Jou nal o Food Composi ion and Analysis 123 (2023) 105629
A ailable online 24 Augus 2023
0889-1575/© 2023 The Au ho (s). Published by Else ie Inc. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
Changes du ing lac a ion in he mine al elemen con en o ma e milk
p oduced in semi-ex ensi e u al a ms
A. Blanco-Do al , L.J.R. Ba on , N. Aldai
*
Lac ike Resea ch G oup, Depa men o Pha macy and Food Sciences, Uni e si y o he Basque Coun y (UPV/EHU), Paseo de la Uni e sidad 7, 01006 Vi o ia-Gas eiz,
Spain
ARTICLE INFO
Keywo ds:
Mac omine als
T ace elemen s
Equid milk
Basque moun ain ho se b eed
G azing
Lac a ion s age
ABSTRACT
This s udy cha ac e izes, o he i s ime, he con en o indi idual mine al elemen s in ma e milk om Basque
Moun ain Ho se b eed, as well as changes ha occu du ing lac a ion and among semi-ex ensi e u al a ms.
Indi idual milk samples om eigh een ma es belonging o h ee comme cial a ms we e collec ed along six
mon hs o lac a ion, and he con en s o ele en mine al elemen s in milk we e de e mined using a iple
quad upole induc i ely coupled plasma mass spec ome e (ICP-MS). Basque Moun ain Ho se milk con ained
calcium (Ca; 958 ±248
μ
g/g milk), po assium (K; 581 ±117
μ
g/g milk), phospho us (P; 454 ±160
μ
g/g milk),
sodium (Na; 141 ±38
μ
g/g milk), magnesium (Mg; 64.1 ±15.7
μ
g/g milk), zinc (Zn; 2.50 ±0.88
μ
g/g milk) and
coppe (Cu; 0.343 ±0.179
μ
g/g milk) amoun s simila o hose epo ed in o he ho se b eeds. Con e sely, low
sulphu (S; 156 ±42
μ
g/g milk) and chlo ine (Cl; 49.3 ±33.4
μ
g/g milk), and high i on (Fe; 2.43 ±2.44
μ
g/g
milk) con en s we e ound. Mn was only quan i iable in 12% o he samples. The con en o all mac omine als,
excep Na and Cl, dec eased a he end o lac a ion, while ha o ace elemen s (Zn, Cu and Fe) ei he inc eased
o luc ua ed. In addi ion, he con en o Ca, P, S and Na di e ed signi ican ly among semi-ex ensi e u al a ms,
p obably due o di e ences in eeding managemen . F om a mul i a ia e pe spec i e, he mine al elemen
composi ion o he milk made i possible o di e en ia e samples om ea ly, mid and la e lac a ion s age.
1. In oduc ion
Mine als a e ino ganic compounds ha play a key ole in he unc-
ioning o human o ganism. Calcium (Ca), chlo ine (Cl), cobal (Co),
coppe (Cu), iodine (I), i on (Fe), magnesium (Mg), manganese (Mn),
molybdenum (Mo), phospho ous (P), po assium (K), selenium (Se), so-
dium (Na), and zinc (Zn) a e known o be essen ial o human physio-
logical unc ions (Hun and Nielsen, 2009); besides, sulphu (S) is
de imen al o he syn hesis o se e al me abolic in e media es (Nimni
e al., 2007). O e all, mine al elemen s con o m s uc u al issues and
ake pa in nume ous me abolic eac ions, so de iciencies can lead o
se ious heal h diso de s. Thei bioa ailabili y depends on many ac o s,
and e en wi h an adequa e consump ion o mine als, de iciencies may
appea due o an ine icien abso p ion (Eu opean Food Sa e y Au ho -
i y, 2017).
Mammalian milk, pa icula ly cow milk, and dai y p oduc s, a e he
main ood sou ce o bioa ailable Ca, a good sou ce o P, and a mode a e
sou ce o K, Na, Mg and Cl. Con e sely, dai y p oduc s a e poo in ace
elemen s such as Fe, Cu, Mn and Zn. Despi e he low concen a ion o Zn
in milk and dai y p oduc s, hese a e one o he main sou ces o his ace
elemen o child en (Fan uz e al., 2016; Hun and Nielsen, 2009).
Signi ican di e ences in he concen a ions o mine al elemen s ha e
been epo ed among mammalian species (Claeys e al., 2014). Mo e-
o e , he bioa ailabili y o mine al elemen s a ies acco ding o hei
dis ibu ion among milk ac ions as well as he p esence o o he
compounds such as asco ba e, ci a e, lac ose, o casein-
ophosphopep ides (Bouhallab and Bougl´
e, 2004; Fan uz e al., 2016;
Zambe lin e al., 2012).
The s udies ca ied ou o da e on di e en b eeds showed ha equid
milk is chemically di e en o ha om o he mammalian species, bu
mo e simila o human milk. Compa ed o uminan milk, ma e milk
showed lowe a , p o ein, ash and d y ma e con en s (0.3–4%, 1–3%,
0.3–0.5% and 9–12%, espec i ely), bu i was iche in lac ose (6–7%)
(Claeys e al., 2014; Uniacke-Lowe e al., 2010). Despi e i s lowe
mac onu ien con en , some au ho s ha e epo ed ha ma e milk is
ich in o he nu i ionally signi ican and bioac i e compounds which
may be esponsible o di e en unc ional p ope ies (Do eau and
Ma in-Rosse , 2002; Sheng & Fang, 2009), ye knowledge on his opic
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (N. Aldai).
Con en s lis s a ailable a ScienceDi ec
Jou nal o Food Composi ion and Analysis
jou nal homepage: www.else ie .com/loca e/j ca
h ps://doi.o g/10.1016/j.j ca.2023.105629
Recei ed 22 June 2023; Recei ed in e ised o m 31 July 2023; Accep ed 20 Augus 2023
Jou nal o Food Composi ion and Analysis 123 (2023) 105629
2
emains sca ce. In essence, conside ing he essen ial ac ion o mine al
elemen s in human, hese (o some o hese) could con ibu e o ma e
milk unc ionali y. Howe e , e y limi ed da a is a ailable conce ning
ma e milk, e y especially abou he con en o some o hem such as S,
Fe, Mn and Cl. P e ious s udies ha e epo ed ha , in gene al, he
con en o mine al elemen s in ma e milk was no signi ican ly a ec ed
by he die a y in ake o hese, wi h excep ion o Se and I (Fan uz e al.,
2016; Hun and Nielsen, 2009). On he con a y, lac a ion s age has
shown o a ec he mine al elemen con en o ma e milk, bu only a ew
scien i ic wo ks ha e moni o ed he lac a ion pe iod (G ace e al., 1999;
Sch y e e al., 1986b; Smolde s e al., 1990; Summe e al., 2004; Ull ey
e al., 1966, 1974). To he bes o ou knowledge, only h ee s udies we e
pe o med unde g azing condi ions (G ace e al., 1999; Ull ey e al.,
1966, 1974). Explo ing changes in he chemical composi ion o milk
along lac a ion – and po en ial in e ac ions wi h animal eeding – could
lead o a be e unde s anding o he ac o s ha a ec equine milk
quali y while p o iding ools o op imising he p oduc ion.
The e o e, he aim o his s udy was o cha ac e ize he composi ion
o mac omine als and ace elemen s, and hei concen a ion changes
o e six mon hs o lac a ion in ma e milk om Basque Moun ain Ho se
b eed ea ed in semi-ex ensi e u al a ms. In his espec , i should be
no ed ha hese ma es had ne e been milked be o e. In addi ion, i was
also in es iga ed whe he di e ences in animal managemen among
a ms had any impac on he mine al elemen con en o ma e milk.
Ma e milk om his b eed has ne e been analysed om a scien i ic
pe spec i e be o e and, conside ing ha di e en ho se b eeds ha e
shown a ia ions in milk mine al elemen composi ion (Sheng and Fang,
2009), he cha ac e isa ion o milk om Basque Moun ain Ho se is o
g ea in e es .
2. Ma e ials and me hods
2.1. Expe imen al design and milk sampling
Eigh een lac a ing ma es om h ee di e en comme cial equine
a ms (six ma es pe a m; om now on, a m I, a m II and a m III)
loca ed in he same egion o Spain (A aba) we e milked. All ma es
belonged o Basque Moun ain Ho se b eed, and a e aged ( ±s anda d
de ia ion) 9.53 ±3.76 yea s old and 5.68 ±3.56 pa u i ions. Ma es
we e milked only in he sampling days. Pa u i ions we e no synch o-
nized, and happened wi hin a ange o 39 days om la e Ma ch o ea ly
May 2021; he e o e, lac a ion ime o each ma e was es ablished ac-
co ding o pa u i ion day. Indi idual milk samples we e collec ed
du ing he lac a ion pe iod, which was om May 4 h un il Oc obe 13 h,
2021. Du ing he i s hal o he lac a ion (un il end July), samples we e
collec ed e e y 7 days, whe eas du ing he second hal ( om ea ly
Augus o mid-Oc obe ), collec ions happened e e y 14 days. In o al,
304 milk samples we e ob ained ( a m I, n =75; a m II, n =125; a m
III, n =104).
Table 1 shows he eeding managemen ollowed by he h ee com-
me cial a ms du ing he lac a ion pe iod. In a m I, ma es g azed on
pas u es only du ing May and hey we e ed wi h a mix u e (15–20 kg
pe ma e) o al al a, silage, hay, ui s and po a oes a e wa ds. Fa ms II
and III kep ma es on pas u e all along he lac a ion pe iod, and om he
beginning o July, onwa ds, when g ass a ailabili y was sca ce, a m II
supplemen ed ma es wi h hay (4–5 kg pe ma e) and a m III wi h g ass
and al al a silage (9–10 kg pe ma e). All he h ee a ms p o ided a
mix u e (2–3 kg pe ma e) o ce eals and al al a o pea in he milking
pa lou in o de o ease and ge he animals accus omed o he milking
p ocess. Fa m I pe o med his accus oming managemen only a he
sampling days, whe eas a ms II and III go he ma es o he milking
pa lou and epea ed he p ocess e e y day du ing he lac a ion pe iod.
All a ms supplemen ed ma e eeding wi h sal blocks.
Ma es we e milked as comple ely as possible using ei he au oma ic
o manual milking. Fa m I used a po able milking machine (model
Cana ias-1, Ul amilk, Socu´
ellanos, Spain) i ed wi h wo sheep ea -
cups, whe eas a ms II and III used manual milking. In he h ee a ms,
milking happened in he p esence o oal a e suckling dep i a ion o
2–3 h. A e milk sampling a he a m, hese we e anspo ed in
po able coole s o he labo a o y and subsequen ly subsampled and
ozen a −80 ºC un il analysis. In o de o a oid deg ada ion o com-
pounds, milk samples we e hawed a 25 ºC o 60 min in a wa e ba h
o he analysis o di e en compounds, including mine al elemen s in
his s udy.
2.2. Chemicals
Ni ic acid (HNO
3
; 68–70%, / ) was pu chased om Fishe
Chemical (Mad id, Spain) and edis illed in he labo a o y o pu i ica-
ion; hyd ogen pe oxide (H
2
O
2
; 30–32%, / ) was Op ima™ g ade and
pu chased om Fishe Chemical; CMS-5 s anda d solu ion (in HNO
3
; 5%
/ ) con aining 10 µg/mL each o he mine al elemen s Ca, Mg, Na, K,
Fe, Cu, Zn and Mn was pu chased om Ino ganic Ven u es (Ch is-
iansbu g, USA); s anda d solu ions (1000 µg/mL in wa e ) o S, P and
hodium (Rh) we e pu chased om SCP Science (Quebec, Canada); and
ul apu e (99.9999%) oxygen, hyd ogen and helium we e pu chased by
Ai Liquide (Mad id, Spain). Millipo e Milli-Q g ade wa e (18.2 MΩ/
cm) was used o dilu ions.
2.3. Sample p epa a ion and mine al elemen analysis
Fo diges ion o milk samples, 5 mL o 65% ( / ) HNO
3
and 1 mL
H
2
O
2
we e added o 0.20 ±0.01 mg o liquid milk, and he mix u e was
submi ed o a Speedwa e- ou mic owa e assis ed diges e (Be gho
P oduc s +Ins umen s, Be lin, Ge many) a a maximum o 190 ºC and
80% powe . Diges ed samples we e dilu ed in Milli-Q wa e , and
Table 1
Feeding managemen used by each comme cial a m du ing he lac a ion pe iod o ma es ( om May o Oc obe ).
G azing egime Supplemen s*
Fa m Pas u e ype Quan i y Time
pe iod
Meadow
size
Type Quan i y (pe ma e) Time pe iod
I Ve ch, yeg ass, oa Ad
libi um
May 2–3 ha Al al a (20%), silage (20%), hay
(20%), ui s (20%), po a o (20%)
Daily a ion o 15–20
kg o mix u e
June-Oc obe
Al al a, oa and co n 2–3 kg o mix u e Du ing he
milking p ocess
II Al al a, clo e , yeg ass, Fes uca
spp., o cha d g ass, dandelion
Ad
libi um
May-
Oc obe
28 ha Hay Daily a ion o 4–5 kg July-Oc obe
Oa (60%), whea (20%) and pea
(20%)
2–3 kg o mix u e Du ing he
milking p ocess
III Al al a, clo e , yeg ass, Fes uca
spp., o cha d g ass, dandelion
Ad
libi um
May-
Oc obe
28 ha G ass and al al a silage Daily a ion o 9–10
kg
July-Oc obe
Oa (60%), whea (20%) and pea
(20%)
2–3 kg o mix u e Du ing he
milking p ocess
*
Pe cen ages and weigh s o eeding ing edien s a e app oxima e.
A. Blanco-Do al e al.
Jou nal o Food Composi ion and Analysis 123 (2023) 105629
3
analysed in an 8900 iple quad upole induc i ely coupled plasma mass
spec ome e (ICP-MS; Agilen , San a Cla a, USA). The ICP-MS sys em
was equipped wi h a Mic oMis concen ic nebulize and a Fassel- ype
o ch wi h an in e nal diame e o 2.5 mm. Plasma was gene a ed
wi h a gon gas as ollows: 1550 W adio equency powe , 1.2 V adio
equency ma ching, 15 L/min plasma gas low, 1.05 L/min nebulize
gas (a gon) low, 0.9 L/min auxilia y gas (a gon) low. An MS/MS scan
was used o P and S wi h oxygen as cell gas ( low 30%), and o Ca wi h
hyd ogen as cell gas ( low 4.7 mL/min). Fo he es o he elemen s, a
single Quad scan was pe o med using helium a a low- a e o 4.6 mL/
min. Rh was used as in e nal s anda d, and o mine al elemen quan-
i ica ion, Na, Mg, K, Ca, P, S, Mn, Fe, Cu and Zn s anda d solu ions we e
analysed by iplica e in a ange g ea e han 0.05 and less han 2400
ng/mL. These concen a ions we e p epa ed om comme cial s anda d
solu ions using 1% ( / ) HNO
3
in Milli-Q g ade wa e . Table 2 shows he
analy ical pa ame e s e alua ed o me hod alida ion. Linea i y was
assessed by calcula ing he de e mina ion coe icien (R
2
) o he cali-
b a ion cu es adop ing be ween i e and eigh calib a ion le els
depending on each mine al elemen . P ecision was es ima ed as he
pe cen ela i e s anda d de ia ion (RSD) o six eplica es o s anda d
solu ions a he lowes and highes concen a ion o each mine al
elemen (Table 2). In he same way, accu acy was e alua ed in e ms o
eco e y by analysing six eplica es o mine al elemen s anda d solu-
ions a hese concen a ions. Limi s o quan i ica ion (LOQ) we e
de e mined, o each elemen , by analysing en eplica es o he lowes
concen a ion o he mine al elemen s anda d solu ion ha ob ained an
RSD alue less han 20%. LOQ alues we e exp essed as µg/g e e ing o
he we weigh o he milk sample, conside ing he dilu ion s ep
occu ed in he sample p epa a ion. Da a in Table 2 showed a high
linea i y wi h R
2
- alues highe han o equal o 0.9940 o all he min-
e al elemen s s udied, as well as a good p ecision wi h RSD alues less
han 8.50% excep o S a high concen a ion. Reco e y alues (accu-
acy) anged be ween 95.39% and 105.13% o all mine al elemen and
concen a ion, which was an accep able alue o mac omine al and
ace elemen quan i ica ion. Al hough LOQ alues o mac omine al
elemen s we e p o ided in ma e milk, hese elemen s we e a abo e
hei LOQ alues. On he o he hand, hose limi s we e o in e es o
hose ace elemen s anging be ween 0.650 and 0.044 µg/g milk ( o Fe
and Mn, espec i ely).
The amoun o Cl in he milk samples was es ima ed by using he
semi-quan i a i e mode o ICP-MS. This me hod is based on p ede ined
de aul alues, and he app oxima e concen a ion o he mine al
elemen is calcula ed om he coun s pe second measu ed by ICP-MS
(K zciuk e al., 2016).
2.4. S a is ical analysis
So wa e e sion 28.0 o IBM-SPSS S a is ics (IBM, New Yo k, USA)
was used o pe o m s a is ical da a ea men . Th ee signi ican igu es
we e used o exp ess he mine al elemen con en o he milk samples.
Mine al elemen con en da a we e log ans o med, and no mali y,
homoscedas ici y and esidual andomness we e checked. Ou lie s
( alues highe o lowe han 3 imes he in e qua ile ange) checked by
box-plo s we e excluded om he da a se . In o de o make mo e
comp ehensible he s udy o he changes in he mine al elemen con en
o ma e milk samples du ing lac a ion, he da a was pooled in se e al
lac a ion anges o 14 days wi h he ea lies lac a ion ange co e-
sponding o weeks 3 and 4, and he las ange o weeks 25 and 26. Da a
(excluding Mn) we e analysed using he Linea Mixed Model (LMM)
p ocedu e o analysis o a iance including indi idual animal as subjec ,
a m as ixed ac o and lac a ion ime as epea ed measu e ac o . The
in e ac ion e m be ween a m and lac a ion ime was also included in
he model. The pa ame e s o he LMM we e es ima ed using he
Res ic ed Maximum Likelihood Me hod and he compound symme y
ma ix was selec ed o he epea ed measu es co a iance s uc u e
ollowing he Akaike in o ma ion c i e ion. Leas squa e means o
dependen a iables o he le els o he ixed ac o ( a m) we e
compa ed using he Tukey’s es . In addi ion, a S epwise Disc iminan
Analysis (SDA) was applied o he mine al elemen composi ion
(excluding Mn and Cl con en s) o he milk samples om he comme cial
a ms o classi y milks acco ding o ma e lac a ion s age. Fo his pu -
pose, milk samples we e labelled as ea ly lac a ion ( om week 3–10),
mid lac a ion ( om week 11 o week 18) o la e lac a ion ( om week
19–26). Signi icance le el was decla ed a P ≤0.05.
3. Resul s
In ma e milk samples analysed om all comme cial a ms o e
lac a ion, Ca, K and P we e he majo mine al elemen s wi h an a e age (
±s anda d de ia ion) con en o 958 ±248, 581 ±117 and 454 ±160
μ
g/g milk, espec i ely, and an a e age Ca:P a io o 2.26 ±0.60.
Among o he mac omine als, an a e age o 156 ±42
μ
g S, 141 ±38
μ
g
Na, 64.1 ±15.7
μ
g Mg and 49.3 ±33.4
μ
g Cl pe g o milk we e ound
(Table 3). Excep Mn, he ace elemen s we e ound abo e he LOQ in
mos milk samples collec ed du ing he lac a ion pe iod. The a e age
con en o ace elemen s was o 2.50 ±0.88
μ
g Zn, 2.43 ±2.44
μ
g Fe
and 0.343 ±0.179
μ
g Cu pe g o milk. Mn was only p esen abo e he
LOQ in 35 ou o he 304 samples analysed, wi h an a e age o 0.118 ±
0.230
μ
g/g milk. Milk samples con aining quan i iable amoun s o Mn
we e om di e en animals and we e andomly dis ibu ed along
lac a ion and among a ms; he e o e, no u he s a is ical da a ea -
men was pe o med o Mn.
Table 3 shows he con en o all he mac omine als and ace ele-
men s by a m, as well as he e ec o he ac o s lac a ion ime and
a m. Fa m e ec was signi ican (P ≤0.05) o Ca, P, S and Na con en s
in ma e milk samples. Fo hese mine als, milk samples om a m I
(ma es g azing only du ing he i s mon h o lac a ion) con ained
highe amoun s han hose om a m II and III (ma es g azing along
lac a ion and supplemen ed a e July wi h hay o silage, espec i ely).
On he o he hand, lac a ion ime signi ican ly (P ≤0.05) in luenced he
Table 2
Valida ion pa ame e s o he ICP-MS analy ical me hod used o quan i y mac omine als and ace elemen s in ma e milk samples.
RSD (%) Reco e y (%)
Mine al elemen Calib a ion ange (ng/mL) R
2
LOQ (µg/g milk) LConc HConc LConc HConc
Ca 43.00 – 2147.00 0.9999 26.875 2.12 0.11 95.76 101.86
K 12.36 – 2074.91 0.9999 7.725 2.89 3.86 99.29 98.82
P 1.54 – 2336.11 1.0000 0.963 2.86 7.79 103.66 104.84
S 1.40 – 2074.91 0.9999 0.875 11.20 8.29 103.11 97.42
Na 12.36 – 746.49 0.9940 7.725 3.83 5.70 103.37 104.51
Mg 0.37 – 372.70 0.9998 0.231 5.46 6.05 95.39 98.65
Zn 0.70 – 372.70 0.9999 0.438 7.73 4.85 103.01 99.22
Fe 1.04 – 372.70 0.9998 0.650 3.01 4.00 105.13 100.40
Cu 0.13 – 175.14 0.9999 0.081 5.88 5.81 98.57 100.93
Mn 0.07 – 57.80 1.0000 0.044 1.53 5.43 97.25 102.37
RSD, ela i e s anda d de ia ion; R2, de e mina ion coe icien ; LOQ, limi o quan i ica ion; LConc; lowes concen a ion; HConc, highes concen a ion.
A. Blanco-Do al e al.
Jou nal o Food Composi ion and Analysis 123 (2023) 105629
4
concen a ions o all he mine als and he Ca:P a io. Figs. 1 and 2 depic
he changes in he mine al elemen con en o milk samples om each
a m. Con en s o Ca, K, P, S and Mg showed a downwa d end
h oughou lac a ion (Fig. 1). Con en s o Mg and K s eadily dec eased
du ing lac a ion. Mo eo e , he e olu ion o Mg con en di e ed among
a ms (signi ican in e ac ion, Table 3): in a m I and a m III, a e age
Mg con en con inuously dec eased excep o one ime poin in a m I
(weeks 17–18), whe eas he dec ease in a m II was discon inuous
(Fig. 1). The e olu ion o Ca, P and S con en s in milk samples along
lac a ion ollowed simila pa e ns in all a ms, wi h a sligh educ ion
du ing he i s weeks o lac a ion, peaking a mid-lac a ion (Ca: a
weeks 15–16; P and S: a weeks 13–14), and no iceably dec easing a -
e wa ds. In a m I, he peaking ime o Ca and S appea s one o wo
weeks delayed. On he o he hand, Na con en s sligh ly inc eased
(2.77% on a e age om weeks 3–4 o weeks 25–26) and Cl con en s
luc ua ed conside ably in all a ms du ing lac a ion (Fig. 1). Howe e ,
Na con en changed di e en ly in milk om he di e en a ms wi h
signi ican (P ≤0.05) in e ac ion be ween a m and lac a ion ime
(Table 3). In ac , Na con en inc eased du ing he second hal o
lac a ion in a ms I and III, emaining mo e cons an in a m II.
The con en o he ace elemen s ei he inc eased (Cu and Zn) o
luc ua ed (Fe) along he lac a ion pe iod (Fig. 2). Bo h Cu and Zn
con en s emained mo e o less cons an un il hey inc eased om weeks
9–12 un il weeks 15–20, wi h a la e dec easing end. The con en o Fe
did no show a clea pa e n du ing lac a ion apa om inal con en s
being o e all lowe han ini ial ones. An in e ac ion be ween a m and
lac a ion s age was seen in Fe concen a ion as well (Table 3), since he
e olu ion along lac a ion appea s one o wo weeks delayed in a m III
compa ed o a ms I and II. On a e age, Ca:P a io dec eased om 2.22
o 1.68 du ing he i s hal o lac a ion, and hen inc eased o he
second hal , achie ing alues as high as 2.78 (Fig. 2).
4. Discussion
Acco ding o he esul s ob ained (Table 3), he majo mine al
elemen in Basque Moun ain Ho se b eed milk was Ca, ollowed by K
and P in app oxima ely hal he amoun o Ca (as con i med by he Ca:P
a io). A e age mac omine al con en ound in his s udy alls wi hin he
ange epo ed in li e a u e o o he equine b eeds: 261–1380
μ
g Ca,
341–701
μ
g K, 152–884
μ
g P, 107–220
μ
g Na, and 29–104
μ
g Mg pe g
o mL aw milk (Alipou e al., 2023; Ande son, 1991; Bilandˇ
zi´
c e al.,
2014; Csap´
o-Kiss e al., 1995; G ace e al., 1999; Sch y e e al., 1986a,
b; Smolde s e al., 1990; Summe e al., 2004; Ull ey e al., 1966) o
ma e milk samples collec ed a 14 o mo e days o lac a ion. The con en
o S was on a e age lowe in Basque Moun ain Ho se b eed milk han in
pas u e- ed Tho oughb ed b eed milk epo ed by G ace e al. (1999),
who ound 196–230
μ
g S/mL milk. This could be a consequence o lowe
p o ein con en ound in Basque Moun ain Ho se b eed milk samples in
he p esen s udy (1.84 s. 2.10 g p o ein/100 g milk, on a e age). In
ac , p o eins, pa icula ly he sulphu con aining amino acids me hio-
nine and cys eine, a e he main sou ce o die a y S (Nimni e al., 2007).
In addi ion, Cl con en in he milk samples o he p esen s udy was 3–4
imes lowe han ha epo ed by Summe e al. (2004) in Ha linge
b eed milk (150–198
μ
g Cl/g milk). Changes in he chlo ine con en in
bo ine and human milks a e known o be mainly due o pa hological
p ocesses o mic obial con amina ion, as well as o lac a ion s age, bu
no o die a y ac o s (Chen e al., 2018; Eu opean Food Sa e y Au-
ho i y, 2019; Gauche on, 2005). Howe e , being in ol ed – oge he
wi h lac ose, Na and K – in he balancing o he osmo ic p essu e o milk,
Cl con en has been also co ela ed wi h he con en o lac ose and/o Na
in bo ine milk (Bijl e al., 2013).
Rega ding ace elemen s, Zn and Cu con en s (Table 3) we e in
acco dance o hose ound in he li e a u e (1.1–4.1
μ
g Zn and
0.07–0.64
μ
g Cu pe g o mL milk), whe eas Fe con en s in milk samples
om Basque Moun ain Ho se b eed we e highe han hose epo ed in
o he equine b eeds (0.18–1.30
μ
g/g o mL) (Alipou e al., 2023;
Ande son, 1992; Bilandˇ
zi´
c e al., 2014; Csap´
o-Kiss e al., 1995; G ace
e al., 1999; Sch y e e al., 1986a, b; Ull ey e al., 1974). Repo s on Mn
concen a ion a y om less han 0.01
μ
g/g milk up o 0.05
μ
g/g milk
(Alipou e al., 2023; Ande son, 1992; Bilandˇ
zi´
c e al., 2014; Csap´
o-Kiss
e al., 1995). In he p esen wo k, Mn con en s lowe han 0.0438
μ
g/g
milk we e below he LOQ es ablished in he ICP-MS me hodology
Table 3
A e age concen a ion (
μ
g/g milk) and ange (minimum-maximum) o mac omine als and ace elemen s in ma e milk samples om he comme cial a ms o e he
lac a ion pe iod ( om May o Oc obe ), and s a is ical signi icance (P- alue) o he p incipal ac o s ( a m and lac a ion) and hei in e ac ion. The o al numbe o milk
samples collec ed in comme cial a ms we e 75, 125 and 104 o a m I, II and III, espec i ely.
Fa m P- alue
Mine alelemen I n II n III n SEM Fa m Lac a ion Fa m * Lac a ion
Ca 104⋅10
1 a
75 928
b
125 936
a,b
104 18 0.025 <0.001 0.857
(654–169⋅10
1
) (504–153⋅10
1
) (503–146⋅10
1
)
K 582 75 560 125 604 104 9 0.461 <0.001 0.104
(448–856) (385–755) (414–103⋅10
1
)
P 502
a
75 431
b
125 447
a,b
104 12 0.013 <0.001 0.471
(201–837) (193–781) (202–806)
S 165
a
75 153
b
125 154
a,b
104 3 0.015 <0.001 0.493
(96.7–287) (86.6–259) (90.1–253)
Na 179
a
75 134
b
125 123
b
104 4 <0.001 <0.001 0.005
(111–304) (89.6–180) (69.0–198)
Mg 65.4 75 61.6 125 66.2 104 1.1 0.493 <0.001 0.013
(38.2–118) (29.2–114) (41.5–103)
Cl 41.2 75 52.3 125 51.5 104 2.4 0.193 <0.001 0.895
(16.5–143) (18.1–202) (19.6–262)
Zn 2.26 75 2.67 125 2.47 103 0.06 0.061 <0.001 0.491
(1.15–4.06) (1.18–7.43) (1.21–5.13)
Fe 2.75 73 2.31 118 2.32 100 0.18 0.427 0.006 0.035
(0.709–24.5) (0.707–8.97) (0.651–18.0)
Cu 0.329 70 0.327 121 0.372 98 0.013 0.056 <0.001 0.080
(0.120–1.06) (0.101–1.03) (0.134–0.956)
Mn 0.0665 5 0.0735 20 0.200 15 0.040
(0.0477–0.0903) (0.0450–0.127) (0.0441–1.39)
Ca:P 2.24 – 2.30 — 2.24 — 0.04 0.794 <0.001 0.707
(1.29–3.54) (1.14–4.45) (1.00–3.55)
a,b
Di e en le e supe sc ip s indica e signi ican di e ences (P ≤0.05) among a ms.
n, numbe o milk samples om each a m in which mine al elemen s we e ound o be abo e he limi o quan i ica ion (see Table 2); SEM, s anda d e o o he mean.
A. Blanco-Do al e al.
Jou nal o Food Composi ion and Analysis 123 (2023) 105629
5
1.5
1.6
1.7
1.8
1.9
2.0
2.1
2.2
Log (μg/g milk)
Week o lac a ion
Magnesium (Mg)
2.5
2.6
2.7
2.8
2.9
3.0
Log (μg/g milk)
Week o lac a ion
Po assium (K)
2.6
2.7
2.8
2.9
3.0
3.1
3.2
3.3
(goL μ)klimg/g
Week o lac a ion
Calcium (Ca)
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3.0
(goL μ)klimg/g
Week o lac a ion
Phospho us (P)
1.8
1.9
2.0
2.1
2.2
2.3
2.4
2.5
2.6
Log (μg/g milk)
Week o lac a ion
Sulphu (S)
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
(goL μ)klimg/g
Week o lac a ion
Cho ine (Cl)
1.8
1.9
2.0
2.1
2.2
2.3
2.4
2.5
2.6
2.7
(goL μ)klimg/g
Week o lac a ion
Sodium (Na)
Fig. 1. Changes in mac omine al con en o ma e milk samples om semi-ex ensi e u al a ms du ing lac a ion. , a m I; , a m II; , a m III.
A. Blanco-Do al e al.
Jou nal o Food Composi ion and Analysis 123 (2023) 105629
6
(Table 2). In consequence, compa ison wi h li e a u e da a was di icul .
Anyway, bo h human and animal milks ha e been conside ed a poo
sou ce o Mn (Fan uz e al., 2016).
Resul s (Table 3, Figs. 1 and 2) con i med he signi ican (P≤0.05)
e ec o lac a ion ime on he con en o ma e milk mine al elemen s
p e iously epo ed by o he au ho s (G ace e al., 1999; Summe e al.,
2004). Acco ding o li e a u e, Ca, P, Mg, Na, K, Cu, Fe and Zn con en s
dec ease om week 3 o mid- (Sch y e e al., 1986b; Smolde s e al.,
1990; Ull ey e al., 1966, 1974) o la e-lac a ion (Ma uzzi e al., 2004;
Summe e al., 2004). Con e sely, G ace e al. (1999) did no ind sig-
ni ican di e ences in S, Cu and Fe con en s a e he 3 d week o
lac a ion. Highe mine al elemen con en s wi hin he ini ial s ages o
lac a ion a e in e sely ela ed o lac ose con en (which inc eases
h oughou lac a ion; Fan uz e al., 2016; Uniacke-Lowe e al., 2010).
This nega i e co ela ion be ween mine al elemen s and lac ose con-
en s has also been obse ed in milk om o he mammalian species, and
has been desc ibed as esponsible o osmo ic egula ion. In his sense,
mainly Na
+
, K
+
and Cl
-
ions, and Ca and P associa ed o caseins, con ol
milk olume in ea ly lac a ion, and his ole is aken up by lac ose a -
e wa ds (Kobeni e al., 2020; Shennan & Peake , 2000).
O e all, he e olu ion o mine al elemen s o e he lac a ion pe iod
sligh ly di e ed among ma e milk s udies. The mos a iable mac o-
mine als seemed o be Na and K since he con en o bo h luc ua e along
lac a ion (as he Na con en in he p esen s udy; Fig. 1), bu di e en ly
among s udies (G ace e al., 1999; Sch y e e al., 1986b; Summe e al.,
2004). In he case o K con en , p esen esul s esemble hose epo ed
by Ull ey e al. (1966), wi h a mo e consis en dec ease and less luc-
ua ion du ing lac a ion (Fig. 1). Simila o he p esen s udy, Summe
e al. (2004) ound ha Cl con en luc ua ed wi hou speci ic pa e n. In
he p esen esea ch, mine al elemen changes along lac a ion we e, in
gene al, simila o hose ob ained by G ace e al. (1999) in pas u e- ed
ma e milk, wi h highe con en luc ua ions, mo e p ominen de-
c eases a e mid-lac a ion (pa icula ly in Ca and P), and low con en o
Na and Zn ound a ound weeks 7–9, also epo ed by Ull ey e al. (1974)
o Zn. In e es ingly, he peaking o some mine al elemen s in Basque
Moun ain Ho se b eed milk a mid-lac a ion has no been p e iously
desc ibed in o he ma e milk s udies.
F om a mul i a ia e pe spec i e, he esul s o he SDA applied o he
mine al elemen composi ion o ma e milk samples collec ed in com-
me cial a ms made i possible o classi y mo e han 86% o he samples
in o hei lac a ion s age (ea ly-, mid- and la e-lac a ion). The milk
sample dis ibu ion o Fig. 3 displayed he wo canonical disc iminan
unc ions ob ained. As depic ed, he h ee lac a ion s ages we e
conside ably disc imina ed along he unc ion 1 axis, whe eas unc ion 2
mainly con ibu ed o disc imina e be ween mid-lac a ion om he
o he wo lac a ion s ages (ea ly- and la e-). This mul i a ia e app oach
con i med he p e iously men ioned esul s abou he indi idual min-
e al elemen con en changes h oughou lac a ion pe iod. Despi e o
in e - a m a iabili y, p incipally ela ed o eeding di e ences among
semi-ex ensi e u al a ms, he SDA was able o di e en ia e ma e milk
samples om ea ly-, mid- and la e-lac a ion.
The Ca:P a io can a ec bo h Ca and P bioaccessibili y and
bioa ailabili y in humans. This a io anges be ween 1.9 and 2.4 in
human milk, and is gene ally lowe in cow, goa and ewe milk (Bass &
Chan, 2006; Bonjou , 2011), ye he e is high a iabili y among s udies.
The Ca:P a io ob ained in he p esen s udy was, o e all, highe han
ha p e iously epo ed in ma e milk in o he equine b eeds (Alipou
e al., 2023; Sch y e e al., 1986b; Smolde s e al., 1990; Summe e al.,
2004) p obably due o sligh ly highe Ca and sligh ly lowe P concen-
a ions. O he au ho s also ound a e age Ca con en s 1.4–1.9 imes
highe han P con en s in ma e milk (Ande son, 1991; Ma uzzi e al.,
2004; Sch y e e al., 1986a). In acco dance o he p esen s udy (Fig. 2),
li e a u e showed a dec ease in he Ca:P a io om ea ly- o
mid-lac a ion in ma e milk (Sch y e e al., 1986b; Smolde s e al.,
1990; Summe e al., 2004) and a pos e io inc ease a e he 16 h week
(Summe e al., 2004).
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Log (μg/g milk)
Week o lac a ion
I on (Fe)
-1.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
(goL μ)klimg/g
Week o lac a ion
Coppe (Cu)
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
(goL μ)klimg/g
Week o lac a ion
Zinc (Zn)
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Log (Ca:P a io)
Week o lac a ion
Ca:P a io
Fig. 2. Changes in ace elemen con en and Ca:P a io o ma e milk samples om semi-ex ensi e u al a ms du ing lac a ion. , a m I; , a m II; ,
a m III.
A. Blanco-Do al e al.
Jou nal o Food Composi ion and Analysis 123 (2023) 105629
7
O he ac o s (apa om lac a ion) a ec ing ma e milk mine al
elemen composi ion a e poo ly unde s ood and, o ou knowledge,
he e is no p e ious da a analysing he e ec o managemen sys em.
Ac ually, he unde s anding o his e ec migh be ele an when
conside ing ma e milk p oduc ion o human consump ion, speci ically
om a comme cial pe spec i e. Resul s om his s udy showed ha
mine al elemen composi ion o ma e milk coming om di e en semi-
ex ensi e u al a ms was gene ally simila , al hough Ca, P, S and Na
con en s signi ican ly (P≤0.05) a ied (Table 3). These a ia ions
could be ela ed o di e ences associa ed o he managemen p ac ices –
including eeding – ollowed by each a m (Table 1). Excep o Se and I,
i is known ha die a y mine al in ake is unlikely o a ec milk mine al
elemen composi ion (Asai e al., 1995; Fan uz e al., 2016; G ace e al.,
1999; Hun & Nielsen, 2009; Ka azis e al., 2002). Howe e , g azing
could con ibu e o mine al elemen changes in milk. In a s udy by
Soyeu e al. (2022), hey we e able o disc imina e be ween g ass- ed
and non-g ass- ed cows acco ding o p o ein, p edic ed Ca and, in a
lowe ex en , p edic ed P and Mg con en s in milk. Ano he s udy ound
ha p o ein (and casein), Ca, P, Na, Zn, Cu, Mn and Se con en s signi -
ican ly a ied among milks om cows ed concen a es and g azing on
di e en pas u es (Gula i e al., 2018). G azing ac i i y (low, mode a e
o high) also a ec ed p o ein, Ca and P con en s in cow milk (S e giadis
e al., 2021). In his ega d, he esul s o he p esen s udy showed
signi ican ly (P≤0.05) highe con en s o Ca, P and S ( his las one
ela ed o p o eins) in milk samples om low g azing ma es ( a m I) han
in hose samples om ma es ha g azed o a longe ime and we e
supplemen ed wi h hay ( a m II). Signi ican ly highe Na con en s we e
also ound in milk samples om low g azing ( a m I) han in hose om
long- e m g azing ma es ( a ms II and III). Fu he mo e, he peaks o
concen a ion ound in Ca, P and S a mid-lac a ion could be ela ed o
changes in g ass a ailabili y a e July.
O he ac o s such as age o he animal (Alipou e al., 2023; Asai
e al., 1995), numbe o pa u i ions o heal h s a us o he mamma y
gland (Linn, 1988) could also ha e had a signi ican e ec on he con en
o speci ic mine al elemen s in milk, bu scien i ic li e a u e on his opic
emains e y sca ce. Since he p esen s udy was pe o med in com-
me cial a ms, o he managemen p ac ices apa om eeding could no
be con olled. Consequen ly, co ela ion o he a iabili y in milk min-
e al elemen con en among a ms wi h speci ic ac o s was no possible.
As p e iously indica ed, milk is a g ea sou ce o some essen ial el-
emen s desc ibed in he p esen wo k, ye no e e y milk ype con ib-
u es equally o ul il human daily mine al equi emen s. Compa ed o
o he animal species, ma e milk is no he iches milk in e ms o
mine al elemen concen a ions. Despi e his, i s mine al elemen
composi ion is e y simila o ha o donkey (also an equid) and human
milks, con aining, o e all, less mine al elemen s han o he mammalian
species (Claeys e al., 2014; Fan uz e al., 2016; Ga hwal e al., 2023).
On he o he hand, mine al bioa ailabili y o milk di e s among
mammalian species and can be a ec ed by a ious ac o s. The e o e,
no only mine al elemen con en bu also hei bioa ailabili y a ec s
he nu i ional alue o he p oduc , so he con ibu ion o each ype o
milk o he ecommended daily mine al in ake should be ca e ully
conside ed. Fu he esea ch should add ess he bioa ailabili y o min-
e al elemen s p esen in ma e milk in o de o p ope ly e alua e he
con ibu ion o his ood p oduc o human heal h.
5. Conclusions
Milk om Basque Moun ain Ho se b eed ma es con ains simila
a e age mine al elemen concen a ions compa ed o hose desc ibed in
he scien i ic li e a u e in o he equine b eeds, excep o lowe S and Cl,
and highe Fe con en s. The mac omine al (Ca, K, P, S, Na, Mg and Cl)
and ace elemen (Zn, Fe and Cu) con en s s udied, as well as he Ca:P
a io, we e signi ican ly in luenced by lac a ion ime. O e all, he con-
en o all mac omine als excep Na and Cl dec eased, Na, Zn and Cu
inc eased, and Cl and Fe luc ua ed h oughou lac a ion. In addi ion,
he managemen sys em used in a m a ec ed Ca, P, S and Na concen-
a ions in milk. This could be ela ed o di e ences in he eeding
managemen o ma es in semi-ex ensi e u al a ms. These indings can
con ibu e o he p omo ion o ma e milk as a di e si ica ion op ion o
he cu en equine mea p oduc ion sys em pe o med in semi-ex ensi e
u al a ms, and o e all, o li es ock g azing sys em sus ainabili y.
CRediT au ho ship con ibu ion s a emen
Ana Blanco-Do al: Me hodology, Fo mal analysis, In es iga ion,
W i ing – o iginal d a . Luis Ja ie R. Ba on: Concep ualiza ion,
Me hodology, Fo mal analysis, W i ing – e iew & edi ing. Noelia
Aldai: Concep ualiza ion, Me hodology, Fo mal analysis, W i ing – e-
iew & edi ing.
Decla a ion o Compe ing In e es
We wish o con i m ha he e a e no known con lic s o in e es
associa ed wi h his publica ion and he e has been no signi ican
inancial suppo o his wo k ha could ha e in luenced i s ou come.
We con i m ha he manusc ip has been ead and app o ed by all
named au ho s and ha he e a e no o he pe sons who sa is ied he
c i e ia o au ho ship bu a e no lis ed. We u he con i m ha he
o de o au ho s lis ed in he manusc ip has been app o ed by all o us.
We con i m ha we ha e gi en due conside a ion o he p o ec ion o
in ellec ual p ope y associa ed wi h his wo k and ha he e a e no
impedimen s o publica ion, including he iming o publica ion, wi h
espec o in ellec ual p ope y. In so doing we con i m ha we ha e
ollowed he egula ions o ou ins i u ions conce ning in ellec ual
p ope y.
We unde s and ha he Co esponding Au ho is he sole con ac o
he Edi o ial p ocess (including Edi o ial Manage and di ec commu-
nica ions wi h he o ice). She is esponsible o communica ing wi h he
o he au ho s abou p og ess, submissions o e isions and inal
app o al o p oo s. We con i m ha we ha e p o ided a cu en , co ec
Fig. 3. Dis ibu ion o ma e milk samples collec ed du ing ea ly- (
○), mid- (Δ),
and la e- (□) lac a ion s ages acco ding o S epwise Disc iminan Analysis
pe o med using he mine al elemen composi ion (■, g oup cen oid). Ca-
nonical unc ions 1 and 2 explained 85.7% and 14.3% o he o al a iance,
espec i ely.
A. Blanco-Do al e al.
Jou nal o Food Composi ion and Analysis 123 (2023) 105629
8
email add ess which is accessible by he Co esponding Au ho and
which has been con igu ed o accep email om [email p o ec ed].
Da a A ailabili y
Da a will be made a ailable on eques .
Acknowledgemen s
The Depa men o Educa ion o he Basque Go e nmen o he
p edoc o al ellowship o A. Blanco-Do al. This wo k was suppo ed by
he Basque Go e nmen h ough Bio asma (Elka ek, 2019), Beho Esne
(Coope a ion, 2020) and IT944-16 and IT1568-22 p ojec s. Au ho s
hank he comme cial a ms ha pa icipa ed in he s udy, and also he
SGIke – Ad anced Resea ch Facili ies o he Uni e si y o he Basque
Coun y (UPV/EHU) o assis ing in sample analysis.
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