Animal (2019), 13:5, pp 1020–1029 © The Animal Consortium 2018

doi:10.1017/S1751731118002422
animal

Effects of feeding treatment on growth rates, metabolic profiles

and age at puberty, and their relationships in dairy heifers
F. Abeni1, F. Petrera1 and Y. Le Cozler2†
CREA – Research Centre for Animal production and Aquaculture, Via A. Lombardo, 11, 26900 Lodi, Italy; 2PEGASE, AGROCAMPUS OUEST, INRA, 35000 Rennes, France
1

(Received 24 December 2017; Accepted 17 August 2018; First published online 10 October 2018)

Puberty attainment in dairy heifers has been widely studied from a hormonal point of view, but few studies have focussed on
puberty–blood profile relationships during growth. We led experiments to determine the effects of feeding treatments on growth
parameters, age at puberty and plasma biochemical profiles, and the relationships between age at puberty and metabolic profiles
at 6, 9, 12 and 15 months (mo) of age. Blood samples were collected from 67 Holstein heifers, born between September 2011 and
February 2012, every 10 days from 5.5 mo of age until heifers were considered pubertal (plasma progesterone concentration
greater than 1.0 ng/ml) or oestrus synchronisation (November 2012; 11 to 15 mo of age). Heifers born before 30 November were
fed either a standard diet (SD, n = 27) or an intensive-plane diet (ID1, n = 27) from 0 to 6 mo of age. This strategy aimed to reach
190 to 200 kg (SD) or 220 to 230 kg (ID1) BW at 6 mo of age. All heifers born after 1 December received an intensive-plane diet
(ID2, n = 13) from birth until oestrus synchronisation, in order to reach a similar BW at first insemination as heifers born before
1 December. Only 56 heifers reached puberty before oestrus synchronisation, at an average age of 10.3 ± 2.2 mo (6.2 to 14.4 mo)
and a BW of 296 ± 40 kg (224 to 369 kg). There was no difference among the three feeding treatments until 6 mo, but at 9, 12 and
15 mo of age, ID2 (n = 11) heifers weighed 37, 52 and 30 kg more than SD (n = 22) and ID1 (n = 23) heifers (P < 0.001),
respectively. Glucose, non-esterified fatty acids, albumin, aspartate aminotransferase, alkaline phosphatase and tartrate-resistant
acid phosphatase, calcium, phosphorus, potassium and iron decreased with age, whereas β-hydroxybutyric acid, total cholesterol,
creatinine, the creatinine : albumin ratio, alanine aminotransferase and chloride increased. The feeding treatment significantly
affected creatinine, the creatinine : albumin ratio, and phosphorus and sodium levels, which were higher for ID2 heifers compared
with SD and ID1. A logistic regression based on plasma metabolites at 6 mo of age to explain puberty attainment before or at
12 mo of age showed a positive relationship with plasma cholesterol (odds ratio = 9.05). In conclusion, the feeding treatment had
minor consequences on plasma metabolites, but it did affect growth performance.

Keywords: rearing, metabolites, Holstein, female, oestrus

Implications
Increasing the growth rate of dairy heifers decreased their
age at puberty, potentially reducing age at first calving
and ultimately shortening the non-productive rearing per-
iod. Combined monitoring of the growth rates and blood
levels of selected metabolites at a young age (6 months
(mo) of age or earlier) could be a way to better predict
oestrus onset in dairy heifers, rather than regular proges-
terone analysis (every 10 days or less). This information
could be of interest for optimal reproduction (number
of oestrus cycles before insemination for improved
fertility) and/or for oocyte/embryo production in peripu-
bertal donors.
†
E-mail: yannick.lecozler@agrocampus-ouest.fr
Introduction
In seasonal calving systems where heifers first calve at a
young age (around 24 mo), the moment of 1st insemination
may be delayed for those born at the end of the calving
period if an adequate BW is not reached (i.e. 360 to 380 kg
for Holstein heifers in French dairy herds; Le Cozler et al.,
2008). To solve this problem, one strategy is to increase the
feeding intensity and thus the growth rate for late-born
heifers. A high growth rate usually results in a decreased age
at puberty and, consequently, 1st calving may occur as early
as 20 mo of age. Puberty onset is generally a consequence of
an early attainment of a standard BW associated, within
breeds, to a target body condition (Le Cozler et al., 2008).
However, reaching this standard BW alone is probably not
sufficient to attain puberty, and specific metabolic signals
likely act to support this phase of reproductive development

1020
 Blood profiles during growth in Holstein heifers

(Chelikani et al., 2003). There are many studies published on
the effect of nutrition during the rearing-phase on profiles of
reproductive (progesterone, oestradiol, LH, FSH, etc.) and/or
metabolic (IGF, growth hormone, leptin, etc.) hormones
during pre- or post-pubertal phases (Bergfeld et al., 1994;
Evans et al., 1994; Melvin et al., 1999). Other authors have
documented the metabolic, endocrine and/or reproductive
responses with respect to growth or puberty onset in dairy
(Taylor et al., 2004; Brickell et al., 2009), beef (Hall et al.,
1995; Rodríguez-Sánchez et al., 2015) or mixed (Samadi
et al., 2014) heifers. All these studies provide additional
information on the complex interplay among nutrition,
growth and reproduction in growing cattle, but information
on circulating enzymes or minerals involved in metabolism is
scarce. Indeed, when accelerated heifer growth is diet-
induced, it is also necessary to consider the possible outcome
of these elements on metabolic profiles in terms of potential
implications for puberty attainment. The relationship
between plasma metabolites and late puberty attainment
may stem from two factors: nutrient supply inadequately
supporting the heifer’s growth potential, and nutritional or
environmental stressors influencing heifer growth. The first
case would likely involve a metabolic profile characterised by
signals of low energy and/or protein supply, such as low
plasma glucose or urea levels (Abeni et al., 2000). The sec-
ond case would likely involve increased signals of tissue
damage, notably to the liver, as suggested by higher enzyme
activities (Abeni et al., 2014). The fact that rearing proce-
dures can alter hormonal profiles in both dairy (Chelikani
et al., 2003) and beef (Gasser et al., 2006) heifers points to
the hypothesis that modifying both diet composition and
feeding management may also modify plasma profiles and
thus affect puberty onset. If puberty onset is affected by
plasma biochemical parameters, then some metabolites
could serve as indicators of potential early puberty onset and
thus be used to select these heifers.
Here, we designed and led an experiment to determine the
effects of feeding treatments on growth parameters, age at
puberty and plasma biochemical profiles, and to study the
possible relationships between age at puberty and metabolic
profiles at 6, 9, 12 and 15 mo of age in dairy heifers.
Material and methods
General design
Sixty-seven Holstein heifers, born during the 2011–12 cal-
ving season (September to February), were reared and fol-
lowed until oestrus synchronisation (11 to 15 mo of age) at
the INRA’s experimental farm in Le Rheu, France. Calves born
between 1 September and 30 November were alternately
allocated to one of two nutritional treatments (according to
birth order) and fed either a standard diet (SD; n = 27) or an
intensive-plane diet (ID1; n = 27) from 0 to 6 mo of age. It
was expected that, thanks to the feeding intensity chosen,
heifers fed SD and ID1 diets would reach 190 to 200 and 220
to 230 kg at 6 mo of age, respectively. Heifers born after
1 December (ID2; n = 13) received the same intensive-plane
diet as ID1 heifers from 0 to 6 mo of age, to limit the possible
confusion effect between age and treatment during this
period. Thereafter, a complementary diet was formulated for
ID2 heifers in order to reach 380 kg at 1 year of age. The
main objective of this latest procedure was to study the
possibility for late-born heifers to catch up with the rest of
the heifers at 1st artificial insemination (AI) at a minimum BW
of 370 to 380 kg. It was expected that this corresponded to
15 and 12 mo of age for (SD and ID1) and ID2 heifers,
respectively. The diets (Table 1) were formulated for the
different stages of growth: birth to weaning, weaning to
4 mo, 4 to 6 mo, 6 to 9 mo, 9 to 12 mo and 12 to 15 mo,
according to the recommendations and procedures pre-
sented by Agabriel and Meschy (2007). These diets were also

Table 1 Ingredients and chemical composition of the experimental diets

Items TMR1 TMR2 TMR3a TMR3b

Stage of growth (age) (7 days to 4 months) (4 to 6/8 months) (9 to 11 months) (6 to 11 months)

Group of heifers All (SD, ID1, ID2) All (SD, ID1, ID2) SD, ID1 ID2
Ingredient (%)
Corn silage 47.5 72.0 80 80
Soya bean meal – 8.0 20 20
18% CP alfafa pellets 5 –
Concentrate 11 47.5 20.0
Concentrate 22 (kg/head per day) 1 2
Estimated chemical composition
DM (%) 61.5 47.7 44.7 45.6
CP (g/kg DM) 118.3 123.4 161.8 163.9
PDIE (g/kg DM) 89.9 921.2 128.4 124.9
PDIN (g/kg DM) 47.0 82.4 116.7 116.7
UFL(per kg DM) 0.9 1.0 1.0 1.0
TMR = total mixed ration; SD, ID1, ID2: animals fed either on a standard (SD) or increased-plane (ID1 and ID2) feeding rearing programme;
DM = dry matter; PDIE = metabolisable protein supply; PDIN = rumen-degradable nitrogen; UFL = unit of feed for lactation.
1
Chemical composition: DM 88.7%; CP 168 g/kg; PDIE 118 g; PDIN 114 g; UFL 1.05.
2
Chemical composition: DM 87.9%; CP 131 g/kg; PDIE 81 g; PDIN 90 g; UFL 0.96.

1021
Abeni, Petrera and Le Cozler
formulated to reach a targeted average daily gain (ADG) per
period, with respect to the initial BW and feeding treatment
used.
Heifer management
The milk-feeding phase (year 1). At the end of the pre-
experimental phase (0 to 10 days), calves were fed recon-
stituted milk replacer (135 g milk power + 865 g water per
litre) until weaning. The CP and fat content of the milk
powder averaged 23.9% and 19.0%, respectively. Heifers
were group-housed indoors on cumulated straw bedding.
They were reared in dynamic groups, fed with automatic milk
feeding systems (AMFS), with free access to fresh water,
straw and hay. Dynamic group strategy meant that calves
entered the group every week while others left it at weaning
(70 to 77 days). Group size thus varied from 8 to 24 calves
per AMFS. Milk was distributed according to either a stan-
dard (SD) or an increased-plane (ID1 and ID2) milk-feeding
programme (Figure 1). The SD ration was routinely used in
the experimental herd and (ID1 and ID2) rations consisted of
15% more milk. As a supplement to the milk, all calves were
fed ad libitum total mixed ration 1 (TMR1; Table 1).
From weaning until turning out to pasture (year 1). From
weaning to 6/8 mo of age, groups of calves were housed in
separate pens on deep straw bedding with ad libitum access
to fresh water and straw. Until 4 mo of age, the SD group
received TMR1 ad libitum until the concentrate reached 2 kg
DM/head per day, no restriction was applied to ID1 and ID2
heifers. From 4 to 6/8 mo of age (turning out to pasture),
TMR2 (Table 1) was distributed ad libitum until the maximum
daily allowance of concentrate reached 2 and 2.5 kg DM/
head for SD and (ID1 and ID2) heifers, respectively, that is, a
total daily allowance of 10 and 12.5 kg/head of TMR2 for SD
and (ID1 and ID2) heifers, respectively. These amounts did
not change until turn out to pasture.
Turning out to pasture. Starting from mid-May 2012 for SD
and ID1 heifers and mid-June for ID2 heifers, heifers were
turned out to pasture and rotationally grazed on a perennial
ryegrass sward. After a 5-day transition phase and through-
out the pasture season, SD and ID1 groups received a daily
supplement of 1 kg DM/head of concentrate 2, whereas the
10 SD
9 ID1 & ID2
8
7
Milk, kg/d/heifer
6 and the results are not presented in detail in the
5 present paper.
4
3 Blood samples
2
1
0
1 7 13 19 25 31 37 43 49
Age, d
Figure 1 Standard (SD) or increased-plane (ID1 and ID2) milk feeding
programme according to age of Holstein heifers.
1022
ID2 group received 1 kg of corn silage and 2 kg of con-
centrate 2 per DM/heifer per day. Grass availability and/or
quality were insufficient to maintain the desired growth rates
during summer (heat stress). Standard diet and ID1 heifers
then received, per DM/heifer per day, up to 2.5 kg of addi-
tional TMR3a, plus 1 kg of concentrate 2; ID2 heifers received
up to 3 kg of TMR3b, plus 2 kg of concentrate 2. To achieve
380 kg at the end of outdoor season (when oestrus syn-
chronisation started), the expected ADG for SD and ID1
heifers was estimated to be around 600 g/day during this
period, with a feeding regime based on pasture plus 1 kg of
concentrate 2, and 800 g/day when receiving grass plus
TMR3a. For ID2 heifers, it was estimated that grass alone
was not sufficient to reach 900 g/day during the same period
and TMR3b was used (Table 1). This procedure, based on the
feed intake of the heifers, grass composition and expected
growth, was successfully tested during the previous 2010
and 2011 grazing seasons on similar heifers reared on the
same paddocks. During the entire experiment, BW records
(every 2 or 3 weeks) were compared with data collected
during 2011 and 2012. If necessary, feed allowance was
adjusted.
In the pasture area, a permanent headlock barrier (80
places on concrete floor) was used daily to feed SD and ID1
heifers with their concentrate. Heifers were locked for 1 h
while eating. The SD2 group had free access to its ration, but
was not locked, since it usually took several hours to eat the
ration. At the end of the pasture season (6 November),
heifers were group-housed (eight heifers per pen) on deep
straw bedding and received 3.8 kg DM/head per day of a diet
containing 79% corn silage and 21% soya bean meal. They
had free access to fresh water, straw and mineral
complements. After a 2-week adaptation period, oestrous
cycles were synchronised in heifers, and the experiment
ended (22 November).
Measurements and registration
Heifers were weighed every 14 days from birth to weaning,
every 21 days from weaning until turning out to pasture, and
every 28 days until the end of the experiment. BW of ID2
heifers at 12 and 15 mo of age was additionally retrieved and
compared to SD and ID1 heifers BW at these stages. BW was
corrected according to age, and daily gains were calculated.
Daily feed allowances and refusals were also registered, on
either an individual or a group basis. Heifer health and
treatment information was recorded during the experiment;
however, heifers were reared under strict control conditions
From 5.5 mo of age (on average), one 5 ml lithium-heparin
tube was collected from the jugular vein at 10-day intervals
55 61 67 73 to determine plasma progesterone. A second 5 ml lithium-
heparin tube was collected at 6, 9, 12 and 15 mo of age for
the plasma biochemical profile. Metabolite concentrations
such as glucose or non-esterified fatty acids (NEFA) vary

based on the time of feeding (Taylor et al., 2004). To limit
such an effect, samples were performed at 1000 h, just
before feed distribution. Plasma was separated by cen-
trifugation at 3000 rpm for 15 min and stored at − 20°C until
analysis. Laboratory progesterone analysis was performed
every 2 to 3 weeks, when enough samples were available to
run assays. Heifers were considered pubertal once plasma
progesterone concentrations were greater than 1.0 ng/ml
and puberty attainment was declared. Age at puberty cor-
responded to the difference between the date of sample
and birth date. When analysis showed progesterone level
<1 ng/ml, samplings went on until the start of oestrus
synchronisation.
Laboratory analysis
Blood samples were analysed to detect puberty based on
changes in progesterone levels. Plasma samples were first
thawed at 20°C and mixed, then progesterone analyses were
performed with an AIA-360 Automated Immunoassay Ana-
lyzer (Tososh Bioscience Inc., San Francisco, CA, USA).
Intra- and inter-assay variability were 12.3% and 7.5%,
respectively. Blood samples collected at 6, 9, 12 and 15 mo of
age were analysed to study metabolite changes and enzyme
activities. Samples were thawed for metabolite analysis as
above, but reaching 37°C. Analyses were performed on
plasma metabolites (glucose, NEFA, β-hydroxybutyric acid
(BHBA), total cholesterol, triglycerides, urea, total protein,
albumin, creatinine, calcium (Ca), inorganic phosphorus (P),
magnesium (Mg) sodium (Na), potassium (K), chlorine (Cl)
and iron (Fe), and enzymatic activities (aspartate amino-
transferase (AST), alanine aminotransferase (ALT), alkaline
phosphatase (ALP), γ-glutamyltransferase (GGT) and
tartrate-resistant acid phosphatase). An automated bio-
clinical chemistry analyser (ILAB Aries, Instrumentation
Laboratory, Lexington, MA, USA) using dedicated
manufacturer-supplied kits was used, except for NEFA
(NEFA-HR(2), Wako Chemicals GmbH, Neuss, Germany) and
BHBA (Randox Laboratories Ltd, County Antrim, UK).
Calculations and statistical analysis
BW changes and puberty. Data on growth and puberty
attainment were tested by ANOVA (SAS Institute Inc., 2009)
in a randomised block design that included feeding treat-
ments (three levels), age at reaching puberty (three classes)
and their interaction as fixed effects. In these analyses, only
heifers reaching puberty before oestrus synchronisation were
used (n = 56). Results are reported as least squares means
and residual standard deviation with effects declared highly
significant at P < 0.001, significant at P < 0.05 and as a
trend at 0.05 < P < 0.10.
Blood and metabolites. Data on plasma metabolites were
evaluated by repeated-measures ANOVA using the MIXED
procedure of SAS/STAT software in SAS System for Windows
v.9.3 (SAS Institute Inc., 2008). A first analysis was per-
formed on data from all heifers (n = 67) to assess the effect
of dietary treatment. The main effects were diet during the
Blood profiles during growth in Holstein heifers
experimental period (SD, ID1 and ID2), age at time of sam-
pling (6, 9, 12 and 15 mo of age) and their interaction as
fixed effects, with time of sampling as a repeated measure,
and heifer within diet treatment (random effect) as an error
term. A second analysis was performed on plasma data from
heifers reaching puberty before oestrus synchronisation
(n = 56) to assess within-group differences obtained by
stratification of the heifers according to age at reaching
puberty. Three classes of age at reaching puberty were cre-
ated: <9 mo of age (Gr1); 9 to 12 mo of age (Gr2); >12 mo of
age until start of oestrus synchronisation (Gr3). The model
included the three classes of age at puberty (Gr1, Gr2 and
Gr3), age at time of sampling (6, 9, 12 and 15 mo of age),
and their interaction as fixed effects, with time of sampling
as a repeated measure, and heifer within diet treatment
(random effect) as an error term. Block diagonal structure of
the residual covariance matrix used in the analyses was deter-
mined by repeated measures on the same subjects. For each
variable, different covariance structures were tested (simple,
Compound Symmetry, Autoregressive, Ante-Dependence,
unstructured), and the best was chosen by Akaike’s Infor-
mation Criteria (Littell et al., 1998). Least squares means
were computed and pairwise-tested for each effect in each
model. Simple Pearson correlation coefficients between age
at puberty and the other dependent variables at the different
times were calculated.
To identify which main plasma metabolites were able to
predict the probability to be puberal before or at 1 year of
age at 6 mo of age, a logistic regression was performed using
R software (R Core Team, 2016). This way, considering the
statistical significance of each factor within the regression,
the relative odds ratio (OR) was evidenced to be discussed.
Results
Growth performance and puberty attainment
Analysis on growth performances according to feeding
treatment or class of age at puberty from birth to 15 mo of
age showed no significant difference among treatments or
groups until 6 mo of age (Table 2). At 9 mo of age, ID2
(n = 11) heifers were heavier than SD (n = 22) and ID1
(n = 23) heifers (P < 0.001), at 301.2 v. 264.8 and 265.2 kg,
respectively. The difference remained at 12 and 15 mo of age
(+50 and +30 kg, respectively). Regardless of puberty onset
(n = 67; data not presented), no difference among treat-
ments emerged until 6 mo of age. Thereafter, feeding treat-
ment had an effect on growth, with ID2 (n = 13) heifers
having higher BW compared to SD (n = 27) and ID1 (n = 27)
heifers at 9 mo (287 ± 42 v. 262 ± 21 and 263 ± 24 kg;
P < 0.05), 12 mo (366 ± 55 v. 312 ± 24 and 313 ± 28 kg;
P < 0.01) and 15 mo (405 ± 56 v. 382 ± 35 and 384 ± 37 kg;
P < 0.01).
The ADG calculated during these three different periods
(Table 2) were higher for ID2 heifers, which consequently
reached puberty 1 mo earlier than SD and ID1 heifers (9.2 v. 10.5
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Abeni, Petrera and Le Cozler

Table 2 Main characteristics of Holstein heifers groups according to feeding treatment and class of age at puberty (least squares means, n = 56)
Feeding treatment (T)1 Class of age at puberty (Gr)2 P-value

Items SD1 ID1 ID2 Gr1 Gr2 Gr3 rSD T Gr T × Gr

BW (kg)
Birth (0 day) 40.4 42.4 38.9 38.8 41.7 42.1 5.2 ns ns ns
Weaning (77 days) 91.0 93.9 94.4 92.9 94.9 90.2 9.0 ns ns ns
3 months of age (91 days) 106 110 111 108 111 107 11 ns ns ns
4 months of age (122 days)3 142 149 153 145 150 146 14 t ns ns
6 months of age (182 days) 3 213 218 222 218 218 214 19 ns ns ns
9 months of age (274 days) 265b 265b 301a 279a 259b 257b 23 *** * *
12 months of age (364 days) 315b 316b 377a 336a 339a 307b 39 *** * *
15 months of age (455 days) 386b 385b 416a 405 390 381 41 *** ns ns
Average daily gain (g/days)
Birth to weaning 657 668 720 702a 691ab 625b 96 ns * ns
Birth to 4 months 832b 871ab 935a 868 882 855 108 * ns ns
Weaning to puberty 879b 862b 1075a 1 067a 907b 762c 150 * *** *
Weaning to 4 months 1139b 1227ab 1310a 1157a 1216b 1249b 197 ** * ns
Puberty
Age (month) 10.5a 10.8a 9.2b 7.9a 10.3b 12.9c 0.83 *** *** ns
BW (kg) 292 296 306 258a 305b 325b 28 ns *** ns
ns = not significant; rSD = residual standard deviation.
a,b,c
Within feeding treatment or class of age at puberty, values within a row with different superscripts differ significantly at P < 0.05.
1
SD, ID1, ID2: animals fed either on a standard (SD, n = 22) or increased-plane (ID1, n = 23 and ID2, n = 13) feeding rearing programme.
2
Gr1, Gr2, Gr3 correspond to the three classes of age at puberty attainment before oestrus synchronisation: <9 months of age (Gr1, n = 18), 9 to 12 months of age (Gr2,
n = 21), >12 months of age (Gr3, n = 17).
3
4 months: total mixed ration 1 (TMR1) switched to TMR2; 6 months: turn out to pasture.
*P < 0.05; **P < 0.01; ***P < 0.001, t: P < 0.1.

and 10.7 mo of age, respectively; P < 0.001) but without a dif-
ference in BW (306 v. 292 and 296 kg, respectively).
By stratifying heifers on three classes of age at puberty,
puberty was detected in 18 heifers before 9 mo (Gr1), 21
heifers between 9 and 12 mo (Gr2) and 17 after 12 mo and
until oestrus synchronisation (Gr3) at an average age of 7.9,
10.3 and 12.9 mo, respectively (Table 2). Gr1, Gr2 and Gr3
groups of heifers were composed of 8, 7 and 3; 5, 8 and 8;
and 9, 8 and 0 heifers of the SD, ID1 and ID2 feeding treat-
ment groups, respectively. Within each group of age at
puberty, age at cycling was always lower for ID2 heifers. As
shown above, on average, ID2 heifers reached puberty at a
lower age (Table 2) and in accordance with this, the three ID2
heifers in Gr1 were the youngest to show cyclicity (at 6.2 mo
for the youngest). Gr1 heifers had a lower BW at puberty
than Gr2 and Gr3 heifers (258 v. 305 and 325 kg, respec-
tively; P < 0.001). ADG from weaning to puberty significantly
differed among treatments, by 1 067, 907 and 762 g/day on
average for Gr1, Gr2 and Gr3 heifers, respectively.
Metabolic profiles
Except for plasma urea, total protein and GGT, all the other
parameters showed an age effect (Table 3). Glucose, NEFA,
albumin, AST, ALP, Ca, P, K and Fe levels decreased with age,
whereas BHBA, total cholesterol, creatinine, the creatinine:
albumin ratio, ALT and Cl levels increased from 6 to 15 mo.
Feeding treatment (67 heifers, Table 3) only affected
plasma creatinine, the creatinine:albumin ratio, and P and
Na levels, which were higher in ID2 compared with SD and
ID1 heifers. Plasma Fe was lower in ID1 compared with SD
heifers. A trend was observed for plasma urea, with lower
values (P < 0.05) in ID2 compared with SD and ID1. There
was an age × feeding treatment interaction on many plasma
parameters; some data are available in Supplementary Figure S1.
This shows higher cholesterol and creatinine at 12 and 15 mo
(b, d), ALP at 9 mo (e), and BHBA at 15 mo (a), but lower
cholesterol at 9 mo (b), urea at 9 and 12 mo (c) and BHBA at
6 mo (a) in ID2 heifers in comparison with ID1 and SD heifers.
Comparison of groups classed by age at puberty revealed
significant differences on some plasma metabolites and
mineral concentrations (Table 4). Gr3 heifers showed lower
levels of plasma BHBA, total cholesterol, albumin and K, and
higher levels of NEFA and ALP compared with Gr1 and Gr2
heifers. There was a class × age interaction on many plasma
parameters; some data are available in Supplementary Figure S2.
This shows lower glucose at 9 mo (a), but higher NEFA at
12 mo (b) and ALP activity at 6 and 15 mo (d) in Gr3 heifers in
comparison with Gr1 and Gr2 heifers. Urea was lower at
9 and 12 mo (c), but creatinine was higher at 12 mo (c) in Gr2
heifers in comparison with Gr1 and Gr3 heifers.
Pearson correlations and logistic regression on puberty
attainment
As expected, age at puberty correlated with BW at puberty
(r = 0.68; P < 0.001, data not shown). The correlation
between age at puberty with BW and plasma parameters
(Table 5) was negative at 6 mo with glucose and cholesterol;
at 9 mo with BW, glucose and albumin; at 12 mo with BW

1024
 Blood profiles during growth in Holstein heifers

Table 3 Plasma parameters in each group of Holstein heifers according to age and feeding treatment during the rearing period (least squares means;
all animals, n = 67)
Feeding treatment (T)1 Age (A) (month) P-value

Items Unit/l SD ID1 ID2 6 9 12 15 rSD T A T×A

Glucose mmol 4.37 4.29 4.40 4.94a 4.30ab 4.04c 4.03c 0.03 ns *** ns
NEFA mmol 0.11 0.11 0.09 0.13a 0.12a 0.09b 0.08b 0.01 ns ** ns
BHBA mmol 0.60 0.61 0.61 0.50c 0.55bc 0.78a 0.61b 0.03 ns *** *
Total cholesterol mmol 2.13 2.06 2.17 1.93c 2.11b 2.05bc 2.38a 0.03 ns *** ***
Triglycerides mmol 0.25 0.23 0.24 0.23b 0.27a 0.23b 0.22b 0.01 ns *** ***
Urea mmol 3.63 3.57 3.16 3.44 3.33 3.44 3.61 0.06 t ns ***
Creatinine µmol 84.5b 82.8b 93.7a 78.1c 80.8c 89.1b 100.0a 0.74 ** *** ***
Total protein g 69.3 68.8 68.2 68.8 69.0 68.5 68.6 0.42 ns ns *
Albumin g 33.3 32.3 33.3 35.25a 31.8c 31.6c 33.3b5 0.208 ns *** ns
Globulins g 35.9 36.4 34.8 33.6c 37.2a 36.8ab 35.2b 0.33 ns *** *
Creatinine:albumin µmol/g 2.55b 2.59b 2.83a 2.22d 2.56c 2.82b 3.03a 0.02 ** *** *
ALT U 19.8 19.9 20.2 19.0b 19.77b 19.8b 21.2a 0.21 ns ** ns
AST U 76.6 76.3 75.6 88.1a 81.78ab 71.9bc 65.9c 1.49 ns *** ns
GGT U 19.1 18.2 17.9 18.0 18.3 18.8 18.5 0.27 ns ns **
ALP U 111.7 111.2 114.8 140.4a 98.2b 104.8b 106.8b 2.12 ns *** ***
TRAP U 2.98 2.97 3.13 3.18a 3.00ab 2.9b 3.06ab 0.04 ns *** ns
Ca mmol 2.51 2.47 2.56 2.62a 2.52b 2.48bc 2.45c 0.014 ns *** ns
P mmol 2.54b 2.56b 2.78a 2.83a 2.55b 2.61b 2.53b 0.02 * *** t
Mg mmol 0.98 0.979 1.00 1.00a 0.97bc 0.93c 0.98ab 0.01 ns ** *
Na mmol 143.9ab 141.7c 144.4a 144.1a 141.5c 143.6ab 144.1ab 0.36 * * ns
K mmol 4.19 4.28 4.35 4.51a 4.21b 4.16b 4.22b 0.03 ns *** **
Cl mmol 102.2 100.9 101.8 100.6b 100.9b 101.9ab 103.0a 0.28 ns * ns
Fe µmol 28.5 25.9 26.1 31.2a 25.0b 25.3b 25.8b 0.44 t *** **
rSD = residual standard deviation; NEFA = non-esterified fatty acids; BHBA = β-hydroxybutyric acid; ALT = alanine aminotransferase; AST = aspartate aminotransferase;
GGT = γ-glutamyltransferase; ALP = alkaline phosphatase; TRAP = tartrate-resistant acid phosphatase; Ca = Calcium; P = inorganic phosphorus; Mg = Magnesium;
Na = Sodium; K = potassium; Cl = Chlorine; Fe = iron.
a,b,c,d
Within feeding treatment or age, values within a row with different superscripts differ significantly at P < 0.05.
1
SD, ID1, ID2: animals fed either on a standard (SD, n = 27) or an increased-plane (ID1, n = 27 and ID2, n = 13) feeding rearing programme.
Significant differences: *P < 0.05; **P < 0.01; ***P < 0.001, t: P < 0.1; ns: not significant.

and BHBA, and at 15 mo with BW, albumin and BHBA. On
the contrary, positive correlations were observed at 6 mo for
ALP, at 12 mo for glucose, NEFA, Ca and Mg, and at 15 mo
for ALP.
The logistic regression analysis used to estimate the
probability that heifers reached puberty before or after 12
mo based on BW and plasma parameters at 6 mo of age
(Supplementary Table S1) indicated positive coefficients
for NEFA and cholesterol (P < 0.05), but negative coeffi-
cients for Fe concentrations (P < 0.05). A trend was found
for BHBA (P = 0.052), urea (P = 0.07), ALT (P = 0.09) and
Ca (P = 0.09). The most significant predictor measured at
6 mo of age for puberty attainment at 12 mo, with an
OR = 9.05 for each unit of increase in plasma, was
cholesterol.
Discussion
Feeding treatment: growth performance and plasma profile
BW at the different stages of growth was considered ade-
quate for a first calving at 24 mo of age or less, as a result of
feeding treatments used to reach targeted weights. However,
a huge variation in BW, as expressed by its standard varia-
tion, was noted. This is not uncommon in most studies
dealing with animal growth, and often originates from indi-
vidual variation in ADG during the growing period (Brickell
et al., 2009). This may compromise the targeted BW for 1st AI
at 15 mo or less and 1st calving at around 24 mo of age,
which is around 360 kg in United States (Hoffman, 1997) or
360 to 380 kg in France (Le Cozler et al., 2008) for Holstein
cows. A high growth rate from birth to 1st AI at 15 mo of age
is generally recommended for first calving at 24 mo of age,
and ADG is close to 750 g/day (Wattiaux, 1997). When first
inseminated at 12 mo of age and for a similar BW, ADG
should then be around 950 g/day. In the present study, hei-
fers reached the target BW at the start of the reproductive
period, even those born late during the previous calving
season (ID2). In this study, the ADG from birth to weaning
was higher in heifers reaching puberty at an early age (Gr1),
but without any effect due to the feeding treatment. As feed
availability was not limited in any groups, it is suggested that
genetic and/or feed efficiency components are probably
involved in such variations and differences (Pryce et al.,
2014). However, the results indicate that for an early age at

1025
Abeni, Petrera and Le Cozler

Table 4 Plasma parameters in each group of Holstein heifers according to age and class of age at puberty attainment (least squares means; n = 56)
Class of age at puberty (Gr)1 Age (A) P-value

Items Unit/L Gr1 Gr2 Gr3 6 9 12 15 rSD Gr Age Gr × A

Glucose mmol 4.37 4.34 4.34 5.04a 4.24b 4.11bc 4.01c 0.031 ns *** **
NEFA mmol 0.09 0.10 0.12 0.13a 0.12a 0.09b 0.08b 0.005 t *** *
BHBA mmol 0.68 0.61 0.57 0.53c 0.54bc 0.80a 0.61b 0.020 t *** ns
Total cholesterol mmol 2.12 2.25 2.05 1.99d 2.22ab 1.98cd 2.36a 0.036 t *** ns
Triglycerides mmol 0.24 0.25 0.23 0.22b 0.29a 0.24b 0.21bc 0.006 ns *** *
Urea mmol 3.80 3.35 3.56 3.41 3.54 3.81 3.52 0.070 t ns *
Creatinine µmol 87.23 88.12 82.32 78.0c 81.2c 85.6b 98.8a 0.896 ns *** **
Total protein g 68.24 69.76 68.27 68.7 69.3 68.9 68.2 0.493 ns ns ns
Albumin g 33.7 33.3 32.2 35.7a 32.0bc 31.5c 33.1b 0.218 t *** ns
Globulins g 34.61 36.48 36.07 33.1c 37.4a 37.4a 35.1b 0.373 ns *** ns
Creatinine : albumin µmol/g 2.60 2.67 2.59 2.19d 2.55c 2.72b 3.02a 0.028 ns *** **
ALT U 19.98 19.15 20.54 18.9b 19.9b 19.4b 21.4a 0.233 ns *** ns
AST U 72.30 73.89 75.24 79.5a 78.6ab 71.2c 66.0c 1.161 ns *** ns
GGT U 19.61 17.83 18.36 18.3 18.3 18.9 18.9 0.279 ns ns ns
ALP U 99.2b 114.1ab 124.9a 147.4a 93.9c 104.0bc 105.6b 2.294 ** *** **
TRAP U 3.07 3.09 2.91 3.18a 2.99abc 2.83c 3.08ab 0.042 ns *** ns
Ca mmol 2.49 2.52 2.47 2.60a 2.50b 2.46bc 2.42c 0.016 ns *** ns
P mmol 2.56b 2.76a 2.51ab 2.86a 2.50b 2.60b 2.49b 0.027 * *** ns
Mg mmol 0.96 1.00 0.99 1.01a 0.99ab 0.95b 1.00a 0.007 ns * *
Na mmol 142.6 144.4 141.9 144.4a 140.9c 143.3ab 143.4ab 0.426 t *** ns
K mmol 4.23ab 4.35a 4.14b 4.49a 4.19b 4.07b 4.21b 0.032 * *** t
Cl mmol 101.1 102.1 101.2 100.6 100.9 101.6 102.8 0.331 ns t ns
Fe µmol 26.88 26.37 27.54 31.8a 23.7c 26.9b 25.3bc 0.495 ns *** *
rSD = residual standard deviation; NEFA = non-esterified fatty acids; BHBA = β-hydroxybutyric acid; ALT = alanine aminotransferase; AST = aspartate aminotransferase;
GGT = γ-glutamyltransferase; ALP = alkaline phosphatase; TRAP = tartrate-resistant acid phosphatase; Ca = Calcium; P = inorganic phosphorus; Mg = Magnesium;
Na = Sodium; K = potassium; Cl = Chlorine; Fe = iron.
a,b,c,d
Within class of age at puberty or age, values within a row with different superscripts differ significantly at P < 0.05.
1
Gr1, Gr2, Gr3 correspond to the three classes of age at puberty attainment before oestrus synchronisation: < 9 months of age (Gr1, n = 18), 9 to 12 months of age (Gr2,
n = 21), >12 months of age (Gr3, n = 17).
Significant differences: *P < 0.05; **P < 0.01; ***P < 0.001; t: P < 0.1; ns: not significant.

Table 5 Pearson’s correlations between age at puberty with BW and selected plasma parameters at different stages of growth in Holstein heifers
(based on the 56 animals presenting oestrous cyclicity before hormonal synchronisation)
BW Glucose Cholesterol Albumin ALP BHBA NEFA Ca Mg

6 months of age – − 0.24t − 0.49* – 0.35** – – –

9 months of age − 0.43** − 0.31* – − 0.31* – – – –
12 months of age − 0.44** 0.27* – – – − 0.26t 0.42** 0.32* 0.41**
15 months of age − 0.34* – – − 0.35** 0.38** − 0.30* – − 0.27*
ALP = alkaline phosphatase; BHBA = β-hydroxybutyric acid; NEFA = non-esterified fatty acids; Ca = Calcium; Mg = Magnesium.
Significant differences: **P < 0.01; *P < 0.05; tP <0.1.

puberty and consequently, at first insemination, the ADG
during the milk-feeding phase is of crucial importance. In
complement to optimal development, puberty was also
reached at an early age for most heifers. The concept of
puberty attainment as a function of reaching a specific fixed
proportion, not only of a mature BW but also of a mature
body size (height), has been stressed by several authors (Fox
et al., 1999; Duplessis et al., 2015). Mature BW and mature
body height can be estimated by random regression within
each breed (Cue et al., 2012). Puberty onset at 9 to 10 mo of
age or less meant that three or four oestrous cycles occurred
before insemination, which is generally consistent with good
fertility results in many species (Lin et al., 1986; Byerley et al.,
1987; Robinson, 1990; Le Cozler et al., 1998). Regardless of
calving strategy, lowering the age at puberty and, conse-
quently, the age at first insemination is an efficient way to
shorten the non-productive period before calving. However,
as suggested by Meyer et al. (2006), it might reduce pre-
pubertal mammary gland development by shortening the
allometric phase of mammary gland growth and, in some

1026

cases, impair further milk production. Investigations on these
aspects are still ongoing and not presented or discussed here.
Until 6 mo of age, BW was not affected by the feeding
treatment. Indeed, during the milk-feeding phase, all heifers
had free access to TMR1 and thus, SD heifers probably
compensated for the lower milk allowance (−15% compared
with ID1 and ID2) by feeding higher TMR1. The level of
feeding (milk and/or TMR) due to feeding treatments in this
experiment was higher than those observed in many com-
mercial farms and certainly could partially explain the lack of
difference. After 6 mo of age, only ID2 heifers were fed an
intensive diet; as a result, they presented better growth, not
only reflected by BW and/or ADG changes, but also by higher
plasma levels of ALP and P (at 9 mo) and total cholesterol
and creatinine (at 12 mo). The possible anabolic effect of the
season (Tucker et al., 1984) due to photoperiod exposure in
the 1st mo of life was limited in the present study. It is sug-
gested that a limited effect of photoperiod exposure during
the 0 to 6 mo of age stage, when heifers were fed a high level
of nutrients and/or that the effect of a photoperiod at this
time of year, might be limited.
Glucose, NEFA and urea levels were within the range of
those in most published papers (Taylor et al., 2004; Brickell
et al., 2009). When comparing plasma parameters according
to age, in many cases, the highest differences were observed
at 6 mo, confirming that most biochemical variables are age-
dependent in dairy (Abeni et al., 2000; Abeni et al., 2012)
and beef (Rodríguez-Sánchez et al., 2015) heifers, with the
same timing schedule. Throughout the trial, a marked dif-
ferentiation in plasma metabolite concentrations resulting
from the higher supplementation to ID2 after 6 mo of age
was noted. In fact, where SD and ID1 showed similar values
and globally similar changes in energy (BHBA and total
cholesterol) and protein-related metabolites (urea and crea-
tinine), ID2 heifers differed on either plasma values or
changes in time. Plasma glucose and NEFA did not differ
among experimental groups, thus evidencing the lack of
significant difference in energy availability during the mon-
itored period. The decline of glucose levels from 6 to 15 mo
of age, also noted by Samadi et al. (2014), was not affected
by the feeding treatment. Increasing the level of feed during
rearing, which resulted in higher BW and body condition
score, improved metabolite homeostasis for these heifers
and also resulted in a decreasing age at puberty. Many
hormonal changes occurred in this case (insulin, IGF1, Leptin),
in favour of earlier puberty attainment (Samadi et al., 2014).
Urea mean levels (3.4 to 3.6 mmol/l) from 6 to 15 mo of
age were considered to be a moderate value suitable for
dairy heifers (Brickell et al., 2009). According to previous
authors, a higher level would have reflected either too high
an intake or too high a catabolism of the proteins, which
would reflect non-optimal feeding strategies. The lower
plasma urea in ID2 at 9 and 12 mo of age is probably attri-
butable to the limited shift from pasture to TMR intake that
decreased the impact of a generally highly degradable source
of protein (the pasture). Previous papers (Abeni et al., 2000;
Abeni et al., 2012) on growing heifers also reported how
Blood profiles during growth in Holstein heifers
plasma urea mirrors the level of nitrogen availability, both
quantitatively and qualitatively (solubility). The higher
plasma creatinine values of ID2 heifers at 12 and 15 mo of
age were likely due to their higher body mass. If an increase
in plasma creatinine-to-albumin ratio is read as a sign of
body mass development (as confirmed by the trend in time
during growth), then ID2 heifers evidently showed body mass
growth earlier on than the other groups. These results con-
firmed how the supplementation in this trial supported early
development in ID2 group.
The mean plasma P values were outside of the range
reported for adult animals (Kaneko et al., 1997; Boudon
et al., 2018) but consistent for young heifers (Abeni et al.,
2000; Klinkon and Ježek, 2012). The higher plasma levels of
inorganic P in ID2 heifers at 9 mo of age are likely attribu-
table to the higher supplementation during pasture com-
pared to SD and ID1. Similarly, plasma Mg and other mineral
concentrations were also higher in ID2 heifers at 9 mo, and
were within the physiological range reported by Boudon
et al. (2018). To our knowledge, there are no papers speci-
fically describing how to interpret Mg levels in heifers.
Growth performance and class of age at puberty
The stratification according to class of age at puberty evi-
denced that heifers showing puberty at a late age (>1 year)
had poor energy status (higher NEFA), probably due to a
lower feed intake (lower total cholesterol and albumin),
resulting in lower ADG until puberty, and higher ALP activity.
As the main factors that can alter plasma ALP activity are
age, puberty and BW, and the bone isoenzyme is mainly
responsible for this variation, it can be hypothesised that the
higher values observed were a consequence of the delayed
puberty. Even if plasma cholesterol changes are tricky to
interpret, the results of this present study would suggest that
its level at 6 mo of age could be a good predictor of pre-
cocious puberty onset. If this result was masked at group
level in the ANOVA, it was clearer when evaluated at the
individual level in the logistic regression. In fact, the OR =
9.05 (P = 0.015) for plasma cholesterol suggests an impor-
tant effect of this metabolite at the individual level as a
marker of a favourable metabolism for puberty attainment.
At the start of metabolic profiling (6 mo), plasma cholesterol
concentration was similar among the three feeding treat-
ment groups, but overall it was tendentially lower in heifers
with delayed puberty onset. Thus, lower levels of BHBA and
cholesterol for progesterone synthesis at 6 mo in these hei-
fers indicates that cholesterol levels, rather than glucose
concentration, could be considered an interesting indicator
to predict early puberty attainment. Anderson et al. (2015)
also noted that a high level of cholesterol during early stages
of growth, due to feeding strategy, was related to the pre-
cocity of puberty attainment, in agreement with Rodriguez-
Sanchez et al. (2015). For heifers reaching puberty at an early
age, the higher uptake of circulating cholesterol to produce
progesterone (Yart et al., 2014) explains the difference in
circulating cholesterol. Results of odd-ratios obtained from
the logistic regression on puberty onset confirmed that the
1027
Abeni, Petrera and Le Cozler
most significant variable was the concentration of plasma
cholesterol. However, the optimal circulating value is yet to
be found.
In conclusion, the tested feeding strategy had minor con-
sequences on plasma metabolites, but clear effects on
growth performance and age at puberty. This finding has
important implications in commercial heifer herd manage-
ment to reduce dairy herd replacement costs. Plasma meta-
bolic profiles (cholesterol, NEFA and BHBA) at 6 mo of age, in
conjunction with BW monitoring, have potential power to
detect heifers at risk of delayed puberty. An earlier age at
puberty could also be an effective way to produce a larger
number of embryos in young Holstein dairy heifers of high
genetic-merit used as embryo donors, and thus accelerate
the genetic progress by reducing the interval between
generations.
Acknowledgements
The authors would like to thank the technical staff of INRA’s
experimental farm for taking care of the heifers and running
blood samplings. They also thank the GALA association for
providing funding for progesterone analysis.
Declaration of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Ethics statement
Experimental work has been conducted in accordance with
French national legislation on the use of animals for research.
Protocol received agreement (00944-02) from French Ethical
Committee no. 7.
Software and data repository resources
None of the data were deposited in an official repository.
Supplementary material
To view supplementary material for this article, please visit
https://doi.org/10.1017/S1751731118002422
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