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Animal Reproduction Science xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Ovarian activity and oocyte quality associated with the

biochemical profile of serum and follicular fluid from
Girolando dairy cows postpartum
Benner G. Alves a,∗ , Kele A. Alves a , Aline C. Lúcio b , Muller C. Martins b ,
Thiago H. Silva b , Bruna G. Alves b , Lucas S. Braga b , Thiago V. Silva a ,
Marco A.O. Viu a , Marcelo E. Beletti b , José O. Jacomini b ,
Ricarda M. Santos b , Maria L. Gambarini a
a
Center for Studies and Research in Animal Reproductive Biology, College of Veterinary and Animal Science, Federal University of Goiás,
Goiânia, GO, 74001-970, Brazil
b
Laboratory of Animal Reproduction, Faculty of Veterinary Medicine, Federal University of Uberlândia, Uberlândia, MG, 38400-902,
Brazil

a r t i c l e i n f o a b s t r a c t

Article history: This study was designed to evaluate the influence of heat stress (HS) on the metabolic
Received 15 October 2013 profile of serum and follicular fluid (FF), ovarian follicle development, and oocyte quality of
Received in revised form 26 February 2014 Girolando dairy cows. Oocytes, blood, and FF (follicles ≥9 mm) samples were obtained at 30,
Accepted 28 February 2014
45, 60, 75, and 90 days postpartum in the summer and winter seasons. During transvaginal
Available online xxx
follicular aspiration, rectal temperature (RT), body condition score (BCS), number of ovar-
ian follicles, and quality of oocytes were recorded. The ambient air temperature (AT) and
Keywords: relative humidity (RH) were also recorded to calculate the temperature humidity index
Dairy cattle (THI). Glucose, total cholesterol (TC), triglycerides (TG), urea, sodium (Na), potassium (K),
Electrolytes and calcium (Ca) concentrations were determined using serum and FF samples. The RT, THI,
Follicular fluid and BCS loss were greater (P < 0.01) in the summer; however, glucose, Na, and K serum con-
Girolando centrations decreased in the same season (P < 0.05). Degenerated oocytes were positively
Heat stress associated (P < 0.05) with THI (r = 0.14) and AT (r = 0.13), and negatively associated with
Oocyte
glucose (r = −0.12) and K (r = −0.11) serum concentrations. HS induces metabolic changes,
which compromise the number of ovarian follicles and the follicular environment, thus
resulting in morphologically damaged oocytes.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction were generated as a consequence of the greater inci-

NOTE: NOTE: Over the last few decades, advances in the
field of genetics associated with improved management
have led to the selection of highly productive cows to meet
the demand of the dairy industry. These improvements
∗ Corresponding author. Tel.: +55 34 3218 2248; fax: +55 34 3218 2494.
E-mail address: bennervet@gmail.com (B.G. Alves).
dence of hormonal and metabolic disorders, which are
detrimental to oocytes and embryos (e.g., reduced fertil-
ity). Although some studies have revealed a correlation
between such disorders and high milk yield, others have
indicated that a decrease in reproductive performance
is associated with a number of factors, including repro-
ductive diseases, heat stress (HS), and weight loss (Bilby
et al., 2006; Sewalem et al., 2008; Shehab-El-Deen et al.,
2010).

http://dx.doi.org/10.1016/j.anireprosci.2014.02.019
0378-4320/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Alves, B.G., et al., Ovarian activity and oocyte quality associated with the bio-
chemical profile of serum and follicular fluid from Girolando dairy cows postpartum. Anim. Reprod. Sci. (2014),
http://dx.doi.org/10.1016/j.anireprosci.2014.02.019
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HS is one of the most important factors responsible
for decreased fertility in dairy cattle (Dobson and Smith,
2000). It is estimated that ∼60% of dairy cattle are affected
by high temperatures during the summer (Roth et al.,
2001), especially in the tropical and subtropical regions. HS
impairs the estrous cycle of cows, thus resulting in a delay
effect, attenuated follicular dominance, and a decrease
in follicular steroid production (Wolfenson et al., 1997;
Bilego et al., 2013). In Brazil, the majority of dairy produc-
tion systems utilize the Girolando breed of cows because
they combine the high productivity of Holstein cattle
(Bos taurus) with the rusticity and thermal adaptations
of Gir (Bos indicus) (Alves et al., 2013). The management
of Girolando herds is conducted in the tropical savannah
region in Brazil (i.e., the Brazilian Cerrado biome), which is
characterized by a hot, semi-humid, seasonal climate with
rainy summers and dry winters.
Metabolic changes that are induced by lactation and
associated with HS might impair ovarian function and the
environment where the oocyte is located, thus reducing
oocyte competence. Therefore, because of the importance
and relationship between these factors and fertility, this
study was conducted to evaluate the effects of HS on the
serum metabolic profile, ovarian activity, oocyte quality,
and follicular fluid (FF) metabolic status from dairy cows
during the early lactation period.
2. Materials and methods
2.1. Animals and location
Girolando breed cows (B. taurus × B. indicus) from the
third lactation were assessed during the winter (n = 30) and
summer (n = 30) seasons between July 2011 and February
2012. The experiment was conducted at the dairy research
unit of the Federal University of Uberlândia, located in the
tropical Cerrado biome (classified as the Cwa type according
to Köppen), 18◦ 55 08 S latitude and 48◦ 16 37 W longi-
tude, 776 m above sea level (Rubel and Kottek, 2010). The
cows were milked twice a day, and the mean production
of the herd per lactation period (305 days) was 5947.5 kg.
During the summer, the animals were fed Cynodon spp. cv.
Tifton 85 (ad libitum), supplemented with ration and min-
erals [bromatologic composition of total diet in the summer
was as follows: % dry matter (%DM) = 15.1% crude pro-
tein (CP); 62.0% total digestible nutrients (TDN); 23.7% acid
detergent fiber (ADF); 41.5% neutral detergent fiber (NDF);
2.6% ether extract (EE); 1.3% calcium (Ca); 0.6% phospho-
rus (P); 2.3 Mcal/kg DM of metabolizable energy (ME); and
1.4 Mcal/kg DM of liquid energy (LE)]. During the winter,
the cows were confined to feedlots with a ration, sorghum
silage, and minerals (bromatologic composition of total
diet in the winter: %DM = 13.9% CP; 65.0% TDN; 26.2% ADF;
40.7% NDF; 2.2% EE; 1.2% Ca; 0.4% P; 2.4 Mcal/kg DM ME;
and 1.5 Mcal/kg DM LE). The diets were formulated accord-
ing to production nutritional requirements (NRC, 2001).
2.2. Rectal temperature (RT), body condition score (BCS),
and weather variables
The BCS was scored based on a scale of 1–5, with
0.25 increments (Edmonson et al., 1989), and the RT
Please cite this article in press as: Alves, B.G., et al., Ovarian activity and oocyte quality associated with the bio-
chemical profile of serum and follicular fluid from Girolando dairy cows postpartum. Anim. Reprod. Sci. (2014),
http://dx.doi.org/10.1016/j.anireprosci.2014.02.019
was measured prior to follicular aspiration using a digital
thermometer inserted into the rectal area. Environmental
variables were obtained from the weather station of the Cli-
matology Laboratory from Uberlândia Federal University.
The ambient air temperature (AT, ◦ C) and relative humid-
ity (RH, %) were used to calculate the temperature humidity
index (THI) according to Mader et al., 2006:
 RH  
THI = (0.8 × AT) + × (AT − 14.4) + 46.4
100
2.3. Transvaginal follicular aspiration (TFA)–FF and
oocyte collection
We conducted 300 TFA procedures (n = 150, summer;
n = 150, winter) throughout the study. Each cow (n = 30,
summer; n = 30, winter) was subjected to the TFA pro-
cedure at 30, 45, 60, 75, and 90 days after parturition.
These procedures were started, on average, at 31.2 ± 1.2
and 32.5 ± 2.4 days postpartum in the summer and win-
ter seasons, respectively. The TFA was performed using
ultrasound scanner (SSD-500; Aloka, Tokyo, Japan), with
a 5-mHz micro convex transducer connected to a biopsy
guide (WTA; Watanabe Applied Technology, Cravinhos,
Brazil) and a suction line (WTA) with a 16G × 5.2 inch
catheter (1.7 × 1.3 mm; BD, Curitiba, Brazil). Caudal epidu-
ral anesthesia was induced with 4 mL of 2% lidocaine
(Eurofarma, São Paulo, Brazil), the perineum area was
scrubbed, and the aspiration guide with the micro convex
transducer was inserted; the size and number of the fol-
licles from the ovaries were classified as small (2–4 mm),
medium (5–8 mm), or large (≥9 mm). The FF was aspirated
from the large follicles (when present) using a 5-mL syringe
(Descarpack, São Paulo, Brazil) coupled to the aspiration
system. Immediately after collection, the samples were
centrifuged (2100 × g, 30 min, 25 ◦ C), and the supernatant
was stored at −20 ◦ C until analysis. Only the FF samples
that were not contaminated with blood were analyzed.
Oocytes were obtained from follicles (2–8 mm in diam-
eter) using constant vacuum pressure of the equipment,
which was adjusted to a volume of water per minute
(16 mL/min) in a medium with phosphate buffered saline
(PBS), with additions of 10% of fetal bovine serum (FBS; Cul-
tilab, Campinas, Brazil) and 7.5 UI/mL of sodium heparin
(Liquemine; Roche, São Paulo, Brazil). The solution was fil-
tered to hold the oocytes, which were transferred to Petri
dishes (60 × 15 mm, Cultilab) containing a tissue culture
medium (TCM-199; Sigma, St. Louis, USA) supplemented
with HEPES (20 mM; Sigma), sodium bicarbonate (5 mM;
Sigma), pyruvate (4 mM; Sigma), FBS (10%; Cultilab), and
amikacin sulfate (80 ␮g/mL; Sigma).
2.4. Oocyte classification
The cumulus-oocyte complexes (COC) were scored for
quality with the aid of a stereoscope (×40), as described
by Walters et al., 2002, as follows: Score 1 (GI), more than
three compact layers of cumulus-oocyte cells and a homo-
geneous cytoplasm; Score 2 (GII), two compact layers of
cumulus-oocyte cells and a less homogeneous than GI;
Score 3 (GIII), irregular layer with a few cumulus-oocyte
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cells and dark agglomerations in the cytoplasm; and degen-
erated oocyte (DEG), absence or spread of cumulus-oocyte
cells, and irregular and dark cytoplasm agglomerations. For
the present study, oocytes GI, GII, and GIII were considered
viable.
2.5. Blood samples
Blood samples were simultaneously obtained alongside
the TFA from all animals utilized throughout the experi-
ment during the summer and winter seasons. The coccidian
vein was punctured to draw blood through an 18 G × 1.5
inch hypodermic needle (BD, Curitiba, Brazil) in sterile vac-
uum tubes. The samples were kept inside the isothermal
box (4 ◦ C) until transported to the laboratory where they
were centrifuged (2100×g, 30 min, 25 ◦ C). The serum was
stored in aliquots at −20 ◦ C until use in the biochemical
analysis.
2.6. Biochemical analyses–Serum and FF
Glucose concentrations were determined for each blood
and FF sample during the TFA proceedings using test
strips (Accu-Chek Active; Roche, São Paulo, Brazil). Serum
analyses of total cholesterol (TC) and triglycerides (TG)
were performed by an endpoint enzymatic reaction using
commercial kits (Cholesterol Liquiform and Triglycerides
Liquiform; Labtest Diagnostica, Lagoa Santa, Brazil). Urea
was determined using an enzymatic reaction and ultra-
violet photometry using the kinetic method (Urea UV
Liquiform; Labtest Diagnostica). All analyses were per-
formed using the semiautomatic biochemical equipment
Bio-2000 BIOPLUS (Bioplus Laboratory Products Ltd., São
Paulo, Brazil). The dosage of the sodium (Na), potassium (K),
and calcium (Ca) ions were determined by the ion-selective
electrode method using the AVL 9180 electrolytic analyzer
(Roche, São Paulo, Brazil). All procedures were performed
according to the manufacturer’s instructions, and the intra-
and inter-assay coefficients of variation were <5%.
2.7. Statistical analysis
The study followed a completely random design in a
2 × 5 factorial arrangement of treatments. Critical anal-
ysis and data consistency were performed using PROC
UNIVARIATE (SAS Institute Inc., Cary, NC, USA), and data
were transformed when necessary. Analyses of oocyte total
number and viable oocytes, number of small (2–4 mm) and
medium (5–8 mm) follicles, and oocyte quality (GI, GII, GIII,
and DEG) were conducted using the logarithm transforma-
tion [log (X)]. For the number of large follicles (≥9 mm), the
√
best fit was obtained through radix transformation ( x).
Variance analyses were performed using the least-squares
method using PROC GLM (SAS). Adjusted means and t tests
were obtained using the LSMEANS option of PROC GLM
(SAS). To study the association between two variables, the
Pearson correlation coefficient was calculated using the
CORR procedure (SAS). Correlation coefficients were clas-
sified as strong (r > 0.6), moderate (0.6 < r < 0.4), or weak
(r < 0.4). The results were considered significantly different
when P < 0.05.
Please cite this article in press as: Alves, B.G., et al., Ovarian activity and oocyte quality associated with the bio-
chemical profile of serum and follicular fluid from Girolando dairy cows postpartum. Anim. Reprod. Sci. (2014),
http://dx.doi.org/10.1016/j.anireprosci.2014.02.019
3
Table 1
Mean ± (SEM) and variation of ambient air temperature (AT), relative
humidity (RH), and temperature humidity index (THI) during summer
and winter.
Season AT (◦ C) RH (%) THI
Summer 23.8 ± 0.1 a
66.4 ± 1.5
a
71.0 ± 0.04a
(18.6–32.5) (31.0–95.0) (69.2–71.5)
Winter 22.9 ± 0.1 40.5 ± 0.7 67.9 ± 0.1
(13.5–33.2) (14.0–74.0) (64.9–70.1)
a
Differences between seasons (P < 0.01).
3. Results
The results are reported as the least square means and
standard error of the mean (mean ± SEM). It was not pos-
sible to obtain FF with 11% of the TFA procedures due
the absence of a large follicle or blood contamination.
The AT, RH, THI, and RT (38.5 compared with 38.2 ◦ C)
were greater (P < 0.01) for the summer when compared
to the winter, thus characterizing the occurrence of HS
(Table 1). BCS loss occurred in both seasons between 30
and 90 days postpartum (P < 0.05). However, during the
summer, there was a greater loss of points on the BCS
scale when compared to the winter (0.89 ± 0.04 compared
with 0.39 ± 0.32; P < 0.05) up until 90 days of lactation. Fur-
ther, cows from the summer group exhibited no significant
difference between 30 (2.4 ± 0.1) and 90 days (2.6 ± 0.08)
following parturition. The BCS for the winter group was
greater on day 90 (3.0 ± 0.05 compared with 2.6 ± 0.08;
P < 0.01; Fig. 1).
In the experiment, 3805 ovarian follicles were counted
prior to the TFA procedures. The mean number of fol-
licles recorded for each cow regardless of the size was
greater (P < 0.05) during the winter (13.7 ± 0.4 compared
with 11.6 ± 0.6), mainly on days 30 (14.4 ± 0.7 compared
with 9.2 ± 0.5; P < 0.01) and 90 (14.4 ± 0.9 compared with
11.3 ± 0.9; P < 0.05) of the lactation period (Fig. 2a). Cows
showed a greater number of small follicles (2–4 mm) dur-
ing the winter when compared to the summer (8.0 ± 0.4
compared with 7.0 ± 0.6; P < 0.01), which was maintained
until 90 days postpartum. During the summer, the lowest
Fig. 1. Mean ± (SEM) of body score condition (BSC) and rectal tempera-
ture (RT) of postpartum dairy cows in the summer and winter seasons.
Differences between seasons for BCS (* P < 0.01) and RT (+ P < 0.01).
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Fig. 2. Mean ± (SEM) of ovarian follicles recorded before transvaginal follicular aspiration, between 30 and 90 days postpartum, during the summer and
winter seasons. (a) Total ovarian follicles, (b) small follicles (2–4 mm), (c) medium follicles (5–8 mm), and (d) large follicles (≥9 mm). Differences between
seasons (* P < 0.05; + P < 0.01).

number of small follicles was observed at 30 days postpar-
tum (4.3 ± 0.5; P < 0.05; Fig. 2b).
The number of medium follicles (5–8 mm) was greater
(P < 0.05) during the winter (4.2 ± 0.2 compared with
3.5 ± 0.2); however, this number decreased (P < 0.05) dur-
ing both seasons after 45 days of lactation. The number of
follicles increased (P < 0.05) and there was a similar number
of follicles at 60 days postpartum in the winter, which was
not observed for the animals in the summer (i.e., recovery
after 75 days of lactation; Fig. 2c).
The number of large follicles (≥9 mm) was greater
during the winter (1.5 ± 0.06 compared with 1.1 ± 0.07;
P < 0.01); at 30 days postpartum, twice the number
(P < 0.01) of large follicles (1.5 ± 0.1) was observed when
compared to the summer (0.7 ± 0.1). Despite the greater
amount of large follicles recorded during the winter, the
difference was not significant (P > 0.05) at 45, 60, and 90
days of lactation (Fig. 2d).
Following the TFA procedure, 1936 oocytes (recovered
index: 50.8%) were obtained. The number of oocytes per
cow did not differ between the seasons (summer: 7.0 ± 0.8
compared with winter: 5.9 ± 0.3; P > 0.05), and the com-
parison of the means of oocytes between days within the
season did not show any difference (P > 0.05) between
the summer (day 30: 4.7 ± 0.4 compared with day 90:
5.3 ± 0.5) and winter (day 30: 6.0 ± 0.6 compared with day
90: 6.6 ± 0.6) seasons (Fig. 3a). The means of viable oocytes
did not differ between the seasons (summer: 3.16 ± 0.5
compared with winter: 3.14 ± 0.2). The number of viable
oocytes was greater (P < 0.05) during the winter when com-
pared to that in the summer at 30 (3.8 ± 0.4 compared
with 1.7 ± 0.3), 60 (4.0 ± 0.9 compared with 2.2 ± 0.5), and
90 days (3.1 ± 0.5 compared with 1.6 ± 0.3) postpartum
(Fig. 3b).
The number of GI and GII oocytes did not differ between
the summer and winter seasons (Fig. 4a, b). However, it
was possible to recover more GIII oocytes in the winter
when compared that in the summer (2.1 ± 0.2 compared
with 1.9 ± 0.3; P < 0.05) (Fig. 4c). The mean of degenerated
oocytes was higher during the summer (3.9 ± 0.3 compared
with 2.7 ± 0.2; P < 0.01), with variation in oocyte quality
observed throughout the study (Fig. 4d). The effect of sea-
son was demonstrated for the mean of GIII oocytes during
the winter (P < 0.05) and degenerated oocytes in the sum-
mer (P < 0.01). The proportions of GI, GII, GIII, and DEG
oocytes throughout the study were 4.3%, 11.4%, 28.3%, and

Please cite this article in press as: Alves, B.G., et al., Ovarian activity and oocyte quality associated with the bio-
chemical profile of serum and follicular fluid from Girolando dairy cows postpartum. Anim. Reprod. Sci. (2014),
http://dx.doi.org/10.1016/j.anireprosci.2014.02.019
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Fig. 3. Mean ± (SEM) of total oocytes (a) and viable oocytes (b) recovered by transvaginal follicular aspiration in postpartum dairy cows during the summer
and winter seasons. There were differences between seasons (* P < 0.05; + P < 0.01).

Fig. 4. Oocyte quality (mean ± SEM) of dairy cows between 30 and 90 days of lactation, during the summer and winter seasons. (a) Oocyte score 1 (GI), (b)
oocyte score 2 (GII), (c) oocyte score 3 (GIII), and (d) degenerated oocytes (DEG). There were differences between seasons (* P < 0.05; + P < 0.01).

Please cite this article in press as: Alves, B.G., et al., Ovarian activity and oocyte quality associated with the bio-
chemical profile of serum and follicular fluid from Girolando dairy cows postpartum. Anim. Reprod. Sci. (2014),
http://dx.doi.org/10.1016/j.anireprosci.2014.02.019
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(r = −0.11; P < 0.05) and a positive correlation with Ca

(r = 0.17; P < 0.01) were also observed.

4. Discussion

The FF samples in the current study were collected every

Fig. 5. Oocyte quality distribution of dairy cows between 30 and 90 days
of lactation in different seasons (summer and winter). GI: oocyte score 1;
GII: oocyte score 2; GIII: oocyte score 3; DEG: degenerated oocytes. There
were differences between seasons for oocyte quality (* P < 0.05; + P < 0.01).
56% for the summer and 6.8%, 10.1%, 35.6%, and 47.5% for
the winter, respectively (Fig. 5).
Metabolites and electrolytes in the serum (glucose, urea,
TG, Na, K, and Ca) and FF (TC, TG, urea, K, and Ca) were
influenced by seasonal variations (Table 2). Correlation
coefficients from clinical and weather variables are shown
in Table 3. The main results observed were a negative asso-
ciation (P < 0.01) between the total of ovarian follicles with
RT (r = −0.14), RH (r = −0.18), and THI (r = −0.16); nega-
tive correlations between viable oocytes with THI (r = 0.19;
P < 0.01) and AT (r = −0.13; P < 0.05); and positive corre-
lations between degenerated oocytes with THI (r = 0.14;
P < 0.01) and AT (r = 0.13; P < 0.05).
Correlations between TC, urea, and Na, and the num-
ber of ovarian follicles, oocyte quantity, and oocyte quality
were not observed (Table 4). Serum TG (r = 0.11) and K
(r = 0.11) were, however, positively correlated (P < 0.05)
with the number of ovarian follicles. Serum glucose con-
centration exhibited a positive correlation (P < 0.05) with
the number of small (r = 0.12) and large follicles (r = 0.13),
and a negative association (P < 0.05) with degenerated
oocytes (r = −0.12). Furthermore, a negative correlation
between the number of degenerated oocytes with K
15 days. Thus, the experimental design had some limita-
tions. For example, some FF samples (≥9 mm) may have
been collected from atretic follicles. Follicular deviation
occurs when the follicle reaches an average of 8.5 mm in
diameter (Ginther et al., 2001). However, it was decided in
the present study to use all the samples due to the fact that
the concentrations of glucose in the FF (mg/dL) during the
summer (76.1 ± 2.8) and winter (77.1 ± 1.9) seasons were
similar to concentrations described for active dominant fol-
licles (Shehab-El-Deen et al., 2010; Moallem et al., 2011;
Aller et al., 2013). In addition, some studies have shown
that the stage of the estrous cycle does not affect the num-
ber of aspirated follicles or oocyte quality (Arlotto et al.,
1996; Acar et al., 2013).
Favorable conditions for HS development occurred dur-
ing the summer in the present study, with greater weather
variable values for AT, RH, and THI. Greater BCS loss and
elevated RT occurred during the postpartum period in the
summer. At the beginning of lactation, the energy require-
ment for maintenance and milk production is greater than
the energy that cows gain from dry matter intake (DMI).
These differences result in greater mobilization of the body
tissue reserves, which is associated with a lesser DMI and
a decrease in metabolic heat production due to an increase
in body temperature imposed by THI that can contribute
to reproductive dysfunctions (Dikmen and Hansen, 2009;
Wolfenson et al., 2000).
The effects of HS on follicles were evident during
the summer because the number of follicles classified as
small (2–4 mm), medium (5–8 mm), or large (≥9 mm) was
greater during the winter. In general, negative correlations
between the total number of follicles and RT, RH, and THI
were identified. Changes in body temperature caused by
HS during follicular development inhibited growth and
ovulation due to a decrease in the receptors for LH, estra-
diol, and aromatase activity (Ozawa et al., 2005). Lactating
dairy cows experiencing HS had a decrease in plasma

Table 2
Seasonal effect (summer and winter) of the metabolites concentration (mean ± SEM) on serum and follicular fluid (FF) of dairy cows between 30 and 90
days postpartum.

Metabolites Serum Follicular Fluida

Summer Winter P-valueb Summer Winter P-valuec

Glucose (mg/dL) 56.1 ± 0.7 63.5 ± 0.9 0.01 76.1 ± 2.8 77.1 ± 1.9 0.78
TC (mg/dL) 136.9 ± 7.7 129.2 ± 4.2 0.23 83.3 ± 4.4 60.7 ± 3.1 0.01
TG (mg/dL) 13.3 ± 0.7 11.3 ± 0.7 0.05 22.1 ± 1.9 14.3 ± 1.0 0.01
Urea (mg/dL) 23.6 ± 0.9 31.3 ± 1.5 0.01 13.3 ± 0.7 11.2 ± 0.6 0.05
Na (mmol/L) 134.4 ± 1.5 139.2 ± 2.1 0.05 221.5 ± 9.8 206.6 ± 5.9 0.19
K (mmol/L) 4.3 ± 0.1 4.8 ± 0.1 0.01 4.7 ± 0.2 4.3 ± 0.1 0.01
Ca (mmol/L) 0.8 ± 0.01 0.6 ± 0.01 0.01 0.9 ± 0.1 0.5 ± 0.02 0.01

TC: total cholesterol; TG: triglycerides; Na: sodium; K: potassium; Ca: calcium.
a
Follicles ≥9 mm in diameter.
b
The P-values refer to the seasonal comparison of the average concentration of metabolites on serum.
c
The P-values refer to the seasonal comparison of the average concentration of metabolites on FF.

Please cite this article in press as: Alves, B.G., et al., Ovarian activity and oocyte quality associated with the bio-
chemical profile of serum and follicular fluid from Girolando dairy cows postpartum. Anim. Reprod. Sci. (2014),
http://dx.doi.org/10.1016/j.anireprosci.2014.02.019
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Table 3
Correlation coefficients (r) between clinical and climatic variables with ovarian follicles, oocyte number, and oocyte quality from postpartum dairy cows.

Variables RT BCS THI AT RH

Total follicles −0.14** 0.05 −0.16** 0.07 −0.18**

Follicles 2–4 mm −0.12* 0.01 −0.13** 0.02 −0.11*
Follicles 5–8 mm −0.04 0.01 −0.13* 0.09 −0.18**
Follicles ≥9 mm −0.06 0.00 −0.16** −0.05 −0.09
Total oocytes −0.05 0.01 −0.05 0.02 −0.01
Viable oocytes −0.08 0.08 −0.19** −0.13* 0.02
Oocytes GI −0.06 0.03 −0.06 −0.12* 0.02
Oocytes GII −0.07 0.04 −0.14** −0.11* 0.01
Oocytes GIII −0.05 0.11* −0.19** −0.12* 0.03
Oocytes DEG 0.00 −0.06 0.14** 0.13* 0.01

RT: rectal temperature; BCS: body condition score; THI: temperature humidity index; AT: ambient air temperature; RH: relative humidity; GI: oocyte score
1; GII: oocyte score 2; GIII: oocyte score 3; DEG: degenerated oocytes.
*
Values are different (P < 0.05).
**
Values are different (P < 0.01).

estradiol concentration and follicular diameter due to the
lesser steroidogenesis in the granulosa and theca cells
(Wilson et al., 1998). In addition, the difference in antral
follicles recorded among the seasons could be due to indi-
vidual cow variation (Burns et al., 2005). However, HS com-
promises the steroidogenic capacity of follicles, and dairy
cows have fewer ovarian follicles (≥3 mm) which could be
associated with reduced fertility (Mossa et al., 2012).
Serum glucose concentration was less during the sum-
mer and was positively correlated with number of small
(2–4 mm) and large (≥9 mm) follicles. Energy concentra-
tions before and after parturition have a notable influence
on the size and number of follicles. The growth of follicles
was suppressed when a negative energy balance (NEB) was
associated with BCS loss resulting in a decrease in the num-
ber of follicles (Perry et al., 1991). The decrease in energy
concentrations consequently leads to a decrease in the con-
centration of IGF-1 and estrogen in the FF. The daily growth
in size and total number of dominant follicles is influenced
by the variability of energy intake, which is positive for
cows fed diets with greater energy content (Kendrick et al.,
1999).
The average number of oocytes and the number of viable
oocytes recovered in each TFA did not differ between the
seasons; moreover, numbers were not influenced by post-
partum duration. However, the average number of GIII
oocytes was greater during the winter, and the number of
degenerated oocytes was greater in the summer. A neg-
ative correlation was observed between serum glucose
concentration and the number of degenerated oocytes.
The THI and AT variables had a negative association with
the number of viable oocytes and a positive correlation
with the number of degenerated oocytes. Furthermore, was
observed a seasonal effect for metabolic and ion concentra-
tions in serum (glucose, TG, urea, Na, K, and Ca) and FF (TC,
TG, urea, K, and Ca). In a recent study, Matoba et al., 2012
indicated that lactation did not have an effect (up to 80 days
postpartum) on the morphology or development of oocytes
from dairy cows when were in vitro cultured. However,
changes in the biochemical profile due to HS were reported
for glucose concentration, IGF-1, NEFA, urea, and TC, which
could compromise oocyte development and granulosa cell
quality (Shehab-El-Deen et al., 2010).
Serum metabolic TC, Na, and urea were not associated
with the number of follicles or oocyte quality in the present
study. The TG and K concentrations, however, were posi-
tively correlated with the number of follicles. The number
of degenerated oocytes was negatively and positively cor-
related with K and Ca concentrations, respectively. Some
studies reported correlations between concentrations of
metabolic variables (e.g., glucose, TC, TG, and urea) and
ions (e.g., sodium, chlorine, and calcium) in the serum and
FF, which can change the microenvironment for oocyte
development (Leroy et al., 2004; Alves et al., 2013). Studies

Table 4
Correlation coefficients (r) between serum metabolites with ovarian follicles, oocyte number, and oocyte quality from postpartum dairy cows.

Variables Glucose TC TG Urea Na K Ca

* *
Total follicles 0.03 0.03 0.11 0.06 0.05 0.11 0.04
Follicles 2–4 mm 0.12* 0.02 0.07 0.06 0.00 0.04 0.05
Follicles 5–8 mm −0.04 0.06 0.06 0.03 0.09 0.18** −0.02
Follicles ≥9 mm 0.13* 0.01 −0.01 0.05 0.00 −0.02 −0.08
Total oocytes 0.05 0.00 0.02 0.02 −0.02 −0.03 0.01
Viable oocytes 0.06 −0.06 0.03 0.00 −0.02 0.00 0.00
Oocytes GI 0.01 0.03 −0.03 0.04 −0.06 0.00 −0.09
Oocytes GII 0.07 −0.01 0.07 −0.05 0.04 0.04 0.00
Oocytes GIII 0.01 −0.06 0.05 0.00 −0.01 0.00 0.00
Oocytes DEG −0.12* 0.05 0.06 −0.08 −0.04 −0.11* 0.17**

TC: total cholesterol; TG: triglycerides; Na: sodium; K: potassium; Ca: calcium; GI: oocyte score 1; GII: oocyte score 2; GIII: oocyte score 3; DEG: degenerated
oocytes.
*
Values are different (P < 0.05).
**
Values are different (P < 0.01).

Please cite this article in press as: Alves, B.G., et al., Ovarian activity and oocyte quality associated with the bio-
chemical profile of serum and follicular fluid from Girolando dairy cows postpartum. Anim. Reprod. Sci. (2014),
http://dx.doi.org/10.1016/j.anireprosci.2014.02.019
G Model
ANIREP-4931; No. of Pages 9 ARTICLE IN PRESS
8 B.G. Alves et al. / Animal Reproduction Science xxx (2014) xxx–xxx
have already demonstrated that urea may impair meiosis,
fertilization, in vitro embryo development (Sinclair et al.,
2000; De Wit et al., 2001), and receptor expression for IGF-
1 and insulin in the endometrium during uterine involution
(Wathes et al., 2011). However, there are other studies
where the effects of urea on fertility were not observed
in dairy cows (Oliveira et al., 2004; Rehak et al., 2009).
Serum TG concentrations were positively correlated
with the total number of follicles and was influenced by
season; in addition, greater concentrations of TG occurred
in the serum and FF during the summer. In addition to
functioning as an energy source, lipids have an important
role in the composition of membrane cells. The physical
properties of the membranes are regulated by lipid consti-
tution, environmental factors, and diet (Kim et al., 2001;
Wonnacott et al., 2010). Zeron et al., 2001 observed infe-
rior quality of cumulus oocyte complexes obtained during
the summer with lesser cleavage indices and blastocyst
development during in vitro production, and a greater
percentage of saturated fatty acids in the oocytes, granu-
losa cells, and FF. However, in the present study there was
not a relationship between TG concentrations and oocyte
quality.
Potassium concentration was greater in the FF during
the summer and was negatively correlated with the num-
ber of degenerated oocytes. All electrolytes evaluated in
the present experiment were affected by seasonal varia-
tion in both the FF and serum, except sodium, which was
only affected in the serum. Sodium and potassium are con-
stituents of sweat, and lesser serum concentrations of these
elements could be found in the summer due to transpira-
tion, which is the main thermoregulatory mechanism of
zebuines (B. indicus; Kadzere et al., 2002).
In maturation medium, different proportions of Na/K
(16 and 24) were used (Iwata et al., 2004), and this vari-
ation in proportions did not influence oocyte competence
or nuclear maturation in cattle. However, in mice, an
increase in hatching rates of blastocysts in vitro was ver-
ified when the Na/K ratio was between three and 10 (Jin
et al., 1994). In the present study, there was a ratio of
47 and 48 for Na/K in the FF during the summer and
winter, respectively. However, the effect of this relation-
ship on the reproductive performance of dairy cows is not
clear.
Serum Ca concentrations were positively correlated
with the number of degenerated oocytes. Furthermore,
serum and FF concentrations of Ca were greater during the
summer. Ca has an important role in acquired meiotic com-
petence and polyspermy block (He et al., 1997). Changes
in Ca intracellular regulation occur during folliculogene-
sis (Rozinek et al., 2006) and oocyte maturation (Carroll
et al., 1994). Lebedeva et al., 1998 identified the accumu-
lation of intracellular Ca reserves in the granulosa cells of
cattle from follicles undergoing atresia. Ca storage is asso-
ciated with the morphologic quality of immature oocytes
and the developmental potential of mature oocytes (Boni
et al., 2002).
In summary, HS modifies the serum profile and FF
metabolic status in lactating dairy cows, thus affecting
oocyte quality. Fluctuations in serum glucose, TG, K, and Ca
are reflected in the follicular environment, thus resulting
Please cite this article in press as: Alves, B.G., et al., Ovarian activity and oocyte quality associated with the bio-
chemical profile of serum and follicular fluid from Girolando dairy cows postpartum. Anim. Reprod. Sci. (2014),
http://dx.doi.org/10.1016/j.anireprosci.2014.02.019
in the production of morphologically damaged oocytes
that can contribute to decreased fertility after parturition.
Conflict of interest
None declared.
References
Acar, D.B., Birdane, M.K., Dogan, N., Gurler, H., 2013. Effect of the stage of
estrous cycle on follicular population, oocyte yield and quality, and
biochemical composition of serum and follicular fluid in Anatolian
water buffalo. Anim. Reprod. Sci. 137, 8–14.
Aller, J.F., Callejas, S.S., Alberio, R.H., 2013. Biochemical and steroid con-
centrations in follicular fluid and blood plasma in different follicular
waves of the estrous cycle from normal and superovulated beef cows.
Anim. Reprod. Sci. 142, 113–120.
Alves, B.G., Alves, K.A., Martins, M.C., Braga, L.S., Silva, T.H., Alves, B.G., San-
tos, R.M., Silva, T.V., Viu, M.A.O., Beletti, M.E., Jacomini, J.O., Gambarini,
M.L., 2013. Metabolic profile of serum and follicular fluid from post-
partum dairy cows during summer and winter. Reprod. Fertil. Dev.,
http://dx.doi.org/10.1071/RD13102.
Arlotto, T., Schwartz, J.L., First, N.L., Leibfried-Rutledge, M.L., 1996. Aspects
of follicle and oocyte stage that affect in vitro maturation and devel-
opment of bovine oocytes. Theriogenology 45, 943–956.
Bilby, T.R., Guzeloglu, A., MacLaren, L.A., Staples, C.R., Thatcher, W.W.,
2006. Pregnancy, bovine somatotropin, and dietary n-3 fatty acids
in lactating dairy cows: II. Endometrial gene expression related to
maintenance of pregnancy. J. Dairy Sci. 89, 3375–3385.
Bilego, U.O., Santos, F.C., Porto, R.N.G., Pires, B.C., Oliveira Filho, B.D., Viu,
M.A., Gambarini, M.L., 2013. Ovarian evaluation of Girolando (Hol-
stein × Gir) heifers submitted to a GnRH–PGF2␣ –GnRH protocol in the
dry or rainy seasons in the tropical savannah. Trop. Anim. Health Prod.
45, 1461–1467.
Boni, R., Cuomo, A., Tosti, E., 2002. Developmental potential in bovine
oocytes is related to cumulus-Oocyte complex grade, calcium current
activity, and calcium stores. Biol. Reprod. 66, 836–842.
Burns, D.S., Jimenez-Krassel, F., Ireland, J.L.H., Knight, P.G., Ireland, J.J.,
2005. Numbers of antral follicles during follicular waves in cattle:
evidence for high variation among animals, very high repeatability in
individuals, and an inverse association with serum follicle-stimulating
hormone concentrations. Biol. Reprod. 73, 54–62.
Carroll, J., Swann, K., Whittingham, D., Whitaker, M., 1994. Spa-
tiotemporal dynamics of intracellular [Ca2+ ]i oscillations during the
growth and meiotic maturation of mouse oocytes. Development 120,
3507–3517.
De Wit, A.A.C., Cesar, M.L.F., Kruip, T.A.M., 2001. Effect of urea during
in vitro maturation on nuclear maturation and embryo development
of bovine cumulus-oocyte-complexes. J. Dairy Sci. 84, 1800–1804.
Dikmen, S., Hansen, P.J., 2009. Is the temperature-humidity index the
best indicator of heat stress in lactating dairy cows in a subtropical
environment? J. Dairy Sci. 92, 109–116.
Dobson, H., Smith, R.F., 2000. What is stress, and how does it affect repro-
duction? Anim. Reprod. Sci. 60–61, 743–752.
Edmonson, A.J., Lean, I.J., Weaver, L.D., Farver, T., Webster, G.A., 1989.
Body condition scoring chart for Holstein dairy cows. J. Dairy Sci. 72,
68–78.
Ginther, O.J., Beg, M.A., Bergfelt, D.R., Donadeu, F.X., Kot, K., 2001. Follicle
selection in monovular species. Biol. Reprod. 65, 638–647.
He, C.L., Damiani, P., Parys, J.B., Fissore, R.A., 1997. Calcium, calcium release
receptors, and meiotic resumption in bovine oocytes. Biol. Reprod. 57,
1245–1255.
Iwata, H., Hashimoto, S., Ohota, M., Kimura, K., Shibano, K., Miyake, M.,
2004. Effects of follicle size and electrolytes and glucose in maturation
medium on nuclear maturation and developmental competence of
bovine oocytes. Reproduction 127, 159–164.
Jin, Z., Jin, M., Roomans, G.M., 1994. Effect of extracellular K+ on hatching
and outgrowth of mouse blastocysts in vitro. Cell Biol. Int. 18, 897–901.
Kadzere, C.T., Murphy, M.R., Silanikove, N., Maltz, E., 2002. Heat stress in
lactating dairy cows: a review. Livest. Prod. Sci. 77, 59–91.
Kendrick, K.W., Bailey, T.L., Garst, A.S., Pryor, A.W., Ahmadzadeh, A., Akers,
R.M., Eyestone, W.E., Pearson, R.E., Gwazdauskas, F.C., 1999. Effects of
energy balance on hormones, ovarian activity, and recovered oocytes
in lactating Holstein cows using transvaginal follicular aspiration. J.
Dairy Sci. 82, 1731–1740.
G Model
ANIREP-4931; No. of Pages 9 ARTICLE IN PRESS
B.G. Alves et al. / Animal Reproduction Science xxx (2014) xxx–xxx
Kim, J.Y., Kinoshita, M., Ohnishi, M., Fukui, Y., 2001. Lipid and fatty acid
analysis of fresh and frozen-thawed immature and in vitro matured
bovine oocytes. Reproduction 122, 131–138.
Lebedeva, I.Y., Denisenko, V.Y., Lebedev, V.A., Kuzmina, T.I., 1998. Prolactin
in follicular fluid and intracellular store calcium in follicular cells are
related to morphological signs of ovarian follicle atresia in cows: work
in progress. Theriogenology 49, 509–519.
Leroy, J.L.M.R., Vanholder, T., Delanghe, J.R., Opsomer, G., Van Soom, A.,
Bols, P.E.J., Kruif, A., 2004. Metabolite and ionic composition of follic-
ular fluid from different-sized follicles and their relationship to serum
concentrations in dairy cows. Anim. Reprod. Sci. 80, 201–211.
Mader, T.L., Davis, M.S., Brow-Brandl, T., 2006. Environmental factors
influencing heat stress in feedlot cattle. J. Anim. Sci. 84, 712–719.
Matoba, S., O’Hara, L., Carter, F., Kelly, A.K., Fair, T., Rizos, D., Lonergan,
P., 2012. The association between metabolic parameters and oocyte
quality early and late postpartum in Holstein dairy cows. J. Dairy Sci.
95, 1257–1266.
Moallem, U., Blanck, R., Lehrer, H., Livshitz, L., Zachut, M., Arieli, A., 2011.
Effects of high dietary crude protein on the characteristics of preovu-
latory follicles in dairy heifers. J. Dairy Sci. 94, 785–792.
Mossa, F., Walsh, S.W., Butler, S.T., Berry, D.P., Carter, F., Lonergan, P.,
Smith, G.W., Ireland, J.J., Evans, A.C.O., 2012. Low number of ovarian
follicles ≥3 mm in diameter are associated with low fertility in dairy
cows. J. Dairy Sci. 95, 2355–2361.
NRC, 2001. Nutrient Requirements of Dairy Cattle, 7th rev. ed. National
Academic Science, Washington, DC.
Oliveira, M.M.N.F., Torres, C.A.A., Costa, E.P., Carvalho, G.R., 2004. Ureia
para vacas leiteiras no pós-parto: Teor plasmático de ureia e pH
uterino. R. Bras. Zootec. 33, 123–127.
Ozawa, M., Tabayashi, D., Latief, T.A., Shimizu, T., Oshima, I., Kanai, Y., 2005.
Alterations in follicular dynamics and steroidogenic abilities induced
by heat stress during follicular recruitment in goats. Reproduction
129, 621–630.
Perry, R.C., Corah, L.R., Cochran, R.C., Beal, W.E., Stevenson, J.S., Minton,
J.E., Simms, D.D., Brethour, J.R., 1991. Influence of dietary energy on
follicular development, serum gonadotropins, and first postpartum
ovulation in suckled beef cows. J. Anim. Sci. 69, 3762–3773.
Rehak, D., Rajmon, R., Kubseva, M., Stipkova, M., Volek, J., Jilek, F., 2009.
Relationships between milk urea and production and fertility traits
in Holstein dairy herds in the Czech Republic. Czech J. Anim. Sci. 54,
193–200.
Roth, Z., Meidan, R., Shaham-Albalancy, A., Braw-Tal, R., Wolfenson, D.,
2001. Delayed effect of heat stress on steroid production in medium-
sized and preovulatory bovine follicles. Reproduction 121, 745–751.
Please cite this article in press as: Alves, B.G., et al., Ovarian activity and oocyte quality associated with the bio-
chemical profile of serum and follicular fluid from Girolando dairy cows postpartum. Anim. Reprod. Sci. (2014),
http://dx.doi.org/10.1016/j.anireprosci.2014.02.019
9
Rozinek, J., Rajmona, R., Petr, J., Rohlik, J., Jeseta, M., Sedmikova, M., Rehak,
D.R., Jilek, F., 2006. Ultrastructural localisation of calcium deposits in
pig ovarian follicles. Anim. Reprod. Sci. 91, 123–132.
Rubel, F., Kottek, M., 2010. Observed and projected climate shifts
1901–2100 depicted by world maps of the Köppen–Geiger climate
classification. Meteorol. Z. 19, 135–141.
Sewalem, A., Miglior, F., Kistemaker, G.J., Sullivan, P., Van Doormaal,
B.J., 2008. Relationship between reproduction traits and functional
longevity in Canadian dairy cattle. J. Dairy Sci. 91, 1660–1668.
Shehab-El-Deen, M.A.M.M., Leroy, J.L.M.R., Fadel, M.S., Saleh, S.Y.A.,
Maes, D., Van Soom, A., 2010. Biochemical changes in the follic-
ular fluid of the dominant follicle of high producing dairy cows
exposed to heat stress early post-partum. Anim. Reprod. Sci. 117,
189–200.
Sinclair, K.D., Kuran, M., Gebbie, F.E., Webb, R., McEvoy, T.G., 2000. Nitro-
gen metabolism and fertility in cattle: II. Development of oocytes
recovered from heifers offered diets differing in their rate of nitrogen
release in the rumen. J. Anim. Sci. 78, 2670–2680.
Walters, A.H., Bailey, T.L., Pearson, R.E., Gwazdauskas, F.C., 2002. Parity-
related changes in bovine follicle and oocyte populations, oocyte
quality, and hormones to 90 days postpartum. J. Dairy Sci. 85,
824–832.
Wathes, D.C., Cheng, Z., Fenwick, M.A., Fitzpatrick, R., Patton, J., 2011.
Influence of energy balance on the somatotrophic axis and matrix
metalloproteinase expression in the endometrium of the postpartum
dairy cow. Reproduction 141, 269–281.
Wilson, S.J., Marion, R.S., Spain, J.N., Spiers, D.E., Keisler, D.H., Lucy, M.C.,
1998. Effects of controlled heat stress on ovarian function of dairy
cattle. 1. Lactating cows. J. Dairy Sci. 81, 2124–2131.
Wolfenson, D., Lew, B.J., Thatcher, W.W., Graber, Y., Meidan, R., 1997. Sea-
sonal and acute heat stress effects on steroid production by dominant
follicles in cows. Anim. Reprod. Sci. 47, 9–19.
Wolfenson, D., Roth, Z., Meidan, R., 2000. Impaired reproduction in heat-
stressed cattle: basic and applied aspects. Anim. Reprod. Sci. 60–61,
535–547.
Wonnacott, K.E., Kwong, W.Y., Hughes, J., Salter, A.M., Lea, R.G., Garnswor-
thy, P.C., Sinclair, K.D., 2010. Dietary omega-3 and -6 polyunsaturated
fatty acids affect the composition and development of sheep granulosa
cells, oocytes and embryos. Reproduction 139, 57–69.
Zeron, Y., Ocheretny, A., Kedar, O., Borochov, A., Sklan, D., Arav, A., 2001.
Seasonal changes in bovine fertility: relation to developmental com-
petence of oocytes, membrane properties and fatty acid composition
of follicles. Reproduction 121, 447–454.