-Gómez, M.; García-Contreras, C.; Astiz, S.; Torres-Rovira, L.; Pesantez-Pacheco, J.; Heras-Molina, A.; Madrigal, T.C.;
López-Bote, C.; Óvilo, C.; González-Bulnes, A.; et al. Effects of L-Glutamine Supplementation during the Gestation of Gilts and
Sows on the Offspring Development in a Traditional Swine Breed. Animals 2021, 11, 903. [CrossRef] [PubMed]
52. Bennett, M.J. Textbook of Biochemistry with Clinical Correlations, 6th ed.; Thomas, M.D., Ed.; Wiley-Liss, John Wiley & Sons: Hoboken,
NJ, USA, 2006; p. 1208. ISBN 0-471-67808-2.
53. Baker, D.H. Advances in protein-amino acid nutrition of poultry. Amino Acids 2009, 37, 29–41. [CrossRef]
54. Hou, Y.; Wu, Z.; Dai, Z.; Wang, G.; Wu, G. Protein hydrolysates in animal nutrition: Industrial production, bioactive peptides, and
functional significance. J. Anim. Sci. Biotechnol. 2017, 8, 24. [CrossRef]
55. Shaw, G.; Lee-Barthel, A.; Ross, M.L.; Wang, B.; Baar, K. Vitamin C-enriched gelatin supplementation before intermittent activity
augments collagen synthesis. Am. J. Clin. Nutr. 2017, 105, 136–143. [CrossRef] [PubMed]
56. Tomlinson, C.; Rafii, M.; Sgro, M.; O Ball, R.; Pencharz, P. Arginine is synthesized from proline, not glutamate, in enterally fed
human preterm neonates. Pediatr. Res. 2011, 69, 46–50. [CrossRef]
57. Wu, G.; Bazer, F.W.; Burghardt, R.C.; Johnson, G.A.; Kim, S.W.; Knabe, D.A.; Li, X.; Satterfield, M.C.; Smith, S.B.; Spencer, T.; et al.
Functional amino acids in swine nutrition and production. Dyn. Anim. Nutr. 2010, 69, 98.
58. Bauchart-Thevret, C.; Cottrell, J.; Stoll, B.; Burrin, D.G. First-pass splanchnic metabolism of dietary cysteine in weanling pigs. J.
Anim. Sci. 2011, 89, 4093–4099. [CrossRef]
59. Escobar, J.; Frank, J.W.; Suryawan, A.; Nguyen, H.V.; Kimball, S.R.; Jefferson, L.S.; Davis, T.A. Physiological rise in plasma
leucine stimulates muscle protein synthesis in neonatal pigs by enhancing translation initiation factor activation. Am. J. Physiol.
Endocrinol. Metab. 2005, 288, E914–E921. [CrossRef]
60. Le Floc’h, N.; Gondret, F.; Matte, J.J.; Quesnel, H. Towards amino acid recommendations for specific physiological and patho-
physiological states in pigs. Proc. Nutr. Soc. 2012, 71, 425–432. [CrossRef]
61. Wiltafsky, M.K.; Pfaffl, M.W.; Roth, F.X. The effects of branched-chain amino acid interactions on growth performance, blood
metabolites, enzyme kinetics and transcriptomics in weaned pigs. Br. J. Nutr. 2010, 103, 964–976. [CrossRef]
Vet. Sci. 2023, 10, 298 12 of 12

62. Mason, G.F.; Gruetter, R.; Rothman, D.L.; Behar, K.L.; Shulman, R.G.; Novotny, E.J. Simultaneous Determination of the Rates of
the TCA Cycle, Glucose Utilization, α-Ketoglutarate/Glutamate Exchange, and Glutamine Synthesis in Human Brain by NMR. J.
Cereb. Blood Flow Metab. 1995, 15, 12–25. [CrossRef] [PubMed]
63. Wu, G.; Knabe, D.A.; Yan, W.; Flynn, N.E. Glutamine and glucose metabolism in enterocytes of the neonatal pig. Am. J. Physiol.
1995, 268, R334–R342. [CrossRef]
64. Pardo, B.; Rodrigues, T.; Contreras, L.; Garzón, M.; Llorente-Folch, I.; Kobayashi, K.; Saheki, T.; Cerdan, S.; Satrústegui, J. Brain
glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation. J. Cereb. Blood Flow Metab.
2011, 31, 90–101. [CrossRef]
65. Wang, W.; Wu, Z.; Lin, G.; Hu, S.; Wang, B.; Dai, Z.; Wu, G. Glycine stimulates protein synthesis and inhibits oxidative stress in
pig small intestinal epithelial cells. J. Nutr. 2014, 144, 1540–1548. [CrossRef] [PubMed]
66. Flynn, N.E.; Knabe, D.A.; Mallick, B.K.; Wu, G. Postnatal changes of plasma amino acids in suckling pigs. J. Anim. Sci. 2000, 78,
2369–2375. [CrossRef] [PubMed]
67. Nie, C.; He, T.; Zhang, W.; Zhang, G.; Ma, X. Branched Chain Amino Acids: Beyond Nutrition Metabolism. Int. J. Mol. Sci. 2018,
19, 954. [CrossRef] [PubMed]
68. Tsabouri, S.; Douros, K.N.; Priftis, K. Cow’s milk allergenicity. Endocr. Metab. Immune Disord.-Drug Targets (Former. Curr. Drug
Targets-Immune Endocr. Metab. Disord.) 2014, 14, 16–26. [CrossRef]
69. Maines, E.; Gugelmo, G.; Tadiotto, E.; Pietrobelli, A.; Campostrini, N.; Pasini, A.; Ion-Popa, F.; Vincenzi, M.; Teofoli, F.; Camilot,
M.; et al. High-protein goat’s milk diet identified through newborn screening: Clinical warning of a potentially dangerous dietetic
practice. Public Health Nutr. 2017, 20, 2806–2809. [CrossRef] [PubMed]
70. Turck, D. Cow’s milk and goat’s milk. World Rev. Nutr. Diet. 2013, 108, 56–62. [PubMed]
71. Verduci, E.; D’elios, S.; Cerrato, L.; Comberiati, P. Cow’s Milk Substitutes for Children: Nutritional Aspects of Milk from Different
Mammalian Species, Special Formula and Plant-Based Beverages. Nutrients 2019, 11, 1739. [CrossRef]
72. Prosser, C.G. Compositional and functional characteristics of goat milk and relevance as a base for infant formula. J. Food Sci.
2021, 86, 257–265. [CrossRef]
73. Brenes-Soto, A.; Dierenfeld, E.S.; Bosch, G.; Hendriks, W.H.; Janssens, G.P. Gaining insights in the nutritional metabolism of
amphibians: Analyzing body nutrient profiles of the African clawed frog, Xenopus laevis. PeerJ 2019, 7, e7365. [CrossRef]
74. Van Riet, M.M.; Millet, S.; Langendries, K.C.; Van Zelst, B.D.; Janssens, G.P.; Nutrition, A. Association between methylation
potential and nutrient metabolism throughout the reproductive cycle of sows. J. Anim. Physiol. Anim. Nutr. 2019, 103, 858–867.
[CrossRef]
75. Okai, D.B.; Aherne, F.X.; Hardin, R.T. Effects of creep and starter composition on feed intake and performance of young pigs. Can.
J. Anim. Sci. 1976, 56, 573–586. [CrossRef]
76. Hurley, W. Composition of sow colostrum and milk. Gestating Lact. Sow 2015, 193–230.
77. Heo, P.S.; Kim, D.H.; Jang, J.C.; Hong, J.S.; Kim, Y.Y. Effects of different creep feed types on pre-weaning and post-weaning
performance and gut development. Asian-Australas. J. Anim. Sci. 2018, 31, 1956–1962. [CrossRef] [PubMed]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.