Available online at www.sciencedirect.

com

Theriogenology 69 (2008) 688–699

www.theriojournal.com

The effect of feeding propylene glycol to dairy cows during

the early postpartum period on follicular dynamics and
on metabolic parameters related to fertility
D. Rizos, D.A. Kenny, W. Griffin, K.M. Quinn, P. Duffy, F.J. Mulligan,
J.F. Roche, M.P. Boland, P. Lonergan *
School of Agriculture, Food Science and Veterinary Medicine, College of Life Sciences,
University College Dublin, Belfield, Dublin 4, Ireland
Received 5 July 2007; received in revised form 17 November 2007; accepted 4 December 2007

Abstract
Postpartum dairy cows (n = 35) were used to determine the effects of feeding propylene glycol (PG) on metabolic variables
related to ovarian function and on oocyte developmental competence. Starting on Day 7 postpartum, each animal received an oral
dose (500 ml) of either PG or water once daily. Blood samples were collected on Days 5, 15, 25 and 35 pp to measure insulin, non-
esterified fatty acids (NEFAs), beta-hydroxybutyrate (BHB) and glucose concentrations. Oocytes were recovered by ultrasound
guided follicular aspiration starting on approximately Day 30 postpartum and submitted to in vitro fertilization. Ovarian follicular
activity was examined daily by ultrasonography from Day 7 until ovulation or Days 35–40 postpartum. Animals receiving PG had
elevated insulin concentrations over the subsequent 90 min following dosing (P < 0.05) compared to control animals. Glucose
concentrations followed a similar pattern. Irrespective of treatment, concentrations of NEFA declined significantly from Days 15 to
35 postpartum. Administration of PG resulted in a decrease in NEFA (P < 0.001) and BHB (P < 0.001) over the subsequent 90 min
compared to control animals. Treatment with PG had no effect on follicular dynamics, mean days to emergence of the first cohort of
follicles postpartum, or days to dominance and duration of dominance for any follicular wave recorded postpartum. There was also
no difference in mean days to first ovulation or in size of the preovulatory follicle between treatments. Oocyte quality as measured
by blastocyst development after IVF was not affected by treatment. These results suggest that administration of PG has the ability to
positively alter the systemic concentrations of a number of metabolic variables which have been related to fertility. However, we did
not observe an effect of PG treatment on follicular dynamics or the length of the postpartum interval. An effect on oocyte
developmental competence remains to be proven.
# 2008 Elsevier Inc. All rights reserved.

Keywords: Fertility; Oocyte quality; Metabolic status; Dairy cow

1. Introduction

Over the past 30 years there has been a significant

* Corresponding author at: School of Agriculture, Food Science and
Veterinary Medicine, College of Life Sciences, University College
Dublin, Belfield, Dublin 4, Ireland. Tel.: +353 1 7166206/6012147;
fax: +353 1 6288421.
E-mail address: pat.lonergan@ucd.ie (P. Lonergan).
worldwide decline in conception rate to first service in
Holstein cows [1,2]. This reduction in conception rate,
in the order of 0.45–1% per annum, has been
concomitant with increased milk production per cow.
High milk yields have been associated with lower

0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2007.12.001
 D. Rizos et al. / Theriogenology 69 (2008) 688–699
reproductive efficiency, though it is not a cause and
effect relationship [3] and it is more likely mediated
through an induction of a negative state of energy
balance during early lactation [4]. However, the
mechanisms involved have not been elucidated.
In the postpartum dairy cow, energy balance is the
difference between the dietary intake of utilisable energy
and energy expenditure for body maintenance and milk
synthesis [1]. Parturition results in an abrupt shift in
metabolic demands from nutrient accrual (body reserves
and foetal mass) to rapid mobilisation of lipid and protein
stores in support of the sudden onset of high milk
production [5,6]. The modern high genetic merit dairy
cow prioritises nutrient supply towards milk production
in early lactation and this demand takes precedence over
the provision of optimal conditions for reproduction [7].
In the period after parturition, dry matter intake must
increase four- to sixfold in order to meet the high nutrient
demands of lactation. However, owing to a lower rate of
increase in feed energy intake compared with the energy
output associated with milk production, the high
producing dairy cow typically experiences a variable
period of negative energy balance (NEB) during early
lactation. This NEB is characterised by a loss of body
weight and mobilisation of body reserves in the form of
fat and protein and may persist for 10–12 weeks of
lactation [5]. The largest part of this mobilisation takes
place in the first week of lactation [8].
Severe NEB in the early postpartum period may affect
follicular development and (or) oocyte competence and,
as a consequence, embryo survival. While negative
energy balance during the first 3–4 weeks postpartum is
highly positively correlated with the interval to first
ovulation [6], follicular growth and recruitment of a
dominant follicle (DF) seem to be independent of energy
status [9]. It appears that the ultimate diameter and
steroidogenic capacity of dominant follicles is influenced
more by metabolic factors such as insulin and insulin-like
growth factor (IGF-I).
Negative energy balance delays the time of first
ovulation through inhibition of LH pulse frequency and
low levels of blood glucose, insulin and IGF-I that
collectively restrain oestrogen production by dominant
follicles [6]. Both insulin and IGF-I enhance follicle cell
function in vitro in several species including cattle
[10,11]. Summarising the results of several studies,
Beam and Butler [1] concluded that dominant follicles
of lactating cows in NEB have lower oestradiol output
per unit of gross follicular size compared with non-
lactating cows in positive energy balance. Beam and
Butler [9] reported that ovulation failure during the first
postpartum follicular wave was accompanied by lower
689
peak plasma oestradiol concentrations, smaller DF
maximum diameter and lower concentrations of IGF-I.
Indeed, systemic IGF-1 concentrations in the first 2–3
weeks of lactation have been associated with increased
probability of ovulation of the first postpartum
dominant follicle [3] and increased conception rate to
first service [3,12].
Low insulin concentrations are associated with NEB
in the early postpartum period [13]. Cows fed glycerol
and Ca-propionate, both glycogenic agents, had a
shorter interval between calving and last insemination
[14]. Artificially increasing insulin concentrations by
dietary manipulation has been associated with advance-
ment of initiation of oestrous cyclicity in the postpartum
period [15] and enhancement of early embryo devel-
opment in beef heifers [16].
Propylene glycol, also known as 1,2-propanediol, is
a 3-carbon compound (C3H8O2) derived from propy-
lene. PG is frequently used as an oral drench in order to
increase the molar percentage of ruminal propionate in
the treatment of ketosis in postpartum dairy cattle
probably because of its ability to lower non-esterified
fatty acid concentrations [17]. Its use may offer an
alternative to conventional dietary manipulation such as
increased inclusion of starch or sugars. Ruminal
administration of propylene glycol produced a greater
molar percentage of ruminal propionate and elicited a
greater insulin response compared with dietary starch
[18]. Grummer et al. [17] found that increasing amounts
of PG linearly increased glucose and insulin and
decreased beta-hydroxybutyrate and NEFA in blood;
they found that 296 ml of PG was nearly as effective as
887 ml in reducing lipid mobilisation during restricted
feed intake; however, the increase in insulin was lower
with 296 ml versus 887 ml of PG.
After oral administration, a portion of PG is
metabolized to propionate but the majority escapes
the rumen untransformed to be converted to glucose by
the liver, primarily via the lactaldehyde pathway and
subsequent oxidation to lactate. Propionate is trans-
ported to the liver through the portal system where it is
transformed into pyruvate and eventually glucose via
oxaloacetate. Plasma concentrations of both glucose
and insulin are known to increase in response to PG
[18,19]. The downstream effect of such induced
changes in metabolite concentration on oocyte devel-
opmental competence has not been investigated.
The aim of this study was to determine the effect of a
daily oral drench of PG in the early postpartum period on
follicular dynamics and metabolic parameters related to
fertility. In addition, the downstream effects of PG
administration on oocyte developmental competence was
690 D. Rizos et al. / Theriogenology 69 (2008) 688–699
assessed using ovum pick-up (OPU) and in vitro
fertilization. Our hypothesis was that PG would increase
serum insulin and glucose thereby stimulating ovarian
function and oocyte quality.
2. Materials and methods
2.1. Animals and treatments
Holstein–Friesian spring-calving cows (n = 35) were
paired by calving date, parity (mean parity 3, range
from 1 to 5) and prior milk production (PG:
6584  258 kg; control: 6286  278 kg) into two
groups. One member of each pair was assigned
randomly to PG or control groups. The study was
carried out over 2 years: in Year 1, 13 multiparous (6
treated, 7 control) and 12 primiparous (6 treated, 6
control) dairy cows were used, while in Year 2, 22
multiparous dairy cows were used (11 per group).
Animals were allocated to treatment on Day 7
postpartum and remained on their respective treat-
ment until completion of ovum pick-up in Year 1 or
day of first ovulation in Year 2. In both years, all cows
were fed a standard ration following calving consist-
ing of 50:50 maize silage:grass silage forage ad
libitum plus 6 kg concentrates per day at milking
(07:00 and 16:00). Each cow in the PG group (n = 17)
was given an oral dose (500 ml) of PG once daily at
2.5 h after morning milking for the period of the
experiment while control animals (n = 18) were given
an oral dose (500 ml) of water at the same time. Cows
were housed indoors on cubicles for the duration of
the experiment.
2.2. Liveweight and body condition score (BCS)
Liveweight and body condition score were recorded
on the day of allocation to treatment, midway through
the experiment and again at the end of the experimental
period for treated and control animals, respectively.
Body condition was assessed by the same technician
and was based on the six-point scale described by
Lowman et al. [20], in which a score of 0 indicates
severe emaciation and a score of 5 indicates obesity.
2.3. Milk yield and composition
Milk yield and composition was recorded for each
cow during both years. Only mean milk yield during the
experimental period is presented here. Milk fat and
protein concentrations were measured on Days 5, 15,
25, 35 and 45 of lactation for all animals.
2.4. Follicular dynamics/ovarian activity
In Year 2, ovarian follicular activity was examined
daily by ultrasonography (Aloka SD900 with 7.5 MHz
linear transducer, Aloka, Japan) in PG and control
animals from Day 7 until ovulation or Days 35–40
postpartum, at which time animals returned to the
breeding herd. All follicles greater than 2 mm were
counted and measured. A dominant follicle was defined
as a follicle >8.5 mm that outgrew other subordinate
follicles present at the same time.
2.5. Blood sampling and analysis
Blood samples were obtained by jugular venepuncture
on approximately Days 5, 15, 25 and 35 postpartum. On
each day, serum concentrations of insulin, NEFA, BHB
and glucose were measured at 0, 30, 60 and 90 min
following administration of PG or water.
Serum insulin was analysed using a fluorimmu-
noassay kit (DELFIA, Walloc Oy, Turku, Finland) as
described by Løvendahl and Purup [21]. At a
concentration of 3.80 and 14.30 mIU/ml, the intra-
assay coefficients of variation were 3.96 and 0.68%,
respectively, and the inter-assay coefficients of variation
were 8.91 and 0.02%, respectively. NEFA, BHB and
glucose concentrations were determined in an auto-
analyser (Bayer opera) using kits supplied by Randox
Laboratories (Crumlin, UK) for NEFA (catalogue no:
FA115), BHB (catalogue no: RB1008) and glucose
(catalogue no: GL2623).
In Year 2, concomitant with ultrasound scanning of
the ovaries, daily blood samples (serum) were taken for
oestradiol concentration (Adaltis MAIA kit product
code 37001, Biostat) (Fig. 1). In addition, all animals
were blood sampled to determine serum LH concentra-
tion on the day following establishment of the first
dominant DF (DF1; between Days 10 and 13 pp). An
indwelling jugular catheter was inserted and blood was
harvested every 12 min over an 8 h period.
2.6. Oocyte development
In Year 1, immature oocytes were collected from PG
and control cows by ovum pick-up carried out over four
sessions within a 2-week period starting around Days
30–35 postpartum. Before the first OPU session follicle
ablation was carried out in all animals. Beginning 2
days later, three injections of FSH (Pluset1, Calier,
Barcelona, Spain, total dose of 150 IU FSH + 150 IU
LH) were given at 12 h intervals. OPU was carried out
12–15 h after the last injection. Beginning the day after
 D. Rizos et al. / Theriogenology 69 (2008) 688–699
Fig. 1. Experimental design. Holstein–Friesian cows were allocated to treatment on Day 7 postpartum and were given a once daily oral dose
(500 ml) of either propylene glycol or water. Follicular dynamics were followed by daily ultrasound scanning and blood metabolites measured as
indicated.
OPU animals received three injections of FSH again
followed by another session of OPU. The recovered
oocytes were morphologically graded and only those
showing a uniform cytoplasm and compact cumulus
investment were submitted to in vitro maturation,
fertilization and culture as described previously [22].
3. Statistical analyses
All data from both experiments were checked for
adherence to a normal distribution (PROC UNIVARI-
ATE, SAS v. 6) [23]. Log transformation of the data was
carried out where appropriate. Data for each experiment
were analysed separately. Data on postpartum follicular
dynamics were analysed using one-way ANOVA fitting
the effect of treatment. The effect of propylene glycol
on oestradiol concentrations and on LH pulses were
analysed using two-way ANOVA including treatment,
ovulation status and the interaction between treatment
and ovulation status in the model. A split-plot design
including the above effects and the effect of cow was
used to analyse the LH concentration data. A split-split
plot design was used to analyse plasma concentrations
of insulin, NEFA, glucose, and BHB. Main effects of the
whole plot were treatment and year and the treat-
ment  year interaction, main effect of the split plot
was days postpartum, and main effect of the split-split
plot was sampling time. The whole plot effects were
tested using cow nested within treatment and year. The
split plot effects were tested using interaction between
days postpartum and cow nested within treatment and
year and the split-split plot effects were tested against
residual error. Values for follicle counts were analysed
using a split-plot design, with treatment and date of
691
OPU nested within animal. Linear (PROC REG, SAS)
and multiple regression (PROC STEPWISE, SAS)
analyses were used to determine relationships between
days to first ovulation and plasma analytes. Unless
stated otherwise, results are presented as the untrans-
formed means and S.E.M.
4. Results
4.1. Liveweight and body condition score
There were neither treatment  year nor treatment
by day interactions for liveweight or BCS (P > 0.05).
Body condition score declined from allocation to
treatment (2.69  0.12) to the end of the experimental
period (2.36  0.12) but was not affected by treatment
(P > 0.05).
4.2. Milk yield and composition
There was no treatment  year interaction on milk
yield (P > 0.05). Mean  S.E. daily milk yield across
both years was 24.87  2.078 and 27.99  1.133 kg for
PG and control and did not differ between treatments.
Similarly there was no treatment  year or treat-
ment  sample time interactions for either milk fat or
protein concentration. Mean  S.E. milk fat across
tended to be lower in PG treated compared with control
animals (4.80  0.168 vs. 4.29  0.194, P = 0.06).
Milk protein concentration was not affected by
treatment (3.26  0.076 vs. 3.22  0.093 for PG and
water, respectively). There was an effect of sample time
on milk protein concentrations with concentrations
decreasing linearly with days postpartum (P < 0.001).
692 D. Rizos et al. / Theriogenology 69 (2008) 688–699

Table 1
Effect of propylene glycol treatment on follicular dynamics in the postpartum period
Parameter PG CON P value
Emergence of cohort 1 8.45  0.43 (11) 7.64  0.31 (11) 0.14
First day of dominance DF1 12.09  0.67 (11) 10.64  0.53 (11) 0.10
Duration of dominance DF1 6.91  1.67 (11) 9.0  1.45 (10)b 0.18
Emergence of cohort 2 17.0  0.93 (8) 18.63  1.71 (8) 0.44
First day of dominance DF2 20.14  0.55 (8) 22.0  2.16 (8) 0.45
Duration of dominance DF2 7.14  1.24a (7) 5.29  0.57 (8) 0.10
Emergence of cohort 3 25.67  1.33 (3) 21.67  0.88 (3) 0.07
First day of dominance DF3 29.33  1.45 (3) 25.0  1.53 (3) 0.11
Duration of dominance DF3 4.0  0.0 (2) 8.0  1.73 (3) 0.17
Maximum size DF1 (mm) 16.68  1.60 (11) 18.3  2.13b (10) 0.45
Maximum size DF2 (mm) 18.07  1.00a (7) 18.88  1.04 (8) 0.61
Maximum size DF3 (mm) 18.0  1.16 (3) 18.67  0.88 (3) 0.67
Days to first postpartum ovulation 25.86  2.69 (7) 28.75  1.16 (8) 0.32
Values refer to days postpartum unless otherwise stated.
a
One animal (no. 555) removed because DF2 became cystic.
b
One animal (no. 586) removed because DF1 became cystic.

4.3. Postpartum follicular dynamics
Administration of PG had no effect (P > 0.05) on the
dynamics of follicular development in the early
postpartum period (Table 1). Overall, irrespective of
treatment, 68.2% (15/22) of cows ovulated during the
trial period; 33.3% (5/15) ovulated DF1, 53.3% (8/15)
ovulated DF2 and 13.3% (2/15) ovulated DF3. Of the
seven animals that failed to ovulate during the
experiment, two had cystic follicles (Fig. 2). Amongst
those animals that ovulated, days to first ovulation was
not affected by treatment (PG: 25.86  2.69; control:
28.75  1.16, P = 0.32).
4.4. Serum insulin
The effect of treatment on serum concentration of
insulin is presented in Fig. 3. There were no interactions
between treatment, sample day or year on serum insulin
concentrations (P > 0.05) and therefore the data were
pooled across years and sampling days. There was a
treatment  time within day interaction on insulin
(P < 0.001); administration of PG resulted in a
significant elevation in insulin concentrations over
the subsequent 90 min compared to control animals
(Fig. 3).
4.5. Serum metabolites
The effect of treatment on serum concentration of
NEFA is presented in Fig. 4. There was no treatment 
year or treatment  day interactions for serum con-
centrations of NEFA (P > 0.05). However, there was a
day effect (P < 0.001); concentrations of NEFA (mM)
declined from Day 15 (0.78 + 0.53) to Day 25
(0.59 + 0.34) to Day 35 (0.40 + 0.26). There was a
treatment  time interaction (P < 0.001); administra-
tion of PG resulted in a significant decrease in NEFA
concentrations over the subsequent 90 min compared to
control animals (Fig. 4).
The effect of treatment on serum concentration of
BHB is presented in Fig. 5. There was no treatment  -
year interaction and no year effect on serum
concentrations of BHB; neither were there any
treatment  day nor year  day interactions and the
data were therefore pooled across both years and
sampling days. There was a treatment  time interac-
tion (P < 0.001); administration of PG resulted in a
decrease in BHB concentrations over the subsequent
90 min compared to control animals (Fig. 5).
The effect of treatment on serum concentrations of
glucose is shown in Fig. 6. Although there was no
treatment  year interaction on serum glucose con-
centrations, there was a treatment  time interaction
(P < 0.001); glucose concentrations increased over the
90 min following administration of PG in contrast the
control animals (Fig. 6).
4.6. Relationship between plasma analytes,
lactation variables and length of the postpartum
period
Regression analysis was used to determine the
relationship between plasma concentrations of insulin
and metabolites and days to first ovulation across
treatments. There were weak but statistically significant
relationships between the following plasma analytes
and days to first ovulation: NEFA (slope = 1.56;
 D. Rizos et al. / Theriogenology 69 (2008) 688–699 693

Fig. 2. Representative graphs of follicular dynamics for cows with one (A), two (B), three (C) or four (D) waves of follicular growth prior to first
ovulation postpartum as well as an animal with a cystic follicle (E). (^) DF1, (&) DF2, (~) DF3, (*) DF4, (*) peripheral oestradiol (E2) concentration.

R2 = 0.03; P = 0.022), BHB (slope = 2.28; R2 = 0.08; showed no additive effects of combining variables to
P < 0.001), glucose (slope = 2.29; R2 = 0.06; P < explain variation in days to first ovulation.
0.01). There was no relationship between insulin and Similar to the plasma analytes, milk yield and
days to first ovulation. Stepwise regression analysis composition data for the first 4 weeks of lactation were
694 D. Rizos et al. / Theriogenology 69 (2008) 688–699
Fig. 3. Effect of oral administration of propylene glycol (^) or water
(&) on peripheral insulin concentrations in postpartum dairy cows.
also regressed against days to first ovulation. There was
no relationship between milk protein and fat concentra-
tions with days to first ovulation. Similarly, no relation-
ship could be established between either average or
cumulative milk yield and days to first ovulation.
4.7. Serum oestradiol
There was no difference (P > 0.05) between treat-
ments in mean (S.E.) concentration of oestradiol (pg/
ml) from day of dominance of DF1 to new wave
emergence for (i) animals that ovulated DF1 (PG:
1.17  0.181 vs. control: 2.03  0.183) or (ii) those that
did not ovulate DF1 (PG: 0.32  0.060 vs. control:
0.70  0.046). For PG-treated animals, oestradiol
concentrations were higher in those that ovulated
Fig. 4. Effect of oral administration of propylene glycol (^) or water
(&) on peripheral non-esterified fatty acid (NEFA) concentrations in
postpartum dairy cows.
Fig. 5. Effect of oral administration of propylene glycol (^) or water
(&) on peripheral beta-hydroxybutyrate (BHB) concentrations in
postpartum dairy cows.
DF1 (1.17  0.120) compared with those that failed to
ovulate DF1 (0.32  0.110, P < 0.05, n = 79 observa-
tions). For control animals this difference approached
statistical significance (2.03  0.136 vs. 0.70  0.094,
P = 0.066, n = 91 observations; for animals that
ovulated or failed to ovulate DF1, respectively).
Overall, animals that ovulated DF1 had higher
concentrations of oestradiol than those that failed to
ovulate (1.60  0.091 vs. 0.51  0.074, P < 0.01).
There was no treatment effect on the maximum
concentration of oestradiol at dominance of DF1 for
either (i) animals that ovulated DF1 (PG: 2.09 + 0.83,
control: 4.08 + 1.17) or (ii) those that did not ovulate
DF1 (PG: 0.53 + 0.25, control 1.17 + 0.23). Within
treatment, there was a significant difference in the
maximum concentration of oestradiol at dominance of
Fig. 6. Effect of oral administration of propylene glycol (^) or water
(&) on peripheral glucose concentrations in postpartum dairy cows.
 D. Rizos et al. / Theriogenology 69 (2008) 688–699
DF1 between animals that ovulated and those that did not
ovulate (PG: 2.09  0.348 vs. 0.53  0.263, P < 0.01;
control: 4.08  0.0.87 vs. 1.17  0.0.436, P < 0.05).
Similar trends were observed for DF2 although the
numbers were much smaller.
4.8. Serum LH
There was no effect of treatment on (i) the mean
concentration of LH over 8 h (PG: 0.38  0.086;
control: 0.32  0.103), (ii) the number of pulses per
8 h (PG: 3.68  0.655; control: 3.44  0.697) or (iii)
the mean amplitude of the pulses (PG: 0.44  0.087;
control: 0.75  0.106).
4.9. Oocyte recovery and development
The number of follicles punctured (13.8  1.02 vs.
10.7  1.04), the number of oocytes recovered
(4.5  0.53 vs. 3.5  0.61) and the number of good
quality oocytes (2.8  0.35 vs. 1.8  0.35) were not
different between treated and control cows. Although,
cleavage rate (68.3% vs. 58.9%) and blastocyst yield on
Day 8 (25.3% vs. 14.4%) were numerically higher for
treated cows, the differences were not statistically
significant (P > 0.05).
5. Discussion
Negative associations between energy status in early
lactation and subsequent reproductive performance
have been reported in a number of previous studies
(see review by Butler [6]). The importance of high
systemic glucose, insulin and IGF-1 concentrations in
early lactation in reducing the incidence of metabolic
diseases the rapid initiation of oestrous cyclicity as well
as the success of subsequent pregnancy have been
reported [3,12]. The objective of the current study was
to identify the usefulness of administration of propylene
glycol, an insulinogenic agent, on metabolic and
endocrinological response, ovarian follicular dynamics,
oocyte quality and embryo development. The results of
this study show that although the treatments imposed
resulted in divergence in a number of important
metabolic variables in both lactating and non-lactating
animals there was no effect on the key reproductive
variables measured.
5.1. Dry matter intake
The cow’s capacity to ingest dry matter rather than
milk yield per se has been shown to be the major
695
regulator of energy balance mediated effects on
reproductive performance [24]. However, increasing
dietary energy intake is constrained by the necessity to
maintain adequate fibre in the diets. While strategies to
appreciably increase dietary energy density such as
lipid [25] or starch [26] supplementation have been
shown to improve energy balance both approaches have
also been associated with negative effects on rumen
function and dry matter intake. Although dry matter
intake was not measured in the current study, we
adopted a strategy to improve the metabolic status of the
cow, through supplementation with an energy substrate,
which has been shown previously not to affect dry
matter intake [27].
5.2. Liveweight and BCS
Excessive loss of body condition in early lactation
and low BCS at the start of the breeding season have
been related to poor cow fertility in previous studies
[28,29]. In the current study, BCS declined during the
first 6 weeks of lactation to a similar extent in both
the supplemented and unsupplemented cows despite the
potential anti-lipolytic effects of higher systemic insulin
concentrations. Perhaps the transitory increases in
insulin were insufficient to appreciably alter lipogenic
pathways and adipocytes may require constant positive
stimulus [30]. We found no relationship between PG
supplementation and BCS in agreement with the
findings of Formigoni et al. [19].
5.3. Milk yield and composition
In the current study there was no association between
either milk yield or composition in early lactation and
any reproductive variable measured, in agreement with
Formigoni et al. [19]. Although, Buckley et al. [28]
reported that reproductive performance declined as
genetic merit for milk yield increased, reproductive
performance was found to be positively associated with
milk yield, after adjustment for genetic merit. Similarly,
both Morton [31] and Patton et al. [24] failed to
establish any difference in reproductive performance as
a consequence of cow milk yield; however, both studies
reported that cows with higher milk protein content in
early lactation had significantly better reproductive
performance. Milk protein content is recognised as an
important indicator of EB, cows with lower milk protein
concentrations indicating a more prolonged and severe
energy deficit [32]. Consistent with this, Patton et al.
[24] reported a positive relationship between milk
protein concentration in early lactation and onset of
696 D. Rizos et al. / Theriogenology 69 (2008) 688–699
cyclicity and subsequent conception rate in moderate
yielding dairy cows. In the current study there was no
evidence of a relationship between concentrations of
milk protein and days to first ovulation but this may
have been a function of the relatively low number of
animals with ovarian records available.
5.4. Metabolites and metabolic hormones
In the current experiment, although daily DMI was
not measured, profiles recorded in BCS, milk yield and
serum metabolites indicate that all cows were in NEB
for at least the first 6 weeks of lactation. Metabolic
profiles are frequently used to assess energy balance,
however, there is a lack of consensus as to their value
[33]. Interactions between homeostatic control mechan-
isms within the animal and factors governing rumen
function have contributed to the complexity of
interpreting metabolic profiles leaving cause and effect
relationships difficult to elucidate, consequently, their
use in assessing energy status is likely to be of greatest
value in association with a complete systemic nutri-
tional assessment [33].
BHB concentrations were lowered in PG-treated
animals throughout the experiment and decreased in
both parity groups in the period following PG treatment.
Similarly, concentrations of NEFA were lower in both
parity groups in PG-treated animals. In agreement,
Grummer et al. [17] reported decreased BHB and NEFA
concentrations in nutritionally restricted pregnant
heifers, and found daily drenching with 296 g PG
nearly as effective as 900 g in reduction of lipid
mobilisation. Hoedemaker et al. [34] reported lower
concentrations of NEFA and BHB in the peripartum
period in cows receiving PG compared with controls.
Formigoni et al. [19] showed that propylene glycol
administration reduced plasma NEFA, increased total
cholesterol but did not affect BHB concentrations.
Butler et al. [27] reported lower systemic concentrations
of both NEFA and BHB in lactating dairy cows
drenched with PG. In agreement with the findings of
Christensen et al. [35] and Butler et al. [27], PG
treatment in this study was effective at increasing blood
glucose and reducing plasma NEFA in animals in NEB
(lactation).
The associations between the metabolites NEFA and
BHB and early-lactation EB are well-established, and
reflect the increased mobilisation of body lipid reserves
and partitioning of nutrients towards milk production.
Addition of NEFA to culture media has been shown to
have negative effects on in vitro oocyte maturation and
fertilization [36]. In a retrospective analysis of two in
vivo studies no relationship could be established,
however, between either NEFA or BHB measured in
the early postpartum period and commencement of
luteal activity or subsequent conception rate to first
service [24].
In the dairy cow, blood glucose concentrations reflect
the contribution of gluconeogenesis, the synthesis of
glucose from non-carbohydrate precursors (lactate,
amino acids and glycerol), and also glycogenolysis,
the mobilisation of glucose from glycogen stored in the
liver and muscle as well as the level and type of dietary
carbohydrate intake. Glucose is the main energy source
of the bovine ovary [37] and is the main energy
substrate of the bovine embryo post-blastulation [38].
Low blood glucose inhibits the GnRH pulse generator
and consequently LH and may contribute to the length
of the postpartum interval in lactating dairy cows in
NEB [6]. In the current study administration of PG led
to transient increases in glucose concentrations in
agreement with other studies [17,18,27,39]. However, it
is possible that the elevated concentrations were not
sufficient to evoke any meaningful effect on reproduc-
tive events.
Propylene glycol-treated animals in the studies
reported here had increased systemic concentrations
of insulin in the immediate post-drenching period which
extended beyond any divergence in serum glucose.
Similarly, other studies have shown rapid postprandial
increases in systemic concentrations of insulin follow-
ing administration of PG to dairy cows [18,27,35,40]
and heifers [17,39]. Furthermore, Christensen et al. [35]
showed that the method and format of administration of
PG is important to its insulinogenic efficacy with
drenching or feeding as part of a bolus concentrate more
effective than offering small, regular quantities as part
of a total mixed ration.
Insulin is involved in the homeostatic control of
blood glucose concentrations and the release of insulin
is responsive to both the absolute as well as the rate of
change in glucose concentrations. Low insulin con-
centration is associated with NEB in the early
postpartum period [13]. Insulin is involved in the
proliferation of and steroidogenesis of granulosa cells
and therefore may influence follicular development [6].
Furthermore, insulin also acts directly on bovine antral
follicles, appearing less potent than IGF-I at stimulating
proliferation but equipotent in relation to oestradiol
production [41]. Beam and Butler [9] reported that cows
ovulating the first postpartum dominant follicle had
higher plasma insulin concentrations during the first
week after parturition than cows that failed to ovulate.
Butler [6] suggested that the most important role of
 D. Rizos et al. / Theriogenology 69 (2008) 688–699
insulin in cow fertility may be its regulatory effect on
blood glucose concentrations. However, in a retro-
spective analysis of two studies Patton et al. [3] failed to
establish any association between early postpartum
insulin or glucose concentrations and either commence-
ment of luteal activity or conception rate to first service.
In agreement, no relationship was observed in the
current study between early postpartum insulin con-
centrations and length of the postpartum anoestrous
interval.
5.5. Follicular dynamics
Many studies which have monitored commercial
dairy herds using milk progesterone profiles have shown
that NEB is associated with a greater incidence of
irregular cycles that can both increase the interval to
first service and reduce conception rates [42]. Some
studies [9], though not all [24] have identified the
interval to EB nadir as being important to the timing of
first ovulation, with dominant follicles emerging after
the nadir having enhanced oestradiol secretion and a
greater likelihood of ovulatory success. In the current
study, there was no difference between treatments in
concentration of oestradiol from day of dominance to
new wave emergence.
Butler et al. [27] reported no difference between
control and PG-treated cows in the fate of the first
postpartum dominant follicle (i.e., ovulation, atresia or
cystic). Similarly in that study, no difference was
detected between treatments in maximum diameter of
the dominant follicle, peak plasma oestradiol concen-
tration, or day of peak plasma oestradiol concentration
[27]. In the current study there was no detectable effect
of treatment on LH pulse frequency or amplitude. This
is in agreement with the study of Butler et al. [27], who
also, despite evidence of improved metabolic status,
failed to show an increase LH pulse frequency, LH pulse
amplitude, or mean circulating LH measured on Day 10
postpartum PG-treated dairy cows. Canfield and Butler
[43] showed that LH pulsatility starts to increase soon
after cows reach their NEB nadir. However, in the study
of Jorritsma et al. [44] no relationship was detected
between LH pulsatility characteristics and days to first
ovulation and neither was time to NEB nadir related to
the occurrence of the first ovulation postpartum.
5.6. Oocyte and embryo development
Britt [45] developed a theoretical model based on the
rates of follicular growth estimated by Lussier et al.
[46]. This theory proposed that follicles that developed
697
during conditions of NEB would have altered gene
expression leading to impaired development. This would
lead to dysfunctional mature follicles, containing oocytes
with a low developmental capacity. Consequently, the
third and fourth ovulatory follicles (which may ovulate
80–100 days postpartum) experience much of their early
development when energy balance is most negative [47].
Short-term fluctuations in energy intake [48] or
prolonged depletion of body reserves during early
lactation [6], can have significant detrimental effects on
resumption of ovarian activity postpartum and concep-
tion rates. Poor energy status has been associated with
lower oocyte recovery rates [36,49,50] and reduced
oocyte competence and subsequent embryo develop-
ment [50] in dairy cows. Nolan et al. [51] reported no
effect of energy level on oocyte quality or develop-
mental competence in vitro; however a higher number
of oocytes developed in vitro to blastocysts from
animals on a low energy allowance compared to those
fed ad libitum. Similarly, in another recent study, early
postpartum dairy cows, fed a low energy diet were
found to yield oocytes with an improved cleavage rate
and morphology and a tendency toward a better
blastocyst formation rate, than cows fed medium or
high-energy diets [52]. Kendrick et al. [49] fed lactating
cows high or low energy diets after calving and NEB
lasted for 3 weeks versus 6 weeks of lactation,
respectively. Cows on the high-energy diet produced
more oocytes and oocytes of a higher quality compared
with cows in greater NEB. In the current study, the
number of oocytes recovered was not affected by
treatment; despite a higher cleavage rate and blastocyst
yield in treated cows, the difference was not significant.
6. Conclusion
The importance of the early postpartum period to
overall reproductive efficiency in the cow cannot be
over stressed; this is the period when maximum energy
output is coupled with minimal energy intake. Events
occurring during this window of time may have both
concurrent and latent effects on the reproductive
process, the consequences of which may only become
apparent during the breeding period and be manifested
either directly or interactively as lowered oocyte
competence, aberrations in the steroidal environment,
or disruptions in the oviductal or uterine environment,
all leading to compromised embryo development and
survival. Despite evidence of altered metabolic status in
response to PG in the present study, we failed to observe
an effect on the measures of fertility assessed. Further
study is warranted.
698 D. Rizos et al. / Theriogenology 69 (2008) 688–699
References
[1] Beam SW, Butler WR. Effects of energy balance on follicular
development and first ovulation in postpartum dairy cows. J
Reprod Fertil Suppl 1999;54:411–24.
[2] Royal MD, Darwash AO, Flint APF, Webb R, Woolliams JA,
Lamming GE. Declining fertility in dairy cattle: changes in
traditional and endocrine parameters of fertility. Anim Sci
2000;70:487–502.
[3] Patton J, Kenny DA, Mee JF, O’Mara FP, Wathes DC, Cook M,
et al. Effect of milking frequency and diet on milk production,
energy balance, and reproduction in dairy cows. J Dairy Sci
2006;89:1478–87.
[4] Grohn YT, Hertl JA, Harman JL. Effect of early lactation milk
yield on reproductive disorders in dairy cows. Am J Vet Res
1994;55:1521–8.
[5] Bauman DE, Currie WB. Partitioning of nutrients during
pregnancy and lactation: a review of mechanisms involving
homeostasis and homeorhesis. J Dairy Sci 1980;63:
1514–29.
[6] Butler WR. Nutritional interactions with reproductive perfor-
mance in dairy cattle. Anim Reprod Sci 2000;60:449–57.
[7] O’Callaghan D, Yaakub H, Hyttel P, Spicer LJ, Boland MP.
Effect of nutrition and superovulation on oocyte morphology,
follicular fluid composition and systemic hormone concentration
in ewes. J Reprod Fertil 2000;118:303–13.
[8] Tamminga S, Luteijn PA, Meijer RGM. Changes in composition
and energy content of liveweight loss in dairy cows with time
after parturition. Livestock Prod Sci 1997;52:31–8.
[9] Beam SW, Butler WR. Energy balance and ovarian follicle
development prior to the first ovulation postpartum in dairy
cows receiving three levels of dietary fat. Biol Reprod 1997;56:
133–42.
[10] Savion N, Lui GM, Laherty R, Gospodarowicz D. Factors
controlling proliferation and progesterone production by bovine
granulosa cells in serum-free medium. Endocrinology
1981;109:409–20.
[11] Spicer LJ, Alpizar E, Ecternkamp SE. Effects of insulin, insulin-
like growth factor 1, and gonadotropins on bovine granulosa cell
proliferation, progesterone production, estradiol production, and
(or) insulin-like growth factor 1 production in vitro. J Anim Sci
1993;71:1232–41.
[12] Taylor VJ, Cheng Z, Pushpakumara PG, Beever DE, Wathes DC.
Relationships between the plasma concentrations of insulin-like
growth factor-I in dairy cows and their fertility and milk yield.
Vet Rec 2004;155:583–8.
[13] McCann JP, Hansel W. Relationships between insulin and glu-
cose metabolism and pituitary-ovarian functions in fasted hei-
fers. Biol Reprod 1986;34:630–41.
[14] Pehrson B, Plym Forshell K, Carlsson J. The effect of additional
feeding on the fertility of high-yielding dairy cows. Zentralbl
Veterinarmed 1992;39:187–92.
[15] Gong JG, Lee WJ, Garnsworthy PC, Webb R. The effect of
dietary induced increases in circulating insulin concentrations
during the early postpartum period on reproductive function in
dairy cows. Reproduction 2002;123:419–27.
[16] Mann GE, Green MP, Sinclair KD, Demmers KJ, Fray MD,
Gutierrez CG, et al. Effects of circulating progesterone and
insulin on early embryo development in beef heifers. Anim
Reprod Sci 2003;79:71–9.
[17] Grummer RG, Winkler JC, Bertics SJ, Studer VA. Effect of
propylene glycol dosage during feed restriction on metabolites in
blood of prepartum Holstein heifers. J Dairy Sci 1994;77:
3618–23.
[18] Studer VA, Grummer RG, Bertics SJ, Reynolds CK. Effect of
prepartum propylene glycol administration on periparturient
fatty liver in dairy cows. J Dairy Sci 1993;76:2931–9.
[19] Formigoni A, Cornil MC, Prandi A, Mordenti A, Rossi A,
Portetelle D, et al. Effect of propylene glycol supplementation
around parturition on milk yield, reproductive performance and
some hormonal and metabolic characteristics in dairy cows. J
Dairy Res 1996;63:11–24.
[20] Lowman BG, Scott N, Somerville S. Condition scoring of cattle.
Rev Ed Bull East Scotl Coll Agric 1976;6.
[21] Løvendahl P, Purup HM. Technical note: time-resolved fluoro-
immunometric assay for intact insulin in livestock species. J
Anim Sci 2002;80:191–5.
[22] Rizos D, Ward F, Duffy P, Boland MP, Lonergan P. Conse-
quences of bovine oocyte maturation, fertilization or early
embryo development in vitro versus in vivo: implications for
blastocyst yield and blastocyst quality. Mol Reprod Dev
2002;61:234–48.
[23] SAS/STAT. User’s guide version 6, vol. 2. Cary, NC, USA: SAS
Institute Inc.; 1989.
[24] Patton J, Kenny DA, McNamara S, Mee JF, O’Mara FP, DiskinF
M.G.. et al. Relationships between milk production, energy
balance, plasma analytes and reproduction in Holstein–Friesian
cows. J Dairy Sci 2007;90:649–58.
[25] Moallem U, Katz M, Arieli A, Lehrer H. Effects of peripartum
propylene glycol or fats differing in fatty acid profiles on feed
intake, production, and plasma metabolites in dairy cows. J
Dairy Sci 2007;90:3846–56.
[26] van Knegsel AT, van den Brand H, Dijkstra J, van Straalen WM,
Jorritsma R, Tamminga S, et al. Effect of glucogenic vs. lipo-
genic diets on energy balance, blood metabolites, and reproduc-
tion in primiparous and multiparous dairy cows in early
lactation. J Dairy Sci 2007;90:3397–409.
[27] Butler ST, Pelton SH, Butler WR. Energy balance, metabolic
status, and the first postpartum ovarian follicle wave in cows
administered propylene glycol. J Dairy Sci 2006;89:2938–51.
[28] Buckley F, O’Sullivan K, Mee JF, Evans RD, Dillon P. Relation-
ships among milk yield, body condition, cow weight, and
reproduction in spring-calved Holstein–Friesians. J Dairy Sci
2003;86:2308–19.
[29] Domecq JJ, Skidmore AL, Lloyd JW, Kaneene JB. Relationship
between body condition scores and milk yield in a large dairy
herd of high yielding Holstein cows. J Dairy Sci 1997;80:
101–12.
[30] Smith SB, Prior RL, Mersmann HJ. Interrelationships between
insulin and lipid metabolism in normal and alloxan-diabetic
cattle. J Nutr 1983;113:1002–15.
[31] Morton JM. High genetic merit and high-producing dairy cows
in commercial Australian herds don’t have substantially worse
reproductive performance. Br Soc Anim Sci 2001;26:
305–11.
[32] Fulkerson WJ, Wilkins J, Dobos RC, Hough GM, Goddard ME,
Davidson T. Reproductive performance in Holstein–Friesian
cows in relation to genetic merit and level of feeding when
grazing pasture. J Anim Sci 2001;73:397–406.
[33] Kelly JM, Whitaker DA. Multidisiplinary approach to dairy herd
health and productivity management, vol. 26. BSAS Occasional
Publication; 2001. p. 209–22.
[34] Hoedemaker M, Prange D, Zerbe H, Frank J, Daxebberger A,
Meyer HHD. Peripartal propylene glycol supplementation and
 D. Rizos et al. / Theriogenology 69 (2008) 688–699
metabolism, animal health, fertility and production in dairy
cows. J Dairy Sci 2004;87:2136–45.
[35] Christensen JO, Grummer RR, Rasmussen FE, Bertics SJ. Effect
of method of delivery of propylene glycol on plasma metabolites
of feed restricted cattle. J Dairy Sci 1997;80:563–8.
[36] Leroy JLMR, Vanholder T, Mateusen B, Christophe A, Opsomer
G, de Kruif A, et al. Non-esterified fatty acids in follicular fluid
of dairy cows and their effect on developmental capacity of
bovine oocytes in vitro. Reproduction 2005;130:485–549.
[37] Rabiee AR, Lean IJ, Gooden JM, Miller BG, Scaramuzzi RJ. An
evaluation of transovarian uptake of metabolites using arterio-
venous difference methods in dairy cattle. Anim Reprod Sci
1997;48:9–25.
[38] Boland MP, Lonergan P, O’Callaghan D. Effect of nutrition on
endocrine parameters, ovarian physiology, and oocyte and
embryo development. Theriogenology 2001;55:1323–40.
[39] Hildago CO, Gomez E, Prieto L, Duque P, Goyache F, Fernandez
L, et al. Pregnancy rates and metabolic profiles in cattle treated
with propylene glycol prior to embryo transfer. Theriogenology
2004;62:664–76.
[40] Miyoshi S, Pate JL, Palmquist DL. Effects of propylene glycol
drenching on energy balance, plasma glucose, plasma insulin,
ovarian function and conception in dairy cows. Anim Reprod Sci
2001;68:29–43.
[41] Wathes DC, Taylor VJ, Cheng Z, Mann GE. Follicle growth,
corpus luteum function and their effects on embryo development
in the postpartum cow. Reproduction 2003;61:219–37.
[42] Wathes DC, Fenwick M, Cheng Z, Bourne N, Llewellyn S,
Morris DG, et al. Influence of negative energy balance on
cyclicity and fertility in the high producing dairy cow. Ther-
iogenology 2007;68 Suppl 1:S232–41.
699
[43] Canfield RW, Butler WR. Energy balance and pulsatile luteiniz-
ing hormone secretion in early postpartum dairy cows. Domest
Anim Endocrinol 1990;7:323–30.
[44] Jorritsma R, Langendijk P, Kruip TAM, Wensing TH, Noord-
huizen JPTM. Associations between energy metabolism, LH
pulsatility and first ovulation in early lactating cows. Reprod
Domest Anim 2005;40:68–72.
[45] Britt JH. Impact of early postpartum metabolism on follicular
development and fertility. Bovine Pract Proc 1991;24:39–43.
[46] Lussier JG, Matton P, Dufour JJ. Growth rates of follicles in the
ovary of the cow. J Reprod Fertil 1987;81:301–7.
[47] Britt JH. Impacts of early postpartum metabolism on follicular
development and fertility. Bovine Pract 1994;24:39–43.
[48] Dunne LD, Diskin MG, Boland MP, O’Farrell KJ, Sreenan JM.
The effect of pre- and post-insemination plane of nutrition on
embryo survival in beef heifers. Anim Sci 1999;69:411–7.
[49] Kendrick KW, Bailey TL, Garst AS, Pryor AW, Ahmadzadeh A,
Akers RM, et al. Effects of energy balance on hormones, ovarian
activity, and recovered oocytes inlactating Holstein cows using
transvaginal follicular aspiration. J Dairy Sci 1999;82:1731–40.
[50] Snijders SEM, Dillon P, O’Callaghan D, Boland MP. Effect of
genetic merit, milk yield, body condition and lactation number
on in vitro oocyte development in dairy cows. Theriogenology
2000;53:981–9.
[51] Nolan R, Duffy P, Wade M, O’Callaghan D, Boland MP. Effect
of quantity and type of diet and frequency of trans-vaginal ovum
aspiration on in-vitro embryo development in heifers. Therio-
genology 1998;49:402.
[52] Lozano JM, Nation DP, Ward FA, O’Callaghan D. Effect of
nutrition on oocyte developmental capacity in dairy cows.
Theriogenology 2000;53:276.