Theriogenology 159 (2021) 153e164

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Theriogenology
journal homepage: www.theriojournal.com

Multistep analysis reveals the relationship between blood indices at

the time of ovum pick-up and in vitro embryo production in heifers
Rasoul Kowsar a, *, Mehdi Komeili a, Nima Sadeghi b, Khaled Sadeghi a
a
Department of Animal Sciences, College of Agriculture, Isfahan University of Technology, Isfahan, 84156e83111, Iran
b
FKA, Animal Husbandry and Agriculture Co., Isfahan, Iran

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

Article history: The inflammatory factors of complete blood count (CBC) are associated with a decrease in the in vitro
Received 24 May 2020 embryo production (IVP) outcome in women. The relation between the blood indices and in vitro
Received in revised form fertilization (IVF) outcomes in bovines remains to be elucidated. Using ovum pick-up (OPU), oocytes were
8 October 2020
retrieved from heifers (n ¼ 60) and inseminated separately with sperm. The blastocyst formation was
Accepted 22 October 2020
Available online 24 October 2020
recorded on day 7 after insemination for each animal and the blood indices were evaluated at the time of
OPU. Then, heifers were classified on the basis of (1) blastocyst formation, cleaved vs. failed, or (2)
inflammation, low-grade inflammation (lymphocyte counts > 5.6  109/L) vs. no inflammation
Keywords:
Blood indices
(lymphocyte counts < 5.6  109/L). Oocytes derived from heifers with higher lymphocytes, red blood
Bovine oocytes cells (RBC), platelets, hematocrit, red cell distribution width (RDW-SD) and plateletcrit values and lower
In vitro embryo production monocytes, eosinophils, mean corpuscular hemoglobin (MCH) and MCH concentration (MCHC) suc-
Lymphocytes cessfully developed to the blastocyst stage. Heifers with low-grade inflammation numerically had a
Eosinophils higher percentage of blastocyst formation than normal heifers. The principle component analysis (PCA)
RDW showed that blastocyst formation had the strongest positive association with RDW-cv and RDW-SD,
while having a strong negative association with mean corpuscular volume (MCV), hemoglobin, MCHC
and MCH. The PCA determined that the number of grade A COCs and the percentage of COCs reached the
cleavage stage had a negative association with white blood cells (WBC), lymphocytes, basophils and
monocytes, and a positive correlation with platelet to lymphocyte ratio, platelet distribution width
(PDW) and plateletcrit. Network mapping detected close similarities between BFR and RDW-SD, MPV,
and lymphocytes. The area under the receiver operating characteristic curve (AUC) identified that, eo-
sinophils (AUC 0.80), RDW-SD (AUC 0.76), monocytes (AUC 0.76) and lymphocytes (AUC 0.76) had a good
predictive ability to detect heifers with high OPU-IVP outcome (60%). In conclusion, these findings
suggest that CBC indices at the time of OPU were associated with the IVF outcome and may be incor-
porated into protocols for the identification of heifers with high potential for blastocyst formation.
© 2020 Elsevier Inc. All rights reserved.

1. Introduction
In recent decades, significant advances in commercial in vitro
embryo production (IVP) and related technologies, such as oocyte
retrieval (ovum pick-up: OPU), have increased the genetic advan-
tage by reducing generation intervals in the dairy and beef in-
dustries [1e3]. The ability to recognize heifers with a high
reproductive capacity for breeding stock is one of the keys to the
efficient production of cattle [4]. Heifers are selected primarily for
the OPU-IVP program on the basis of the milk production capacity
* Corresponding author. Isfahan University of Technology, Iran.
E-mail address: rasoul_kowsarzar@yahoo.com (R. Kowsar).
of the sire and the dam [5]. Heifers are also expected to have good
growth, good health and free from genetic disorders [6]. Due to the
nature of complexity, reproductive and milk production traits are
negatively correlated with each other [5]. Recently, newer selection
approaches have reduced such a negative relationship [7,8], but
there is still little genetic correlation between reproductive traits
[7,9]. These shortcomings make the selection of animals compli-
cated and long, making such biomarkers low-precision.
Peripheral blood monocytes (MON) have been shown to be
activated following ovulation [10] and systemic changes, including
increased levels of granulocytes, MON and lymphocytes (LYM),
have been observed in circulation [11,12]. In fact, the action of sex
hormone, such as luteinizing hormone (LH) and estradiol (E2), can
affect the blood flow to the ovary and its follicles [13,14]. Increased

https://doi.org/10.1016/j.theriogenology.2020.10.026
0093-691X/© 2020 Elsevier Inc. All rights reserved.
R. Kowsar, M. Komeili, N. Sadeghi et al. Theriogenology 159 (2021) 153e164

2.1. Animals and OPU procedure

Abbreviation
The experiment was conducted in a commercial dairy farm, Feka
IVF In vitro fertilization Dairy Farm, Isfahan, Iran. A total of 60 healthy Holstein heifers with
IVP In vitro embryo production similar genetic merit, age (13 ± 1.1 months old), body weight
CBC Complete blood count (345 ± 15 Kg) and body condition score (3.0 ± 0.25, on a scale of
OPU Ovum pick-up 1e5) were used in this experiment. First, using an ultrasound, the
DFR Dominant follicle removal puberty of heifers was verified by the presence of corpus luteum
COCs Cumulus oocyte complexes (CL) and only heifers with CL were used for the experiment. The
FCS Fetal calf serum ovarian cyclicity of heifers was verified by the existence of CL
IVM In vitro maturation during two consecutive ultrasound tests conducted 14 days (d)
FBS Fetal bovine serum apart prior to the OPU procedure [19]. Ovarian examinations were
IGF Insulin like growth factor conducted using a transrectal ultrasound and a portable scanner
LH Luteinizing hormone (SonoSite® 180 Plus, Fujifilm SonoSite Japan, Tokyo, Japan)
FSH Follicle stimulating hormone installed with a 7.5 MHz linear probe. Heifers were subjected to the
BSA Bovine serum albumin OPUeIVP program without prior synchronization of the follicular
HCA Hierarchical clustering analysis wave. All heifers were fed a total mixed ration formulated to meet
ROC Receiver operating characteristic the nutrient requirements recommended by the NRC.
PCA Principal component analysis Fig. 1 shows the OPU protocol used in this study. In the OPU trial,
MIF Migration inhibitory factor the estrus synchronization of the pubertal heifers was carried out
by injection of 500 mg of prostaglandin F2a (PGF2a, Cloprostenol,
Estrumate, Coopers, Berkhamsted, England) twice at a 12-
d interval. After the last injection of PGF2a, the estrus was
blood flow and velocity are also related to the ovulatory process, considered to be d 0 of the protocol. Dominant follicles 10 mm in
which facilitates the delivery of immune cells to the ovulatory diameter were removed (dominant follicle removal, DFR) on d 7
follicle [15]. In the previous study, we observed that the number of [20]. Almost 20% of treated heifers showed no CL and were there-
eosinophils (EOS) increased in the bovine oviduct just after fore excluded from the experiment. Then, 48 h after DFR (d 9),
ovulation [16] in which the concentrations of E2 were still high. heifers received follicle stimulating hormone (FSH, Folltropin®-V)
Importantly, granulosa and theca cells communicate with resident twice daily for three consecutive days (d 9, 10, and 11; first day
and infiltrating immune cells to create paracrine mediators of 150 mg, second and third day 75 mg). Finally, the OPU was per-
ovulation [15]. In addition, sex ovarian steroids, such as E2 and formed 48 h after the last injection of FSH (d 13). It should be
progesterone, affect the immune system. Estrogen and progester- mentioned that we performed OPU on follicles 48 h after the last
one have been shown to stimulate MON [17] and thus it can be injection of FSH, in which some follicles may have ovulated earlier.
deduced that the action/number of the blood cells can vary in Prior to collection of oocytes, heifers were held back in an
response to reproductive events. adjustable squeeze chute and lidocaine hydrochloride (2%) was
Despite a significant increase in commercial IVP of cattle administered for epidural anesthesia. The rectum was then emptied
worldwide, reports on the usefulness of complete blood count and the vagina, vulva and perineum were properly washed and
(CBC) for IVP after OPU in cattle are very rare to date. Some CBC cleaned. The OPU device, including the transducer and the (18-G)
parameters, such as white blood cell (WBC), neutrophil (NEU), NEU needle guidance system, was inserted into the vagina. The ovaries
to LYM ratio (NLR), the platelet (PLT) to LYM ratio (PLR) and mean were rotated per rectum and placed over the transducer face in
platelet volume (MPV), are thought to be inflammatory indicators such a way that the intended follicle was transected by the built-in
[18]. These low-grade chronic inflammatory markers are generally puncture line on the ultrasound monitor, which reflected the di-
accounted for the etiopathogenetic mechanisms of subfertility [18]. rection of the projected needle. Antral follicles (2 mm in diam-
Therefore, it can be deduced that there is a closely regulated eter) on both ovaries were aspirated during each ultrasound session
physiological relationship between the immune and reproductive [20]. After follicle aspiration, the device was rinsed with phosphate
systems [10]. To the best of our knowledge, there is no available buffered saline medium (pH 7.4) containing 3% bovine serum al-
study suggesting any association between the CBC indices at the bumin (BSA) and 2 IU/mL heparin.
time of OPU and the result of bovine IVF. The only study was con- Follicular aspirates were collected by a circuit length of 120 cm
ducted in 2018 [18] and showed a negative association between IVF (with an inner diameter of 1.1 mm) connected directly to a 50 mL
outcomes and CBC inflammatory parameters, including WBC, NLR, conical tube containing 15 mL of Dulbecco phosphate-buffered
PLR and MPV among women with unexplained infertility [18]. saline (D-PBS) and 5000 IU/mL sodium heparin at a temperature
Therefore, we hypothesized that CBC indices, which constitute of 35e37  C. Vacuum attached to the needle was fixed at
cheap and easily measurable tests, are associated with in vitro 85e90 mm Hg. All retrieval procedures were conducted by the
embryo production in heifers under the OPU-IVF system. To this same veterinarian.
aim, we used a multidimensional approach to provide a straight-
forward insight into the relationship between CBC indices and IVF 2.2. Cumulus oocyte complexes processing and maturation
outcomes.
The conical tube containing the follicular aspirates was imme-
2. Materials and methods diately transported to the farm laboratory and cumulus oocyte
complexes (COCs) were retrieved using a filter (75 mm, Watanabe
Animal experiments were carried out according to the Guiding Tecnologia Aplicada) and washed once in D-PBS, supplemented
Principles for the Care and Use of Research Animals promulgated by with 1% fetal calf serum (FCS) at 37  C. The COCs were morpho-
the Isfahan University of Technology, Iran. The protocol and logically examined using a stereomicroscope and morphologically
methods were approved by the Committee on the Ethics of Animal classified as follows on the basis of the number of cumulus cell
Experiments of the Isfahan University of Technology (No. 390132). layers: grade A, more than three layers of compact cumulus cells;
154
R. Kowsar, M. Komeili, N. Sadeghi et al. Theriogenology 159 (2021) 153e164

Fig. 1. The schematic representation of the estrous synchronization, OPU protocol, blood sampling and in vitro embryo production. In the OPU trial, the estrus synchro-
nization of the pubertal heifers was carried out by injection of 500 mg of prostaglandin F2a twice at a 12-d interval. After the last injection of PGF2a, the estrus was considered to be
d 0 of the protocol. Dominant follicles 10 mm in diameter were removed (dominant follicle removal, DFR) on d 7. Then, 48 h after DFR (d 9), heifers received follicle stimulating
hormone twice daily for three consecutive days (d 9, 10, and 11). Finally, the OPU was performed 48 h after the last injection of FSH. Before OPU, whole blood samples for the CBC
assessment were collected from the heifers. Recovered COCs were used for in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro embryo culture (IVC).

grade B, at least one layer of cumulus cells; grade C, denuded; and
grade 4, atretic with dark cumulus cells and signs of cytoplasmic
degeneration [20].
In this study, grades 3 and 4 COCs were not considered suitable
for cultivation and discarded upon assessment. Prior to in vitro
maturation (IVM), COCs were washed three times in HEPES-
buffered TCM-199 (supplemented by 10% FCS and 50 mg/mL of
gentamycin) and once in the maturation medium. The maturation
medium consisted of bicarbonate-buffered TCM-199 (Gibco Life
Technologies) supplemented by Na-pyruvate (2.5 mM), L-gluta-
mine (1 mM), penicillin (100 IU/mL), streptomycin (100 mg/mL),
fetal bovine serum (FBS, 10%, v/v, BioWhittaker, Walkersville, MD,
USA), epithelial growth factor (EGF; 100 mM), insulin like growth
factor (IGF; 100 mM), FSH (10 mg/mL, Folltropin-V, Bioniche Animal
Health, Canada), LH (10 mg/mL, APL, Ayerst, Rouses Point, NY), E2
(1 mg/mL), and cysteamine (0.1 mM). The COCs of each heifer were
cultured separately for 24 h in 50-ml maturation medium drops
under mineral oil at 38.5  C, 5.5% CO2, 20% O2, balanced N2, and
maximum humidity.
2.3. IVP procedures
According to previous studies [21,22], after IVM, oocytes of each
heifer were fertilized separately with single sire sperm (Spermex,
GmbH) at a final concentration of 1  106/mL in IVF medium
supplemented by amino acids (50 mL) in 35 mm cell culture dishes
(Falcon brand, BD Biosciences, San Jose, CA) at 38.5  C under 5% CO2
in the air. For IVF, semen straws were thawed for 30 s in a 35  C

Table 1
Descriptive statistics and confidence intervals (95% CI) of data.

Items Mean Median Min Max SD 95% CI Lower upper Roland et al. [49] Herman et al. [24]

White blood cells, 109/L 8.23 7.73 3.49 15.09 2.85 7.55 8.91 4.9e12.0 2.9e11.6
Neutrophil (NEU), 109/L 2.15 1.84 0.60 6.71 1.47 1.80 2.50 1.8e6.3 (0.6e6.0
Lymphocyte (LYM), 109/L 5.58 5.11 2.57 11.14 2.00 5.11 6.06 1.6e5.6 (1.7e5.5)
NEU:LYM ratio 0.42 0.33 0.11 1.72 0.33 0.34 0.50 e e
Monocyte, 109/L 0.29 0.24 0.05 0.64 0.20 0.24 0.33 0e0.8 0.2e0.4
Eosinophil, 109/L 0.15 0.12 0.03 0.65 0.14 0.11 0.18 0e0.9 0.04e2.3
Basophil, 109/L 0.06 0.02 0.00 0.27 0.08 0.04 0.08 0e0.3 (0.0e0.1
Red blood cells, 1012/L 6.99 7.33 4.40 8.67 1.23 6.70 7.29 5.1e7.6 (4.3e8.8)
Hemoglobin, g/dL 10.64 10.80 8.80 12.00 0.81 10.44 10.83 8.5e12.2 (6.8e14.1)
Hematocrit, % 31.05 32.10 22.40 37.40 4.27 30.03 32.08 22e33 20e41)
MCV, fL 44.98 45.90 37.80 51.60 4.68 43.86 46.10 38e50 (38.9e61.8)
MCH, pg 15.75 14.40 12.70 24.00 3.47 14.92 16.58 14e18 (13.3e20.5)
MCHC, g/dL 34.83 33.30 28.90 48.00 5.03 33.62 36.03 36e39 (30.3e37.2
RDW-cv, % 19.29 20.40 10.50 27.20 4.46 18.22 20.35 15.5e19.7 (17e28)
RDW-SD, fL 34.96 35.70 19.80 48.90 7.58 33.14 36.77 e 28.6e47.1
Platelet (PLT), 109/L 194.22 192.00 39.00 327.00 77.70 175.61 212.83 193e637 e
MPV, fL 6.84 6.80 5.00 8.10 0.65 6.69 7.00 4.5e7.5 e
PDW, fL 14.98 14.90 14.10 16.40 0.57 14.84 15.11 e e
PCT, % 0.13 0.14 0.02 0.24 0.06 0.12 0.14 e e
P-LCC, 109/L 21.48 20.00 0.00 66.00 18.05 17.16 25.80 e e
P-LCR, % 9.78 11.00 0.00 20.30 6.59 8.20 11.36 e e
PLT:LYM ratio 38.29 36.63 7.63 110.10 20.65 33.35 43.24 e e
Follicle size, mm 10.27 10.0 4.0 17.0 2.78 9.87 10.7 e e
Aspirated follicle/Heifer 4.59 4.0 3.0 10.0 1.16 4.23 4.95 e e
Recovered COCs/Heifer 7.51 7.0 1 21 4.99 5.96 9.06 e e
Cleavage rate, % 77.40 81.8 0.0 100 21.69 70.60 84.2 e e
Blastocyst formation, % 27.60 30.80 0.0 80.0 24.9 19.80 35.40 e e

SD: standard deviation; CI: confidence interval.

155
R. Kowsar, M. Komeili, N. Sadeghi et al. Theriogenology 159 (2021) 153e164

water bath, and semen was deposited on a 90%e45% of the Percoll

Category 1: Failed, heifers that their COCs did not reach the blastocyst stage (day 7 post-insemination); Cleaved, Heifers that their COCs reached the blastocyst stage (day 7 post-insemination). Category 2: Low-inflam, Heifers
with lymphocyte >5.6  109 cells/L; Normal, Heifers with lymphocyte <5.6  109 cells/L; FHR: Follicle/Heifer ratio; Rec: recovered; CHR: COC/Heifer ratio. The asterisk (*) indicates p < 0.05 within each column in each category.
Of heifers Of COCs (95% CI) Of heifers Of COCs (95% CI)
gradient prepared with sperm wash medium (modified Tyrodes

(34.4 ± 11.6%)
(41.4 ± 5.8%)*

(31.5 ± 5.8%)
0/92 (0.00%) medium) and centrifuged at 320g for 30 min to separate the
Blastocyst formation

138/332
motile sperm and remove the diluents and seminal plasma. Sperm

65/189

74/235
and COCs were incubated at 38.5  C in humidified air with 5% CO2
for 18e20 h. The IVF medium was Tyrodes albumin lactate pyru-
vate (TALP) supplemented by 10 mg/mL of heparin, 22 mg/mL of
(0.00%)

(76.9%)

(50.0%)
(100%)
37/37

20/26

17/34 sodium pyruvate, 50 mg/mL of gentamycin, 6 mg/mL of fatty-acid-

0/23

free bovine serum albumin (BSA), and penicillin, hypotaurine,

epinephrine solution (2 mM of penicillin, 1 mM of hypotaurine,
(81.9 ± 3.6%)*

(86.8 ± 7.2%)*
(65.2 ± 2.9%)

(74.3 ± 5.2%)

and 0.25 mM of epinephrine).

Approximately 18 h after insemination, zygotes were manually
272/332

163/189

175/235
60/92

stripped of the cumulus cells and the resulting embryos were

transferred into cell culture dishes containing synthetic oviductal
fluid medium supplemented by amino acids and 4 mg/mL BSA.
Cleavage

(65.2%)

(88.8%)

(85.3%)
(100%)

Embryos were then cultured in an incubator at 38.5  C under 5%

15/23

37/37

23/26

29/34

O2 and 6% CO2 for 60 h. Developing embryos were scored using a

stereomicroscope (Olympus, Tokyo, Japan) as embryos reached
Grade A, B COCs

the stage of cleavage on day 3 after insemination and the blasto-

(18.2 ± 2.5%)

(15.5 ± 2.1%)

(19.4 ± 3.5%)

(21.3 ± 5.1%)

cyst stage on day 7 after insemination. The percentage of cleavage

(95% CI)

52/332

37/189

50/235

and the percentage of blastocyst formation were calculated in

17/92

relation to the number of COCs cultured in the maturation

medium.
(81.7 ± 17.0%)

(84.5 ± 12.7%)

(80.5 ± 15.5%)

(78.7 ± 10.1%)
Grade A COCs

2.4. Blood sampling and assays

280/332

152/189

185/235
(95% CI)

75/92

Before OPU, whole blood samples (10 mL) for the CBC assess-
ment were collected from the heifer caudal vein by disposable
CHR (95% Recovery rate, %

syringes containing potassiumeethylenediamine tetra-acetic acid

(EDTA/K3, 1.27 mg EDTA/K3 per mL of blood). Samples were
9.0 ± 1.6* 80.1 ± 3.0*

6.9 ± 1.4 88.3 ± 3.4*

4.0 ± 1.3 75.4 ± 4.0

7.2 ± 0.9 69.7 ± 4.9

transported into the laboratory using an icebox within 2 h at 4  C.

(95% CI)

Hematological analysis was carried out using an automated he-

matology counter (Scil Vet abc Plus, Horiba Medical, Kyoto, Japan).

2.5. Statistical analysis

COCs (n) CI)

A total of 60 heifers were categorized into two groups in

relation to the IVF outcome as (1) a cleaved group (the heifers with
Rec.

332

189

235

COCs formed blastocysts on Day 7, n ¼ 37) and (2) a failed group

92

(the heifers whose COCs did not reach the blastocyst stage on day
FHR (95% CI)

(11.2 ± 1.4)*

(10.4 ± 0.3)

7, n ¼ 23). It was done to investigate how the blood indices vary

(5.3 ± 0.5)

(7.8 ± 0.6)

between heifers with different competent COCs. In addition,

122/23

415/37

271/26

266/34

heifers were grouped into two groups on the basis of low-level

inflammation. Among all the indices, LYM had a potential for
(<7 mm) (7e10 mm) (>10 mm)

grouping heifers since 18 out of the 60 heifers had an elevated

181/415

103/266
(46.7%)

(43.6%)

(36.5%)

(38.7%)
57/122

99/271

number of LYMs (>5.6  109 cells/L), while other indices were

mainly in the normal range. Therefore, heifers were grouped into
two classes: (1) heifers with a low-grade inflammation (n ¼ 18)
and (2) heifers with no inflammation (n ¼ 42). This grouping was
190/415

144/271

128/266
(41.8%)

(45.8%)

(53.1%)

(48.1%)
51/122

performed to determine the impact of the elevated number of

Follicle size (mm) Follicle number

LYMs on the IVF outcome. Data were not normally distributed

using the Anderson-Darling test (EasyFit software, version 5.6,
(11.5%)

(10.6%)

(10.3%)

(13.2%)
14/122

44/415

28/271

35/266

MathWave Technologies, Spokane, WA, USA).

The Mann-Whitney U test was used to compare the medians of
two groups using PAST software (accessible at: http://folk.uio.no/
ohammer/past). Differences at P < 0.05 were considered statisti-
cally significant. Descriptive statistics and confidence intervals (CI)
Failed (n ¼ 23) 10.4 ± 0.7

10.3 ± 0.4

10.2 ± 0.7

10.3 ± 0.5

for the hematological/follicular parameters of Holstein heifers (at

(95% CI)
OPU and IVF outcomes.

CI: confidence interval.

the time of OPU) and IVF outcomes are shown in Table 1.

The bivariate analysis was performed using the Spearman
correlation to assess the relationship between blood parameters
Low Inflam.
(n ¼ 37)

(n ¼ 26)

(n ¼ 34)

and the percentage of grade A COCs, the cleavage rate and the
Cleaved

Normal

formation of blastocysts. Because only the formation of blastocysts

Table 2

had a significant correlation with the blood indices, other pa-

rameters (grade A and cleavage rate) were excluded from the
156
R. Kowsar, M. Komeili, N. Sadeghi et al. Theriogenology 159 (2021) 153e164

Cytoscape analysis. Correlation-based network analysis of blood
indices and blastocyst formation was performed using Cytoscape.
In Cytoscape, data were integrated into interactive nodes or edges,
based on individual attributes, such as the correlations between
blood indices and the blastocyst formation using Spearman
correlation.
Principal component analysis (PCA) was conducted to deter-
mine the correlation of blood indices with the percentage of grade
A COCs, the rate of cleavage and the formation of blastocysts.
Multiple data dimensions were reduced to two dimensions and the
biplot was prepared using PAST software. The hierarchical clus-
tering analysis (HCA) with Spearman distance metric and un-
weighted pair group method algorithm (UPGMA) was performed
using the PAST software [23] to generate clustering patterns of
blood parameters, the percentage of grade A COCs, cleavage rate,
and blastocyst formation. The network analysis was carried out
using the Spearman similarity index and the Fruchterman-Reingold
algorithm as a force-directed layout algorithm. This algorithm or-
ganizes a network based on the frequency of connections to the
nodes. Spearman correlation and network visualization analyses
were conducted using PAST software.
Using the easyROC web-tool (accessible at: http://www.biosoft.
hacettepe.edu.tr/easyROC/), the receiver operating characteristic
(ROC) curve analysis was performed on the basis of network out-
puts that established a link between blood parameters and blas-
tocyst formation. This was done to identify the predictive power
and cut-off point of the network-detected parameters for the
detection of heifers with a high percentage of blastocyst formation
by the area under the ROC curve (AUC). To this aim, the results were
divided into separate groups as follows: (1) low percentage of
blastocyst formation (<60%) and (2) high percentage of blastocyst
formation (60%). The optimal cut-off was calculated by maxi-
mizing the Youden index.
3. Results
3.1. OPU and IVF results
Considering all heifers (Table 1), the follicular size, the aspirated
follicle per heifer, and the recovered COCs per heifer were
10.27 ± 2.78 mm, 4.59 ± 1.16, and 7.51 ± 4.99, respectively. The
mean cleavage rate and blastocyst formation in experimental
heifers was 77.40 ± 21.70% and 27.60 ± 24.9%, respectively.
Grouped into heifers developed (cleaved) or did not develop
(failed) to the blastocyst stage (Table 2), it was found that the
cleaved group had more retrieved follicles (415 vs. 122) and follicle
per heifer ratio (11.2 ± 1.4 vs. 5.3 ± 0.5, p  0.05) than failed heifers.
The number of COCs recovered per heifer was higher (p  0.05) in
the cleaved than in the failed group (4.0 ± 1.3 vs. 9.0 ± 1.6). The
recovery rate (COCs per follicle) was greater (p  0.05) in the
cleaved than failed group (75.4 ± 4.0% vs. 80.1 ± 3.0%). The per-
centage of cleavage and blastocyst formation was higher (p  0.05)
in cleaved heifers than in failed heifers (65.2 ± 2.9% vs. 81.9 ± 3.6%
and 0.0 vs. 41.4 ± 5.8%).
In the case of grouping heifers as low-grade inflammation
(>5.6  109 cells/L) or normal (<5.6  109 cells/L, Table 2), it was
noted that the low-grade inflammation group had a lower recovery
rate (COCs per follicle) than the normal group (69.7 ± 4.9% vs.
88.3 ± 3.4%, p  0.05). However, the percentage of cleavage and the
blastocyst formation was numerically higher in the low-grade
inflammation group than in the normal group (86.8 ± 7.2% vs.
74.3 ± 5.2% and 34.6 ± 11.6 vs. 31.5 ± 5.8%).
3.2. Blood indices in heifers whose oocytes developed or did not
develop to the blastocyst stage
The NEU, WBC, NLR, and PLR values were not statistically
different between heifers whose oocytes developed (cleaved) or
did not develop (failed) to the blastocyst stage (Figs. 2 and 3). Data
showed that oocytes recovered from heifers with higher values of
MON and EOS did not develop into the blastocyst stage (p  0.05,
Figs. 2 and 3). Oocytes recovered from heifers with higher LYM
successfully developed into the blastocyst stage (p  0.05, Figs. 2
and 3).
Data showed that oocytes recovered from heifers with higher
values of MCH and MCHC (at the time of OPU) did not reach the
blastocyst stage (p  0.05, Figs. 2 and 3). Oocytes recovered from
heifers with higher RBC, HCT, RDW-SD, PLT, RDW-cv, and PCT
values successfully reached the blastocyst stage (p  0.05, Figs. 2
and 3).

Fig. 2. White blood cell indices in heifers at the time of OPU. A total of 60 heifers were divided into two groups in relation to their IVF outcome as either a cleaved group (heifers
whose oocytes developed into the blastocyst stage, Day 7 post-insemination, n ¼ 37, brown boxes) or a failed group (heifers whose oocytes did not enter the blastocyst stage, n ¼ 23,
aqua boxes). Boxes range from the 25th to the 75th percentile of the distribution of values for each group. The median is the line inside the box. The whiskers represent the
maximum and minimum data point within 1.5 times the interquartile range. The Mann-Whitney U test was used to compare the median between two groups. The asterisk (*)
indicates p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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R. Kowsar, M. Komeili, N. Sadeghi et al. Theriogenology 159 (2021) 153e164

Fig. 3. Red blood cell indices (a) and (b) platelet indices in heifers at OPU time. A total of 60 heifers were divided into two groups in relation to their IVF outcome as either a
cleaved group (heifers whose oocytes developed into the blastocyst stage, Day 7 post-insemination, n ¼ 37, brown boxes) or a failed group (heifers whose oocytes did not enter the
blastocyst stage, n ¼ 23, aqua boxes). Boxes range from the 25th to the 75th percentile of the distribution of values for each group. The median is the line inside the box. The
whiskers represent the maximum and minimum data point within 1.5 times the interquartile range. The Mann-Whitney U test was used to compare the medians between two
groups. One asterisk (*) indicates p < 0.05. Two asterisks (**) indicate p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web
version of this article.)

3.3. The relationship between blood indices and the blastocyst rate:
Spearman correlation, HCA, and PCA analyses
Data showed that the percentage of grade A COCs tended to be
correlated with MCHC (R ¼ 0.24, p ¼ 0.14), PLT to LYM ratio
(R ¼ 0.23, p ¼ 0.15), PDW (R ¼ 0.27, p ¼ 0.07), P-LCC (R ¼ 0.26,
p ¼ 0.09) and P-LCR (R ¼ 0.26, p ¼ 0.10). The percentage of cleavage
tended to be correlated with WBC (R ¼ 0.26, p ¼ 0.13, Table 3).
The Spearman correlation analysis showed that the percentage
of blastocyst formation was positively correlated with the

Table 3
The Spearman correlation and ROC analysis.

Blood indices Grade A COCs Cleavage rate Blastocyst formation The ROC analysis for
(BF) BF**

R* p value R* p value R* p value AUC p value

Neutrophil (NEU), % 0.10 NS 0.11 NS 0.26 0.11 0.74 0.02

Lymphocyte (LYM), % 0.06 NS 0.13 NS 0.33 0.04 0.76 0.01
Monocyte, % 0.22 NS 0.15 NS 0.41 0.01 0.76 0.01
Eosinophil, % 0.07 NS 0.05 NS 0.33 0.04 0.80 0.002
Basophil, % 0.05 NS 0.06 NS 0.07 NS 0.57 NS
White blood cells, 109/L 0.18 NS 0.24 0.13 0.05 NS 0.51 NS
NEU, 109/L 0.19 NS 0.03 NS 0.18 NS 0.70 0.09
LYM, 109/L 0.12 NS 0.11 NS 0.29 0.07 0.65 0.11
NEU:LYM ratio 0.10 NS 0.08 NS 0.27 0.10 0.74 0.02
Monocyte, 109/L 0.10 NS 0.04 NS 0.30 0.05 0.72 0.06
Eosinophil, 109/L 0.21 NS 0.08 NS 0.22 NS 0.79 0.005
Basophil, 109/L 0.01 NS 0.14 NS 0.13 NS 0.54 NS
Red blood cells, 1012/L 0.05 NS 0.18 NS 0.00 NS 0.52 NS
Hemoglobin, g/dL 0.06 NS 0.02 NS 0.12 NS 0.45 NS
Hematocrit, % 0.18 NS 0.17 NS 0.23 NS 0.65 0.10
MCV, fL 0.11 NS 0.12 NS 0.05 NS 0.56 NS
MCH, pg 0.15 NS 0.20 NS 0.04 NS 0.50 NS
MCHC, g/dL 0.24 0.14 0.21 NS 0.26 0.11 0.58 NS
RDW-cv, % 0.01 NS 0.17 NS 0.23 0.15 0.66 NS
RDW-SD, fL 0.07 NS 0.20 NS 0.40 0.01 0.76 0.01
Platelet (PLT), 109/L 0.02 NS 0.01 NS 0.04 NS 0.64 NS
PLT:LYM ratio 0.23 0.15 0.15 NS 0.19 NS 0.73 0.02
MPV, fL 0.15 NS 0.06 NS 0.19 NS 0.60 NS
PDW, fL 0.27 0.07 0.13 NS 0.02 NS 0.47 NS
PCT, % 0.21 NS 0.02 NS 0.04 NS 0.62 NS
P-LCC, 109/L 0.26 0.09 0.13 NS 0.03 NS 0.58 NS
P-LCR, % 0.26 0.10 0.18 NS 0.02 NS 0.54 NS

One asterisk (*) indicates that the Spearman correlation was used to assess the correlation between the blood indices and number of COCs (grade A), cleavage rate (day 3 after
insemination); and the percentage of blastocyst formation (day 7 post-insemination). Two asterisks (**) indicate that the ROC curve analysis was performed using the easyROC
web-tool. The analysis was accomplished by defining the predictive power of the parameters for the detection of high blastocyst formation by area under the ROC curve (AUC).
In order to counter potential biases in finding that the relationship between blood indices and blastocyst formation could be linear, the results were divided into separate
categories as follows: (1) low percentage of blastocyst formation (<60%); (2) high percentage of blastocyst formation (60%). NS: not significant.

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Fig. 4. Correlation-based network analysis of the blood indices and the percentage of blastocyst formation. (A) All blood indices (white blood indices: blue octagons; red blood
cell indices: red octagons; platelets indices: yellow octagons; blastocyst formation percent: green octagon); (B) white blood indices; (C) red blood cell indices; and (D) platelets
indices. Cytoscape was used for the construction of networks. Using Spearman correlation, data is integrated into interactive nodes or edges in Cytoscape based on individual
attributes, such as correlations between blood indices and blastocyst formation rate. Red and blue arrows indicate negative and positive correlation, respectively. Thickness of the
edges connecting the nodes represents the greater correlation between blood indices and blastocyst rate. (For interpretation of the references to color in this figure legend, the
reader is referred to the Web version of this article.)

Fig. 5. Study of hierarchical cluster analysis (HCA) and principal component analysis (PCA) of the blood indices and the blastocyst formation. (A) HCA was developed by PAST
using Spearman distance measurement and UPGMA. Multi-color boxes (i.e., red, green, and yellow) reflect various clusters. (B) Biplot was developed from the PCA of variables.
Close-angles Vectors (<45 ) indicate a positive association, perpendicular vectors indicate no correlation, and vectors in opposite directions (approaching 180 ) indicate a negative
association. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

159
R. Kowsar, M. Komeili, N. Sadeghi et al. Theriogenology 159 (2021) 153e164

percentage of LYM (R ¼ 0.33, p ¼ 0.04) and RDW-SD (R ¼ 0.40,
p  0.01), but was negatively associated with the percentage of
MON (R ¼ 0.41, p  0.01) and EOS (R ¼ 0.33, p ¼ 0.04, Table 3 and
Fig. 4). Correlations between the blood indices and the percentage
of blastocyst formation are shown in Fig. 4 using the Cytoscape
software.
Next, in order to identify the variables with the greatest impact
on system variability and to consider the parallel relationship be-
tween variables, all variables were simultaneously included in the
multivariate study of factors, including HCA and PCA analysis. The
HCA analysis showed no closeness between the percentage of
cleavage and the blood indices (Fig. 5A). Also, the percentage of
blastocyst formation and the grade of COCs did not form a separate
sub-cluster with other blood indices.
However, the PCA study showed a strong positive association
between the percentage of blastocyst formation, RBC, MPV, RDW-
SD, RDW-cv, HCT, PLT, NLR and PCT. Furthermore, the PCA study
showed that the percentage of blastocyst formation had a strong
negative association with MCH, MCHC and MCV values (Fig. 5B).
The PCA study found that the percentage of cleavage had a strong
positive association with the P-LCC, P-LCR, PDW, and PLT to LYM
ratio, but showed a strong negative association with WBC, BAS,
LYM, and MON (Fig. 5B). The PCA revealed that the first three
principle axis explained (31.0, 16.4, and 12.3%, respectively) a suf-
ficient amount of variance, indicating 59.7% of the total variability
(Supp. Table 1).
3.4. The network structure of the variables
Finally, a network analysis was performed to determine the
relationship between the blood indices and the blastocyst rate. In
the case of a 65% cut-off point, a link between the rate of blastocyst
formation and RDW-SD was found when all parameters were
analyzed together (darker line shown in Fig. 6A). This 65% cut-off
point was chosen since above this level the formation of blasto-
cyst was disconnected from the blood indices in the network.
Next, the network analysis was conducted on different blood
indices using the maximum cut-off point. LYM counts were found
to be associated with the blastocyst formation when WBC indices
were analyzed at a 40% cut-off point (Fig. 6B). The rate of blastocyst
formation was found to be associated with MPV when PLT indices
were analyzed at a 40% cut-off point (Fig. 6C). In addition, the rate
of cleavage was associated with PCT and P-LCC (Fig. 6C). In the case
of a 65% cut-off point, the formation of blastocyst was correlated
with RDW-SD in the event that RBC indices were considered to be
only one (Fig. 6D).
3.5. ROC analysis to identify heifers with a high percentage of
blastocyst formation
The AUC of all blood indices for the identification of heifers with
a high percentage of blastocyst formation (>40) is shown in Table 3.
The AUC of the ROC analysis showed that EOS percentage <1.3%
(AUC 0.80, p ¼ 0.002), EOS number < 0.08  106 (AUC 0.79,
p ¼ 0.005), RDW-SD level > 38.5 fL (AUC 0.76, p  0.01), MON

Fig. 6. Visualization of the relationships between the blood indices and the percentage of blastocyst formation at the highest threshold. All variables (blood indices and
percentage of blastocyst formation) are represented in the circles within the network. Network analysis and visualization was carried out using the PAST software and Fruchterman-
Reingold algorithm as a force-directed layout algorithm. The following thresholds were selected to define the relations between edges (i.e., correlation) and nodes (i.e., variables) as
follows: (A) 65%, if the network is made up of all blood indices; (B) 40%, if the network is made up of white blood cell indices; (C) 40%, in the case that the network was made up of
platelet indices; and (D) 65%, if the network is made up of red blood cell indices. These thresholds were the highest cut-off at which there was no subsequent interaction between
the blood indices and the blastocyst formation. Nodes are the blood indices and the blastocyst formation. Edges are the interactions of all variables. The size of the nodes and edges
refers to the coefficient of clustering and the coefficient of correlation, respectively. Small nodes and thin edges refer to small values. (For interpretation of the references to color in
this figure legend, the reader is referred to the Web version of this article.)

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Table 4
ROC analysis to describe the predictive power of the blood indices to discriminate against low percentage of blastocyst formation.

AUC P- Optimal cut off Sensitivity Specificity Positive predictive Negative predictive Positive likelihood Negative likelihood AUC
value point, % value value ratio (LR) ratio (LR) rank

EOS, % 0.80 0.002 1.3 80.2 75.9 53.3 91.7 3.31 0.26 1
EOS, 109/L 0.79 0.005 0.08 80.2 82.8 61.5 92.3 4.64 0.24 2
RDW-SD, fL 0.76 0.01 38.5 70.0 79.3 53.8 88.5 3.38 0.37 3
MON, % 0.76 0.01 2.9 80.5 82.8 61.5 92.3 4.64 0.24 4
LYM, % 0.76 0.01 79.8 60.4 89.7 66.7 86.7 5.8 0.45 5
NEU:LYM 0.74 0.02 0.20 60.3 86.2 60 86.2 4.35 0.46 6
ratio
NEU, % 0.74 0.02 15.7 60.0 82.8 54.5 85.7 3.48 0.48 7
PLT:LYM 0.73 0.02 31.8 70.5 82.8 58.3 88.9 4.06 0.36 8
ratio
MON, 109/L 0.72 0.06 0.19 80.2 82.5 61.5 92.3 4.64 0.24 9
NEU, 109/L 0.70 0.09 0.99 60.5 86.2 60 86.2 4.35 0.46 10

The analysis was carried out by determining the predictive power and cut-off point of the blood indices for the detection of high percentage of blastocyst formation (60%) by
AUC. The optimal cut-off was calculated by maximizing the Youden index. Objects with the highest AUC (i.e., 70) are shown.

percentage < 2.9% (AUC 0.76, p  0.01), LYM percentage > 79.8% when the higher number of oocytes per OPU was recovered [28].
(AUC 0.76, p  0.01), NEU:LYM ratio < 0.20 (AUC 0.74, p ¼ 0.02), Thus, it seems important to identify donors with a higher oocyte
NEU percentage < 15.7% (AUC 0.74, p ¼ 0.02), and PLT:LYM ra- recovery-per-OPU potential, particularly in cattle breeds with fewer
tio < 31.8 (AUC 0.73, p ¼ 0.02) had a good predictive ability to detect oocytes per OPU, such as Holstein, in order to ensure greater IVP
heifers with high IVF outcomes (60%) as shown in Table 4 and efficiency.
Supplementary Fig. 1. Next, in order to determine how inflammatory indices affect
OPU-IVP results, heifers were classified as low-grade inflammation
(LYM > 5.6  109/L, (95% CI: 6.97 ± 0.77) and normal
4. Discussion
(LYM < 5.6  109/L, (95% CI: 4.32 ± 0.32). The percentage of cleavage
and blastocyst formation was found to be higher in the low-grade
In this study, it was found that the inflammatory markers of
inflammation group than in the normal group. Similar to our
hematological indices, such as WBC count, NLR, MPV and PLR, did
findings, Talo [18] reported a significant and positive correlation
not differ statistically between heifers whose oocytes developed or
between the fertilization rate and LYM (R ¼ 0.23) and found that
fail to develop to the blastocyst stage. It is known that the assess-
LYM was the only positive predictive indicator for normal fertil-
ment of NLR and PLR can be used to evaluate systemic inflamma-
ization. Liu et al. [29] reported that low-dose LYM immunotherapy
tion [18,24], but there is no research on the relationship between
is beneficial for restoring balance in the T helper (Th)1/Th2/Treg
inflammatory markers in the blood and the results of IVF in ru-
paradigm and improving pregnancy outcomes. It has been reported
minants. The present findings showed that heifers with either
that the homeostasis of Th1/Th2 cytokines regulates maternal-fetal
cleaved or uncleaved oocytes had no chronic inflammation [25].
immune tolerance during pregnancy [29]. It seems that the
However, the LYM count was greater than 5.6  109 cells/L in 26 out
marginally higher levels of LYM may support the balance of the
of the 60 heifers with low-grade inflammation. In addition, the
Th1/Th2/Treg in the cleaved heifers. However, further studies are
small sample size in this study may be the cause of a non-
needed to confirm this assumption.
significant discrepancy between heifers that produced or failed to
Data showed that there was a negative correlation between the
produce blastocyst.
number of EOS at the time of OPU and the formation of blastocyst;
In this study, first, we grouped heifers into the cleaved group,
the number and percentage of EOS was lower in heifers with oo-
which their COCs developed to the blastocyst stage, and the failed
cytes that were successfully developed to the blastocyst stage. The
grouped, which their COCs did not develop to the blastocyst stage.
AUC identified EOS as the best predictive index for detecting heifers
This grouping was done to determine how the blood indices vary
with high IVF outcomes; EOS percentage <1.3% (AUC 0.80) as the
based on the success of OPU-IVP. The data showed that the per-
best cut-off point for distinguishing heifers with high IVF outcomes
centage of blastocyst formation was generally lower than the
(60%), which resulted in a sensitivity of 80% and a specificity of
typical levels as 11 out of the 60 heifers did not produce blastocyst
75.9%. FSH has been shown to enhance the development of follicles
on day 7 after insemination, but their COCs developed to the
and stimulate E2 synthesis [30]. Following an increase in the
cleavage stage. It has been known that the number of COCs in IVM
follicular concentration of E2, the final stage of leukocyte infiltra-
affects the rate of oocyte maturation because the cumulus cells and
tion occurs more or less concurrently with ovulation [31]. Large
oocytes maintain the supply of paracrine and autocrine factors that
numbers of EOS are present in the uterus, especially the endome-
are essential for oocyte development [26]. Salvador et al. [27] re-
trial stroma and the endometrial-myometrial junction [32].
ported adverse effects on the percentage of blastocyst when a
Increased numbers of EOS in pre-ovulatory ovarian follicles [33,34],
single or low number of embryos (5-10) were cultured during post-
post-ovulatory infundibulum [16] and endometrium [32,35,36]
fertilization in vitro culture. High paracrine and autocrine activities
mean that they play a role in ovulation-related tissue remodeling
have been reported to be achieved in cultures with more than 20
[34,36]. Moreover, it has been shown that the uterus produces an
COCs [26]. In this study, the culture of a small number of COCs
EOS chemoattractant in response to estrogens [33]. Delays in estrus
retrieved from each heifer, ranging from 1 to 20, may reduce the
onset have been reported in mice with uterine EOS deficiency
positive interactions between COCs for improved competence.
[32,37], suggesting the role of EOS in uterine maturation [37]. The
In addition, the recovery rate (COC/follicle ratio), the cleavage
lower circulatory number of EOS in heifers with cleaved oocytes
rate and the formation of blastocysts were higher in the cleaved
may be due to higher migration of EOS to different parts of the
heifers than in the failed group. It has been shown that in both, beef
body, such as reproductive tissues.
and dairy breeds, the number of blastocysts per OPU was higher
161
R. Kowsar, M. Komeili, N. Sadeghi et al.
The results showed that the number of MON at the time of OPU
was negatively associated with the blastocyst formation; the lower
number of MON was found in heifers whose oocytes were suc-
cessfully developed into the blastocyst stage. The AUC identified
that MON had a good predictive potential to detect heifers with
high IVF outcomes; MON percentage <2.9% (AUC 0.76) was the best
cut-off point for the identification of heifers with high IVF out-
comes (60%), with a sensitivity of 80% and a specificity of 82.8%.
Most experiments have been performed on blood MON obtained
during the third trimester of pregnancy and there is a significant
lack of evidence for other conditions, such as OPU time [38]. An
increase in the number and activation of blood MON has been
observed during normal pregnancy [38]. Inherent production of
pro-inflammatory cytokines by MON has been shown to increase
following ovulation [10]. Estrogen receptors have been reported to
be expressed in the nucleus [10,39] and on the surface [10,40] of the
MON. This indicates that MON can respond directly to estrogens
and is susceptible to cyclical changes in circulating hormone con-
centrations [10]. Peripheral blood MON produces pro-
inflammatory cytokines which, in turn, promote the migration of
cells from the vascular compartment into the endometrium. This
implies the role of peripheral blood MON in a non-specific immune
response to ovulation [10].
The Spearman correlation analysis showed that the percentage
of blastocyst formation was positively correlated with the per-
centage of PLT and LYM; these cells were higher at the time of OPU
in heifers whose oocytes had been successfully developed into the
blastocyst stage. Tola [18] recently reported a positive association
between the fertilization rate and the PLT and LYM counts in
women, a finding that is consistent with our results. The reason for
increased PLT count in heifers with successful IVF is not fully un-
derstood. PLTs are involved in inflammatory processes and release a
variety of cytokines, chemokines and growth factors [41]. More-
over, the physiological roles of PLT in reproductive events can be
speculated. For example, PLTs have been reported to modulate the
function of the hypothalamo-hypophyseal-ovarian system. Follic-
ular cells release PLT activator factor (PAF) that activates the PLT and
increases the accumulation of PLTs in follicular vessels around the
follicle [42e44]. PLT-released soluble molecules (such as chemo-
kines and cytokines) have been shown to affect oocyte maturation
and hormonal secretion [42e44]. These findings may explain, at
least in part, the higher number of PLTs in heifers whose oocytes
have developed to the blastocyst stage.
In addition, the LYM-released macrophage migration inhibitory
factor (MIF) has been reported to be involved in the development of
maternal-fetal immune tolerance during placentation in human
and porcine [45]. MIF has been shown to reduce the cytotoxic ac-
tion of endometrial natural killer cells and immune reactions
against fetal membranes during placentation [46]. The positive
association between LYM and the blastocyst formation implied a
physiological role for LYM in reproductive events. Indeed, LYMs are
involved in maintenance of the sterile environment of the uterine
lumen, providing an optimum condition for reproductive events
[47]. Moreover, as mentioned above, LYMs are involved in the
balance of the Th1/Th2/Treg and the homeostasis of Th1/Th2 cy-
tokines that regulate maternal-fetal immune tolerance during
pregnancy [29]. However, further study remains on the exact role of
LYM in the development and quality of oocytes. In addition, The
AUC identified that LYM had a good predictive ability to find heifers
with high IVF outcomes; the LYM percentage >79.8% (AUC 0.76)
was the best cut-off point for the detection of heifers with high IVF
outcomes (60%), with a sensitivity of 60% and a specificity of
89.7%.
It was found that oocytes recovered from heifers with higher
values of MCH and MCHC measured at the time of OPU did not
162
Theriogenology 159 (2021) 153e164
reach the blastocyst stage. A strong negative association was found
in the Spearman correlation and PCA analysis between the blas-
tocyst formation and the MCH and MCHC. It has been reported that
MCHC only increases in a few rare diseases, such as spherocytosis,
and is therefore rarely used in practice [48]. High scores of MCH are
usually a sign of macrocytic anemia [49]. In this study, 38.5% of
heifers whose oocytes did not reach the blastocyst stage had MCH
values above the normal range (>19 pg) reported by others [24,49]
compared to the MCH value observed for heifers with competent
oocytes (19.2%). Increased MCH occurs when there is a higher
amount of hemoglobin (HBG) is found in RBC, which may be due to
vitamin B12 or folic acid deficiency in the body [50,51] that is
believed to be associated with decreased IVF outcomes [52]. In
addition, chronic vitamin B12 deficiency causes infertility by
inducing changes in the ovulation or development of the ovum
[53].
The results showed that oocytes recovered from heifers with
higher RBC, HCT, RDW-SD, RDW-cv and PCT values (within the
normal range) were successfully developed into the blastocyst
stage. Indeed, the marginal levels of RBC and HBG (mild anemia)
caused by Iron deficiency [54] may lead to a reduction in oocyte
competence and ovulatory infertility [55]. Women who do not have
enough iron have been shown to suffer from anovulation (lack of
ovulation) [55]. The results suggest that there could be a relation-
ship between the amounts of HBG and RBC at the time of OPU and
the result of IVF in heifers.
Moreover, the analysis by Spearman and PCA showed that the
rate of formation of blastocyst was positively associated with RDW-
SD. Using a network analysis, RDW-SD was shown to be associated
with the blastocyst formation when all blood indices were
considered together (Fig. 6B). In addition, the ROC analysis showed
that the RDW-SD had a good predictive value (AUC 0.76) for the
high percentage of blastocyst formation. Moreover, the RDW-SD
value of >38.5 fL was detected as an optimal cut-off for the
detection of high percentage of blastocyst formation, with a
sensitivity of 70.0% and a specificity of 79.3%. The RDW reflects a
variation in the size of RBC and its rise means that the size of RBC is
not uniform [56]. Although RDW has been used in clinical practice
for decades, normal reference ranges in reproductive practices are
not well defined [57]. Li et al. [57] reported that median RDW
values vary significantly between non-pregnant and pregnant
women (12.3 vs.13.5%, respectively) [56]. Higher RDW levels have
been reported to be associated with polycystic ovary syndrome
(PCOS) [58]. Increased RDW may be associated with iron, vitamin
B12, folic acid, and HGB deficiency [59]. In the present study, the
RDW-SD value was higher in heifers whose oocytes developed into
the blastocyst stage than in failed heifers (30.9 vs. 35.9 fL, respec-
tively), but these values matched the normal range [24]. In the
normal range, the upper limits of RDW-SD in heifers seem to have
been associated with successful blastocyst formation because the
ROC analysis indicated that the RDW-SD value of >38.5 fL was the
best cut-off point for the identification of heifers with high per-
centage of blastocyst formation (i.e., 60%).
In conclusion, the results showed that the percentage of blas-
tocyst formation was associated with certain blood indices at the
time of OPU. Our research corroborates the growing evidence of a
close relationship between the reproductive outcome and the
blood indices that can be used for assisted reproductive technology
practice. However, it should be noted that heifers were super-
stimulated by FSH that this is not what usually occurs in
livestock-level OPU programs where OPU can be done on a random
day of the estrous cycle. In addition, the present data was obtained
from heifers with a high concentration of E2. Since blood cells have
E2 receptors that regulate a number of genes [60], our findings
cannot necessarily be generalized to all OPU programs.
R. Kowsar, M. Komeili, N. Sadeghi et al.
Funding
This study was supported by the Isfahan University of Tech-
nology (Grant-in-Aid for Scientific Research, No. 14708).
CRediT authorship contribution statement
Rasoul Kowsar: Conceptualization, Writing - review & editing,
Supervision. Mehdi Komeili: Farm and laboratory tests, Investi-
gation. Nima Sadeghi: Farm and laboratory tests, Methodology.
Khaled Sadeghi: Conceptualization, Methodology.
Declaration of competing interest
The authors report no conflicts of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.theriogenology.2020.10.026.
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