animals
ArticleEffects of Two Commercial Diets on Several Reproductive
Parameters in Bitches: Note One—From Estrous Cycle
to Parturition
Riccardo Orlandi 1 , Emanuela Vallesi 1 , Serena Calabrò 2 , Alessandro Vastolo 2 , Nadia Musco 2, * ,Alessandro Troisi 3 , Angela Polisca 4 , Pietro Lombardi 2 and Monica I. Cutrignelli 2
1

2

3

4

*



Citation: Orlandi, R.; Vallesi, E.;
Calabrò, S.; Vastolo, A.; Musco, N.;
Troisi, A.; Polisca, A.; Lombardi, P.;
Cutrignelli, M.I. Effects of Two

Tyrus Veterinary Clinic, Via A. Bartocci 1/G, 05100 Terni, Italy; riccardo.orlandi83@hotmail.it (R.O.);
manu0391@libero.it (E.V.)
Department of Veterinary Medicine and Animal Production, University of Napoli Federico II,
80100 Napoli, Italy; serena.calabro@unina.it (S.C.); alessandro.vastolo@unina.it (A.V.);
pietro.lombardi@unina.it (P.L.); monica.cutrignelli@unina.it (M.I.C.)
School of Bioscience and Veterinary Medicine, University of Camerino, 62024 Matelica, Italy;
alessandro.troisi@unicam.it
Department of Veterinary Medicine, University of Perugia, 06124 Perugia, Italy; angela.polisca@unipg.it
Correspondence: nadia.musco@unina.it

Simple Summary: Reproductive efficiency is a key aspect of all breeding species. Several factors,
such as infective diseases, hormonal, and nutritional status could affect female fertility. As demonstrated by high numbers of newborns per litter and proper fetal development, nutritional status
at mating, and correct nutritional management during pregnancy are fundamental. In this study,
two diets with different ingredients (protein and lipid sources) and different macro and micronutrients concentrations (crude protein, fatty acids profile, vitamins, and mineral concentration) wereadministered from two months before the expected onset of proestrus to parturition to 18 bitches
divided into two groups. The experimental diet, richer in protein, essential and polyunsaturated
fatty acids and vitamins, when compared to control diet, seems able to guarantee better clinicalpresentation of estrus and embryonic development in the first third of pregnancy with a lower
incidence of a fetal resorption.

Commercial Diets on Several
Reproductive Parameters in Bitches:
Note One—From Estrous Cycle toParturition. Animals 2021, 11, 23.
https://dx.doi.org/10.3390/ani
11010023
Received: 27 October 2020
Accepted: 21 December 2020
Published: 25 December 2020
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claimsin published maps and institutional

Abstract: The close link between nutrition management and reproductive efficiency is well known,
but there is very little data available concerning this topic in canine species. The present study
aimed to compare the effect of two different diets upon the follicular period and gestation in bitches.
Eighteen pluriparus medium and large size bitches were recruited and divided into control (CTR)
and experimental (EX) groups and fed, respectively, with a commercial kibble diet and a speciallyformulated diet from two months before the expected onset of proestrus up to the end of the trial.
It was possible to observe how the EX group had a better clinical presentation of the estrous phase,a higher number of ovarian follicles (p < 0.05), a lower percentage of fetal resorption (p < 0.05),
and lower oxidative status, expressed by d-ROMs (p < 0.01), at the moment of pregnancy diagnosis
compared to CTR group. Moreover, the EX group showed a lower fetal resorption rate and higher
litter size (p < 0.05). These results highlight how a diet characterized by high protein and fat content
and richer in essential fatty acids can improve reproductive performance in dogs.

affiliations.

Keywords: dog; estrous cycle; pregnancy; vaginal cytology; follicular development; fetal resorption

Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This
article is an open access article distributed
under the terms and conditions of theCreative Commons Attribution (CC BY)
license (https://creativecommons.org/
licenses/by/4.0/).

1. Introduction
Nutrition plays a crucial role in satisfying metabolic demands during pregnancy.
The maternal diet must provide sufficient energy and amounts of nutrients to satisfy themother’s requirements, as well as the needs of the growing fetus, and enable the mother tolay down stores of nutrients for fetal development and for lactation [1]. Proper nutrition

Animals 2021, 11, 23. https://dx.doi.org/10.3390/ani11010023

https://www.mdpi.com/journal/animals

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management for reproducing bitches should precede mating and continue throughout
gestation and lactation in order to improve maternal and fetal requirements and health.
Within the first two-thirds of the period of gestation, energy requirements should notincrease [2], while they rise to 1.25–1.50 times maintenance after the 35th–40th days [3].
From this last period up to parturition, due to the volume occupied by the uterus inside theabdomen and to the hormonal status, voluntary feed intake decreases, and several small
meals are recommended to be administered daily [2,3].
The use of a commercial diet, specially formulated for canine pregnancy, is linked to
the necessity to guarantee an efficient reproductive capacity in the bitch and optimal growth
rates in puppies [2]. Essential fatty acids, vitamins, and minerals (particularly calcium,
phosphorus, and magnesium) play an important role in ovarian hormone and uterine
protein production, placentation, and fetal development and, when correctly balancedwith other micro and macronutrients, may improve early embryonic survival, litter size,enhance lactation and neonatal survival [4,5].
Conversely, inadequate nutrition management, characterized by either deficiencies
or excesses, may have a detrimental effect in pregnancy even on healthy animals. Indeed,both conditions have been shown to exert negative effects on fertilization rate and numberof fetuses [4,6]. Moreover, a poor body condition score could also induce metabolic disturbances such as gestational ketosis, impaired endocrine equilibrium, impaired placentation,
increased neonatal death rates, and eclampsia, while obesity is known to contribute to thedevelopment of dystocia [6,7].
Despite the central role of nutrition in improving reproductive efficiency and avoiding maternal and fetal pathologies during pregnancy, very little data regarding canine
species are available and, consequently, many aspects related to the connection between
reproduction and nutrition are still little understood [8,9].
The aim of the present study was to compare two complete diets for bitches different
for ingredients and nutrients content. Each diet was administered to bitches of medium
and large size from two months before the expected onset of proestrus up to parturition.
The effects on estrus vaginal cytology, follicular development, and pregnancy progression
are reported in this paper.
2. Materials and Methods
All the procedures used in the study were approved (PG/2020/0044625) by the EthicalAnimal Care and Use Committee of the University of Naples Federico II in accordancewith local and national law regulations and guidelines (Dlgs 26, 4 March 2014).
2.1. Animals and Diets
For this study, 22 privately owned pluriparus bitches (mean: 1.5 previous pregnancies) at two months before the expected onset of proestrus were recruited from a private
veterinary clinic. The canine breeds of the dogs were represented: Bernese Mountaindog (2), French Bulldog (2), English Bulldog (3), Dobermann (1), Argentine Dogo (2),Pitbull (2), Great Danes (2), Golden Retriever (1), Hovawart (2), Labrador Retriever (2),German Shepherd (1) and Whippet (2). All dogs were judged healthy based on routine
clinical examination and hematological exams. According to the clinical history of eachsubject, the inter-estrus intervals of previous cycles were judged normal. All subjects wereof proven fertility with a history of previous normal pregnancies. Moreover, the studied
population was negative for Brucellosis and was vaccinated against Herpesvirus.
The dogs were equally divided into two groups (11 bitches) and each group was fed
one of two diets which differed as regards their ingredients (starch and protein sources),
energy, protein and folate levels. The two groups of bitches were given the names Control_CTR and Experimental_EX (Table 1).

Animals 2021, 11, 23

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Table 1. Diets characteristics on a metabolizable energy basis (Unit/MJ ME).
Diets Characteristics

Unit

CTR

EX

Crude protein
Fat
Crude fiber
AshCalcium
Phosphorus
Magnesium
Methionine
Cysteine
Linoleic acid
α-Linolenic acidArachidonic acid
EPA+DHA

g
g
g
g
g
g
g
g
g
g
g
mg
g

17.90
11.26
1.427
4.655
0.259
0.226
0.035
0.227
0.150
0.717
0.024
0.016
0.028

19.73
11.28
1.975
5.569
0.501
0.500
0.036
0.246
0.200
0.806
0.049
0.018
0.030

CTR: Control diet (ME 16.47 MJ/kg DM); EX: Experimental diet (ME 17.04 MJ/kg DM); EPA: eicosapentaenoic
acid; DHA: docosahexaenoic acid.

The ingredients of the two diets were:••

CTR: poultry meal, rice, corn, poultry fat, fishmeal, beet pulp, fish oil, seeds oil,
sodium chloride and dehydrated Saccharomyces cerevisiae.
EX: poultry meal, spelt, oat, potato, fishmeal, hydrolyzed fish protein, beet pulp dehydrated eggs, fish oil, flax seed, calcium carbonate, monocalcium phosphate, potassium
chloride, psyllium, MOS, sodium chloride and dehydrated Saccharomyces cerevisiae.

Both diets were supplemented with a mix of vitamins and microminerals in proportion
to their specific energy levels. In addition, both diets were supplemented with different
levels of folate (CTR 12.84 and EX 13.14 µg/MJ Metabolizable Energy ME).
During the trial, four owners decided to drop out of the protocol, and this study,
so we report only the data of the bitches that concluded the protocol. Consequently,
the experimental groups were distributed as follows:••

Control group (CTR): composed of 4 bitches of medium size and 5 of large size
(body weight 31.94 kg ± 13.08; BCS, 5 points scale: 3.30 ± 0.26);
Experimental group (EX): composed of 2 bitches of medium size and 7 of large size
(body weight 29.08 kg ± 14.35; BCS, 5 points scale: 3.63 ± 0.52).

Both the diets were produced by Farmina Pet-foods (Nola, Italy). The first was acommercial kibble diet formulated for adult dogs, whereas the second was specificallyformulated in order to perform the trial.
Each bitch group was fed one of the diets during all the trial, and the daily individual rations were calculated according to NRC [10] in function of body weight, stage ofpregnancy and body condition score. During the first two-thirds of pregnancy, the daily
ratio was divided into 2–3 meals, and in the last third of gestation, the ratio was left at thebitches’ disposal all day long.
2.2. Monitoring of Bitches until Pregnancy Diagnosis
The owners were instructed to measure some parameters of the bitches at home, suchas cardiac and respiratory rate and rectal temperature once a week starting from twomonths before the expected onset of proestrus until the weaning of the puppies.
From the first appearance of vulvar serosanguineous discharges, indicating the onset
of proestrus, the cycle was monitored at veterinary clinic by means of colpocitology,
serum progesterone and ultrasound of the ovaries every 3 days during proestrus andestrus in order to detect ovulation. After 48 h from ovulation, the bitches were matedtwice 24 h apart. Ultrasound examination was carried out every 3 days from the onset ofproestrus until the day of ovulation in order to evaluate the follicular development and,then, to confirm pregnancy on day 18 post-ovulation and, from this time, repeated twice aweek to evaluate embryonic development and viability until parturition.

Animals 2021, 11, 23

4 of 132.3. Vaginal Cytology and Plasma Progesterone Determinations
From the onset of proestrus, vaginal smears, obtained from the cranial portion of thevagina, were taken every 3 days until the day of ovulation from each bitch and promptly
examined using hematoxylin-eosin staining as described by Wright [11]. The evaluation of
the vaginal smears was performed using a light microscope (Nikon eclipse E200, Nikon Instrument Europe Amstelveen, Amsterdam, The Netherlands) at a magnification of 100×to 400× as described Antonov [12]. At the same time, serum progesterone concentrations
were determined by enzyme linked fluorescent assay (Minividas Biomerieux, Marcyl’Etoile,
France). According to Concannon [13], the ovulation time was identified by elevated serum
progesterone concentrations (i.e., >5 ng/mL) and a preponderance of superficial cells inthe vaginal cytology.
2.4. Ultrasound Scanning

Animals 2021, 11, x

All ultrasonographic examinations were performed on un-sedated bitches positioned
in right lateral recumbency using a GE Logiq E9 (GE Medical Systems, Milwaukee, WI,USA) ultrasound machine equipped with 9 L linear transducer (5–9 MHz) coupled via
acoustic gel which was applied directly to the shaved abdominal wall.
From the onset of proestrus, the ultrasound was performed every 3 days until the dayof ovulation in order to evaluate the ovaries. More particularly, the gonads were detectedbehind the caudal pole of the ipsilateral kidney and the number and size of the follicles
present were recorded.
The time of ovulation was determined when, compared to the previous ultrasoundscanning, a clear transformation of the image of the ovaries was recorded and at least someof the follicles present previously had collapsed, as described by Fontbonne [14].
Eighteen days after ovulation, ultrasonographic scans were performed and repeated
twice a week throughout the pregnancy until parturition. At each examination, the following parameters were considered in order to evaluate embryo/fetal viability and development: inner chorionic cavity diameter, crown-rump length, body diameter, biparietal diam5 of 13
eter, fetal heart rate and the appearance of an embryo/fetal organs to the gestational phase(Figure 1).

Figure 1. Representative ultrasonographic scans of biometry measurements recorded during pregFigure 1. Representative ultrasonographic scans of biometry measurements recorded during pregnancy in an Argentine Dogo. (A): inner chorionic cavity diameter at 23 days of pregnancy. (B):nancy in an Argentine Dogo. (A): inner chorionic cavity diameter at 23 days of pregnancy. (B): crowncrown-rump length (distance from the top of the head to the base of the tail) at 40 days of gestation. (C): body diameter in the transverse plane at the level of fetal liver at 53 days of gestation.
(D): biparietal diameter (distance between parietal bones) at 57 days of pregnancy.

2.5. Weight Control and Oxidative Status
The bitches’ body weights and body condition scores (BCS, scale 5 points) were rec-

Animals 2021, 11, 23

5 of 13rump length (distance from the top of the head to the base of the tail) at 40 days of gestation. (C):body diameter in the transverse plane at the level of fetal liver at 53 days of gestation. (D): biparietal
diameter (distance between parietal bones) at 57 days of pregnancy.

2.5. Weight Control and Oxidative Status
The bitches’ body weights and body condition scores (BCS, scale 5 points) wererecorded at the time of recruitment and then weekly. At the moment of recruitment,
the bitches were subjected to blood sampling in order to perform biochemical analysis
by means of an automatic biochemical analyzer AMS AUTOLAB (Rome, Italy) using
reagents from Spinreact (Santa Colomna, Spain) to determine: blood urea nitrogen (BUN),
creatinine (CREA), glucose (GLU) total proteins (TP), albumin (ALB), bilirubin (BIL), aspartate amino transferase (AST), alanineaminotransferase (ALT), gamma-glutamyltransferase(GGT), cholesterol (CHO) and triglycerides (TRI), chloride (Cl) sodium (Na), calcium (Ca)and phosphorus (P) (data reported as Supplementary Material as Tables S1 and S2).
Moreover, blood samples were collected at the moment of pregnancy diagnosis inorder to evaluate the reactive oxygen metabolites (d-ROMs test) and biological antioxidant
potential (BAP test) on serum aliquots which were measured using reagents from DiacronInternational s.r.l. (Grosseto, Italy) validated for canine species.
2.6. Statistical Analysis
The data was statically analyzed by means of one-way ANOVA according to thefollowing model:
yijk = µ +Ti +Sj + (T × S)ij + εijk
where y is the dependent variable, µ is the mean, T is the treatment effect (i: CTR, EX), S isthe dog size effect, j: medium (body weight < 30 kg) and large (body weight > 30 kg), T × S
is the first level of interaction and ε is the error effect. When significant differences were
found in the ANOVA, means were compared using Tukey’s test.
Fetal resorption and litter size data were analyzed using the Wilcoxon non-parametrictest.
All the statistical analyses were performed using JMP 14 software, SAS Institute, Cary,
NC, USA.
3. Results
Monitoring of Bitches until Pregnancy Diagnosis
Nutritional treatment affected only a few parameters of ovarian ultrasound and vaginal cytology (Tables 2 and 3). During anestrus and ovulation, the long ovarian axis was
significantly (p < 0.05) higher in the EX group compared to CTR one. The same trend(p < 0.05) was observed for follicles per ovary during estrus. During estrus, the experimental group showed significantly (p < 0.05) higher values of progesterone and a higher
percentage of superficial cells.
The size of the bitches significantly (p < 0.05) affected only a few parameters. Particularly, bitches of medium size showed higher values of short ovarian axis and progesterone
level during proestrus. During estrus, bitches of large size showed a significantly (p < 0.05)
higher percentage of superficial cells than medium size.

Animals 2021, 11, 23

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Table 2. Ultrasound data.
Group

Anaestrus

Proestrus

LA

SA

LA

SA

cm

cm

cm

cm

EX
CTR

0.96 a0.87 b

0.64
0.62

1.77
1.67

1.06
1.12

M
L

0.89
0.94

0.60
0.66

1.72
1.72

Int
MSE

**
0.007

**
0.009

NS
0.028

Estrusf/o

GR fol

LA

SA

cm

cm

cm

4.27
3.99

Treatment
0.46
0.40

2.17
2.20

1.15
1.24

1.19 a0.99 b

3.82
4.45

Size
0.42
0.43

2.14
2.23

NS
0.027

NS
0.618

NS
0.021

NS
0.102

Ovulationf/o

GR fol

LA

SA

cm

cm

cm

3.84
3.44

0.79 a0.69 b

1.48 a1.21 b

0.90
0.99

1.19
1.20

3.55
3.73

0.73
0.74

1.33
1.36

0.90
0.98

NS
0.019

NS
0.196

NS
0.010

NS
0.040

NS
0.002

EX: experimental diet; CTR: control diet; M: medium; L: large; Int: interaction (treatment*size); MSE: mean square error; LA: long axis; SA:
short axis; f/o Follicle/ovary; GR Fol: great follicle. NS: not significantly, **: p < 0.01; along the column, different lowercase letters indicate
difference for p < 0.05.

Table 3. Monitoring of bitches until pregnancy diagnosis: vaginal cytology and serum progesterone.
Group

Proestrus

EstrusOvulation

Progesterone

Sup cellProgesterone

Sup cellProgesterone

Ng/mL

%

Ng/mL

%

Ng/mL

EX
CTR

0.83
0.78

33.52
35.84

2.89 a2.31 b76.83 a67.00 b

6.70
6.78

M
L

0.92 a0.69 b

34.99
34.37

2.57
2.62

69.39 b74.50 a

7.08
6.40

Int
MSE

*
0.028

NS
7.860

NS
0.243

NS
8.475

NS
1.850

TreatmentSize

EX: experimental diet; CTR: control diet; M: medium; L: large; Int: interaction (treatment*size); MSE: mean squareerror; Sup cell: superficial cells. NS: not significantly, * p < 0.05; along the column, different lowercase letters
indicate difference for p < 0.05.

Figures 2–5 showed the rate of embryonal growth (e.g., vesicle, biparietal and body
diameters and crown-rump length). Each parameter showed a different tendency infunction of nutritional treatment and the bitches’ size:

•

•

•

•

Inner chorionic cavity diameter—EX group showed higher values than CTR one and,in particular, on the 23rd day the difference was significant (1.30 vs. 0.80 cm; p < 0.05).
Regarding size, the larger bitches showed higher values with a significant difference
on the 23rd day (1.27 vs. 0.83 cm; p < 0.05) compared to bitches of medium size.
Biparietal diameter—both effects (diet and size) did not affect this parameter,
except for the 37th day when the diameter value of CTR group was significantly
higher (1.36 vs. 1.13 cm, p < 0.05) than EX group one.
The body diameter of the control group showed higher values than the experimentalgroup even if the difference was significant only on the 41st day (1.91 vs. 1.61 cm,
p < 0.05). Concerning size, until the 46th day, large bitches showed a higher diametercompared to bitches of medium size, while from the 46th day the values became
similar.
Crown-rump length—the control group showed a greater length during the first
50 days of gestation (50th: 10.74 vs. 9.75 cm, p < 0.05), afterward the values became
overlapped. The embryos of large size bitches were always longer than those of
medium-sized bitches.

•••Animals 2021, 11, 23<<<0.05).
0.05).
Concerning
size,
until
the
46th
day,
large
bitches
showedhigherdiameter0.05).Concerning
Concerningsize,
size,until
untilthethe46th
46thday,
day,large
largebitches
bitchesshowed
showedaaahigher
higherdiameter
diametercomparedtobitchesofmedium
size,
while
from
the
46th
day
the
valuesbecamecomparedtobitchesofmedium
size,
while
from
the
46th
day
the
valuesbecamesimcompared to bitches of medium size, while from the 46th day the values becamesimsimilar.
ilar.
ilar.
Crown-rump
Crown-rumplength—thecontrol
groupshowedgreaterlengthduringthefirst
50
Crown-rumplength—thelength—thecontrol
controlgroup
groupshowed
showedaaagreater
greaterlength
lengthduring
duringthe
thefirst
first50
50
daysofgestation(50th:
10.74
vs.
9.75
cm,
p
<
0.05),
afterward
thevaluesbecamedays
overdaysof
ofgestation
gestation(50th:(50th:10.74
10.74vs.
vs.9.75
9.75cm,cm,pp<<0.05),
0.05),afterward
afterwardthe
thevalues
valuesbecame
becameoverover7 of 13
lapped.
lapped.
Theembryos
of
large
size
bitcheswerealwayslonger
than
those
of
mediumlapped.The
Theembryos
embryosof
oflargelargesize
sizebitches
bitcheswere
werealways
alwayslonger
longerthan
thanthose
thoseofofmediummediumsizedbitches.
sizedbitches.
sized bitches.

FigureInnerchorionic
cavityduringgestation
in
of
and
size.
EX:
Figure2.
Innerchorionic
cavityduringgestationinfunctionof dietssize.
EX:experimental
Figure2.2.2.
Inner
chorionic
cavityduringgestationinfunction
of
diets
and
size.
EX:
experimental
FigureInnerchorionic
cavityduringgestation
infunction
function
ofdiets
diets
andandsize.
EX:experimental
experimental
group
CTR:
controlgroup;
a,
b:
p
<
0.05.
groupCTR:
CTR:control
controlgroup;group;a,a,a,b:b:
b:ppp<<<0.05.
0.05.
group
group
CTR:
controlgroup;
0.05.

Figure3. Biparietal
diameterduringgestation
ininfunction
ofofdiets
andexperimental
Figuresize.
EX:FigureBiparietal
diameterduringgestationfunctiondiets
andsize.
size.EX:EX:
experimental
Figure3.3.
3.Biparietal
Biparietaldiameter
diameterduring
duringgestation
gestationin
infunction
functionof
ofdiets
dietsand
EX:experimental
experimental group
group
CTR:
controlgroup;
a,
b:
p
<
0.05.
group
group
CTR:control
controlgroup;
b:pp<<0.05.
0.05.
CTR: CTR:
controlgroup;group;a, b: pa,a,
< b:
0.05.

Animals 2021, 11, x

8 of 13

FigureFigure4.
4.BodyBodydiameter
diameterduring
duringgestation
gestationin
infunction
function
of
diets
and
size.
EX:
experimental
group
Figure
4.4.
Body
diameterduringgestationinfunction
of
diets
and
size.
EX:
experimental
FigureBody
diameterduringgestationinfunctionof
ofdiets
dietsandandsize.
size.EX:
EX:experimental
experimentalgroup
groupCTR:
CTR:
controlgroup;
a,
b:
p
<
0.05.
controlgroup;a, b: a,pa,<b:
CTR:
controlgroup;
pp<<0.05.
CTR:
controlgroup;
b:0.05.
0.05.

FigureFigure5.5.Crown-rump
Crown-rumplength
lengthduring
duringgestation
gestationin
infunction
functionof
ofdiets
diets andand size.
size. EX:
EX: experimental
experimental group
group
controlgroup;
p < 0.05.
CTR: CTR:
controlgroup;a, b: pa,<b:0.05.

Alldogs
dogs showed pregnancy
± 2.0
days)
thatthatfallsfalls
in averagevaluesvalues[11,13]All
pregnancyduration
duration(63(63
± 2.0
days)
in averagewith theexceptionof theoffive
subjects
for whoma cesarean
sectionwas[11,13]withthe exception
thebrachycephalicfive brachycephalicsubjects
for whoma cesarean
sectionplanned,alltheparturitions
were
natural.
The
fetalresorption(Figure6)
recordedby
was planned, all the parturitions were natural. The fetal(Figure 6) recorded byultrasoundcheckswassignificantly
(p
<
0.05)
different
for
EX
and
CTR
group
(7.05%
andultrasound checks was significantly (p < 0.05) different for EX and CTR group (7.05% and11.94%,respectively),respectively),andand
thenumberpuppieslitter(Figure7) wassignificantly
11.94%,
thenumberof ofpuppies
perperlitter(Figure7) wassignificantly(p< 0.05)higher
group
than
CTR
one
(9.11
3.89,
respectively).No
Nomalformations
malformations
< (p
0.05)
higherfor for
EX EX
group
than
CTR
one
(9.11
vs.vs.
3.89,
respectively).
wereobserved
observedininnewborns.
newborns.
wereAnimals 2021, 11, 23

All
All dogs
dogs showed
showed pregnancy
pregnancy duration
duration (63(63 ±± 2.0
2.0 days)days) thatthat falls
falls inin average
average values
values[11,13][11,13] with
with thethe exception
exception ofof thethe five
five brachycephalicbrachycephalic subjects
subjects forfor whomwhom aa cesareancesarean section
sectionwas
was planned,
planned, all
all thethe parturitions
parturitions were
were natural.
natural. The
The fetal
fetal resorption
resorption (Figure(Figure 6)6) recorded
recorded bybyultrasoundcheckswassignificantly
(p
<
0.05)
different
for
EX
and
CTR
group
(7.05%
andultrasound checks was significantly (p < 0.05) different for EX and CTR group (7.05% and11.94%,(p
11.94%, respectively),respectively), andand thethe number
number ofof puppies
puppies per
per litter
litter (Figure(Figure 7)7) waswas significantly
significantly(p
8 of 13<< 0.05)higher
for
EX
group
than
CTR
one
(9.11
vs.
3.89,
respectively).
Nomalformations
0.05) higher for EX group than CTR one (9.11 vs. 3.89, respectively). No malformations
were
were observed
observed inin newborns.
newborns.

Figure 6.
6. Percentage
Percentageof
offetalfetalresorption
resorptionrecorded
recordedby
ultrasoundchecksbetweentwo
groups.
Figurechecksbetweentwo
groups.
EX:
Figure6.
Percentage
of
fetal
resorption
recordedbybyultrasoundultrasoundchecksbetweentwo
groups.
EX:EX:
experimental;
experimental; CTR:
CTR: control;
0.05 (Wilcoxon(Wilcoxon non-parametricnon-parametric test).
test).
experimental;
CTR:
control; a,a, b:b: ppp <<< 0.05
0.05
(Wilcoxon
non-parametrictest).

Figureexperimental;
CTR:
control;
Figure 7.
7. Number
Numberof
ofpuppies
puppiesper
perlitterlitterrecorded
recordedinto
intothethegroups.
groups.EX:EX:
experimental;
CTR:
control;
Numberof
puppiesperlitterrecordedintothegroups.
EX:
experimental;
CTR:
control;
a,
b:
p
<
0.05
(Wilcoxon
non-parametrictest).
a, b: p << 0.05
0.05 (Wilcoxon(Wilcoxon non-parametric test).
Animals 2021, 11, x

9 of 13
Clinical
rate,
heart
rate
and
rectaltemperature
wasClinical datadatacollectedcollectedon
onrespiratory
respiratory
rate,
heart
rectaltemperaturewasdatacollected
onrespiratory
rate,
heart
raterate
andandrectaltemperature
was nornormal
inallsubjects
fortheentirestudy
period.
Nodifferences
betweengroupswere
foundnormalinallsubjects
fortheentirestudy
period.
Nodifferences
betweengroupswere
mal in all subjects for the entire study period. No differences between groups were foundforbitches’body(Figure8)(32.56
vs.
kg
and
EX
found
for bitches’
body weight(Figure8) parturition
afterparturition(32.56
vs. 39.73
kgCTR
for CTRforbitches’body weightweight(Figure8) afterafter
parturition(32.56
vs. 39.73
39.73
kg forfor
CTR
andandEX
EX group,
respectively)even
if theBCS(Figuretheendendofofpregnancy
pregnancywas
was significantly
significantly
group,
respectively)even
if theBCS(Figure9)9)atatthe
0.001) forfor EX
EX groupgroup (3.42/5)(3.42/5)than
thanforforCTR
CTRoneone(2.75/5).
(2.75/5).
higher (p << 0.001)

Figure 8. Bitches
Bitches body
body live
live weightweight at
at thethe beginning
beginning and
andat
atthe
theendendof
ofpregnancy.
pregnancy. EX:
EX: experimental;
experimental;
CTR:
CTR: control;
control; LW
LWii:: initial
initial body
body weight;
weight; LW
LWff:: body
body weightweight after
after delivery.
delivery.

Animals 2021, 11, 23

9 of 13

Figure 8. Bitches body live weight at the beginning and at the end of pregnancy. EX: experimental;
CTR: control; LWi: initial body weight; LWf: body weight after delivery.

Figure9. Bitches
body condition
conditionscore
scoreatatthethebeginning
beginningandendof pregnancy.
experiFigure 9.
Bitches body
and
at at
thetheendof pregnancy.
EX:EX:
experimental;
i: initial Body condition score; BCSf: body condition score after delivery.
mental;CTR:
control;
BCS
CTR: control; BCS : initial Body condition score; BCS : body condition score after delivery.
if

The oxidative statuses of the bitches, which were registered at pregnancy diagnosis,
were affectedaffectedboth
bothbybynutritional
nutritionaltreatment
treatment
andsize(Figurein particular,
d-ROMs
were
and
size
(Figure10):10):in particular,
d-ROMsvalvalues
werehigher
< 0.01)in CTRgroup
thanin theandmedium-sizedbitchesues werehigher(p <(p0.01)
in CTRgroup
thanin theEXEX
oneoneandin inmedium-sizedbitchesasascomparedto largeones< 0.05).
In anycase,significant
differenceswereobserved
comparedto largeones(p <(p0.05).
In anycase,no nosignificant
differenceswereobserved
for
BAPBAPvalues,even
if thetrend
waswasoppositeto thatobserved
for d-ROMs.
forvalues,even
if thetrend
oppositeto thatobserved
for d-ROMs.

Figure 10.
10. Oxidative
Oxidative status
Figurestatus evaluatedevaluated at
at thethe moment
momentof
ofpregnancy
pregnancydiagnosis
diagnosisininfunction
functionofofdiets
dietsandand size.
EX: experimental
group
controlgroup;
d-ROMS:
Reactive
OxygenMetabolitessize.
EX: experimental
group
CTR:CTR:
controlgroup;
d-ROMS:
Reactive
OxygenMetabolites-derived
derived compounds;
BAP: Biological
Antioxidanta, b: pA,< B:
0.05;
B: p < 0.01.
compounds;
BAP: Biological
Antioxidant
Potential.Potential.
a, b: p < 0.05;
p <A,
0.01.

4.
Discussion
4. Discussion
Poor
Poor nutrition,
nutrition, either
either deficiencies
deficiencies oror excesses,
excesses, adversely
adversely affects
affects pregnancy
pregnancy even
even inin
healthyanimals.
Excellent
nutritioncanimproveearlyembryonic
survival,
improve
healthy animals. Excellent nutrition can improve early embryonic survival, improve litter
litter size and birth weight, and neonatal survival [6,7]. Macro and micronutrients havevarious effects on ovarian hormone production, uterine protein production, placentationand fetal development. To take full advantage of the potential benefits of good nutrition,a diet appropriate for pregnancy and lactation should be fed from the time of breeding
onward [15].
The Association of American Feed Control Officials [16] and the European Pet Food
Industry Association (FEDIAF) [17] have established minimal requirements for the constituents of diets formulated for reproduction. This ensures adequacy, but not necessarily
optimal nutrition. The quality and chemical form, as well as the quantity, of the constituents, are important. Therefore, the results of feeding trials are more important than thestated “diet formulation”.
Overall, both the experimental and control group of bitches enrolled in the follicularphase progressed normally. The weight and body condition scores registered during
pregnancy for all bitches fall into the range indicated as optimal for the species, with aweight gain of 15–25% of the initial body weight before whelping [2,4,5].
From a clinical point of view, the greater number of superficial cells and the higher
value of progesterone found in the EX group during estrous make this phase more typical and cytologically easy to recognize as is referred to in the literature [18]. Moreover,Animals 2021, 11, 23

10 of 13the higher number of preovulatory follicles found in the bitches fed with EX diet suggests
a potential increase in fertility linked to the higher allowances of macro and micronutrients.
The trend was confirmed by the higher number of puppies per litter registered in EX groupcompared to CTR one.
Resorption is defined as early embryonic or fetal death within the first 45 days ofpregnancy [15]. When resorption occurs in a bitch, there is absence of external signs.
There are no contractions, and the fetus is not being expelled out, but the fetus is dissolved
into the uterus without externally visible signs. As reported by Sharma et al. [19] the sizeof the breed was found to have a significant effect on the incidence of fetal resorption
in bitches, the incidence being significantly higher in medium and large sized breeds.
However, in our study, no differences between the bitches’ size were detected for litter
size. Gaytàn et al. [20] reported that the bitches found a significant effect (p < 0.01) of% vaginal cornified epithelium at the moment of artificial insemination and litter size:
the bitches with >75% of vaginal cornified epithelium showed greater (p < 0.01) whelping
rate (85.1% vs. 51.6%, respectively). Considering that in our study we found significant
differences (p < 0.05) for the number of vaginal superficial cells comparing the groups(76.8% vs. 67.0% for EX and CTR group, respectively) and for litter size (9.11 and 3.89,
for EX and CTR group, respectively), it is possible that the availability of the different
nutrients could affect both parameters.
In any event, both the fetal resorption and number of puppies per litter registered inboth groups were in the physiological ranges for the species, indicating an adequate intake
of nutrients with both diets [21].
The progesterone profile through the follicular period was normal, and the difference
within groups, found at the beginning of estrus, can be linked to the broad variability
in the rise of this hormone [17]. In contrast to Hollinshead and Hanlon [22], we found asignificant (p < 0.05) difference of progesterone levels in proestrus between medium andlarge-sized bitches.
The better reproductive outcome registered in EX group could be related to the higher
supplementation of essential fatty acids of EX diet. Indeed, their deficiencies are responsible
for host symptoms and disorders, including reproductive disorders, newborns abnormalities in kidney and liver; for this reason, essential fatty acids must be present in pregnant
dog diet to ensure that hormones production and egg cells development. Indeed, increased
essential fatty acids content led to an increase in Arachidonic acid in phospholipids ofovarian follicular granulosa cells stimulating prostaglandin production in ovarian follicular
cells and prostaglandins, in turn, are known to stimulate ovarian steroidogenesis [23].
In dog Linoleic (LA, C18:2 n6) and α-Linolenic (ALA, C18:3 n3) are considered essential
fatty acids (EFA) because they cannot be synthesized ex novo in the body end consequently
the dogs have to assume LA and ALA with the diet. These EFA are considered themain precursors of n6 and n3 fatty acids, such as EPA and DHA. Moreover, as suggested
by Bauer [24], there is a competition between LA and ALA metabolism. In particular,
these authors compared the effect on coats and skin of two iso-energy and iso-fat diets
formulated with different fat sources: sunflower seed (rich in omega-6 linoleic acid, LA)
vs. whole ground flaxseed (rich in omega 3, ALA). They observed a significantly higher
dermatological improvement using flaxseeds and postulated a sparing effect of ALA on
LA. Considering the similar fat content of the diets administered to EX and CTR group in
this study, it seems possible that the better performance obtained by EX group could be dueto this effect. Indeed, CTR diet was characterized by a higher LA:ALA ratio (29.87 vs. 16.44
in CTR and EX groups, respectively). Moreover, the NRC [10] recommends an LA:ALA
ratio of between 2.6 and 16.0.
In the first third of gestation, the diameter of the embryo vesicles was greater in the
EX group, suggesting a faster growth rate at the beginning of gestation related to theexperimental diet. These results could be due to the higher protein content of EX dietcompared to CTR one (17.90 and 19.73 g/MJ ME) [24].

Animals 2021, 11, 23

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During pregnancy, different percentages of fetal resorption were recorded between
groups (11.94% vs. 7.5% for CTR and EX group, respectively). The percentages of fetal
death recorded in this trial are closer to the lower percentages commonly reported in theliterature and ranged from 10 to 15% [25,26]. Our study population gives rise to someconsiderations. In fact, among the causes of embryo/fetal resorption, the following must be
considered: infectious diseases, physical trauma, hormonal dysfunction, fetal abnormalities,
or genetic and chromosomal alterations [14,25]. However, many of these causes can be
excluded in our case considering the history and the method of recruitment used for the
study. In this context, taking into account that during pregnancy there is a high demandfor energy, protein and vitamins which may affect the survival of the embryos if notavailable [27], the low percentage of fetal resorption of the experimental group should be
related to the specific diet, even if it is difficult to define which nutrients (e.g., crude protein,
LA:ALA, folate) play a more decisive role. Indeed, nutritional factors were considered a
non-infectious potential cause of fetal resorption [19]. Because dietary ingredients vary inprotein content (range, 27–34% as fed) and amino acid profile, diets with 29–32% animalbased protein sources are recommended for pregnant bitches [28]. Similarly, both theoverall fat content and the fatty acid profile affect pregnancy rates, litter size and thenumber of newborns. Conversely, obesity is known to contribute to the development ofdystocia and increased neonatal mortality [15]. Despite the calcium and phosphorous
content of the experimental diet was double compared to CTR diet, we did not observe
significant differences in terms of serum calcium and phosphorous levels between groups.
These are probably ascribable to the superimposable Ca/P ratio of the diets and/or tothe high ability of the bitches to regulate the deposition and mobilization of calcium formilk production [29]. Considering that our observations were limited to blood profile
analysis, we are not able to exclude long-term bone demineralization phenomena due tothe dramatically elevated calcium requirements at the initiation of lactation.
The oxidative status of the bitches measured at the moment of pregnancy diagnosis
testifies a good antioxidant action of reactive species and/or a decrease of the efficiency ofantioxidant system [30]. Indeed, the d-ROMs test provides a measure of the whole oxidant
capacity of plasma and its decrease is considered an indicator of cellular health. Even if thed-ROMs levels observed in both groups fall into the normal range of values for the canine
species proposed by Sechi et al. [30] and Pasquini et al. [31], EX group showed significantly
lower values compared to CTR ones, indicating a general decrease of reactive oxygen
species (ROS) production. This result could be related to the dietary factors involved in
the prevention of the negative effects of oxidative stress, such as feed manufacturing andstorage conditions, use of antioxidant supplements [32]. Indeed, both diets used in thistrial were supplemented with tocopherols as antioxidants; the ether extract concentrationof the diets was similar although the fatty acid profile was different. The EX diet showed
higher concentrations of Linoleic, α-Linolenic acids and Eicosapentaenoic acid (EPA) plus
Docosahexaenoic acid (DHA). This could be due to the presence in EX diet of flaxseeds
which were rich in Linoleic, α-Linolenic acids and in γ-tocopherol, which preserves cells
from oxidation [33].
5. Conclusions
It is possible to highlight that the administration of the experimental diet allowed an
improvement of the clinical presentation of estrus, an increase in the number of ovarianfollicles, a faster rate of early embryonic growth, a lower percentage of fetal resorption
and a higher litter size. Despite the non-homogeneous distribution between the groups ofbitches of large and medium size, which represents the limit of this trial, our results confirm
how a diet characterized by a different distribution of protein, fat and carbohydrates and adiet richer in essential fatty acids can improve dog’s reproductive performances.
Further studies are needed to better identify the interaction between the different
components of the diet and a peculiar metabolic phase such as canine pregnancy.

Animals 2021, 11, 23

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Supplementary Materials: The following are available online at https://www.mdpi.com/2076-261
5/11/1/23/s1, Table S1: Bitches’ serum biochemical parameters (m ± sd) at recruitment, Table S2:
Bitches mineral blood profile (m ± sd) at recruitment.
Author Contributions: Conceptualization R.O. and M.I.C.; Formal analysis R.O., E.V., A.P. and A.T.;
Methodology S.C., P.L., N.M., A.V. and R.O.; Data curation A.V.; Supervision R.O., P.L. and M.I.C.;
Writing—original draft R.O., E.V., M.I.C., N.M., P.L. and A.V.; Writing—review and editing N.M. and
A.V. All authors have read and agreed to the published version of the manuscript.
Funding: This trial was partially supported by Farmina Pet Food (Nola, Italia) and by DMVPA
founding.
Institutional Review Board Statement: All the procedures used in the study were approved(PG/2020/0044625) by the Ethical Animal Care and Use Committee of the University of Naples Federico II in accordance with local and national law regulations and guidelines (Dlgs 26, 4 March 2014).
Data Availability Statement: The data presented in this study are available on request from thecorresponding author.
Acknowledgments: The authors would like to thank Farmina Pet Food (Nola, Italia) for the technicalsupport in formulating and for providing the used diets.
Conflicts of Interest: The authors declare no conflict of interest.

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