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ORIGINALRESEARCH
published: 12 July 2022
doi: 10.3389/fvets.2022.908763
Influence of Fat-Soluble Vitamin
Intramuscular Supplementation on
Kinematic and Morphometric Sperm
Parameters of Boar Ejaculates
Josué Calderón-Calderón1, Francisco Sevilla1, Eduardo R. S. Roldan2, Vinicio Barquero1,3
1
andAnthonyValverde *
1 Animal Reproduction Laboratory, School of Agronomy, Costa Rica Institute of Technology, Alajuela, Costa Rica,
2 Department of Biodiversity and Evolutionary Biology, National Museum of Natural Sciences, Spanish National Research
Council (CSIC), Madrid, Spain, 3Faculty of Agri-Food Sciences, Alfredo Volio Mata Experimental Station, University of Costa
Rica, Cartago, Costa Rica
Ejaculate quality can be regarded as multifactorial, with nutrition being a factor that
could directly influence sperm parameters. The present study aimed to evaluate
seminal quality associated with seasonal fat-soluble vitamin supplementation of boars.
Seven sexually mature boars were randomly allotted to one of the three groups,
and fed one of the three supplementary diets for 32 weeks: (1) control treatment
(COD), without supplementation of fat-soluble vitamins, (2) treatment containing 100%
Edited by: fat-soluble vitamin supplementation administered intramuscularly, which was based on
Elisabeth Pinart, fat soluble vitamin supplementation (A, D3, E) (FVD1), and (3) treatment containing
University of Girona, Spain
Reviewedby: 50% of fat-soluble vitamin supplementation (FVD½). Semen was collected at 7-day
Francesca Mercati, intervals. Semen samples were analyzed to assess several sperm parameters using
University of Perugia, Italy ®
Luis J. Garcia-Marin, the Computer-Assisted Semen Analysis (CASA) ISAS v1 system. Results showed that
Universidad de Extremadura, Spain groupsreceivingFVD1andFVD½supplementationhadanincreasedsemenvolume.The
*Correspondence: percentagesofmotileandprogressivelymotilespermwereincreasedbyFVD1treatment.
Anthony Valverde A statistically significant interaction between treatment and season was found in the
anvalverde@tec.ac.cr percentageofmotilityandprogressivemotility(p<0.05).Spermconcentrationsshowed
Specialty section: significant differences (p < 0.05) between treatments. Velocity variables (VSL, VCL, and
This article was submitted to VAP)werehigher(p<0.05)inboarsthatreceivedfat-solublevitaminsupplementationin
Animal Reproduction - comparison to controls receiving no supplementation. The FVD1 treatment presented
Theriogenology,
a section of the journal spermatozoa with greater head size and more elongated heads (p < 0.05). Overall,
Frontiers in Veterinary Science the utilization of dietary fat-soluble vitamin supplementation significantly improved the
Received: 31 March 2022 semen quality of boar ejaculates. This highlights the importance of fat-soluble vitamin
Accepted: 20 June 2022
Published: 12 July 2022 supplementation in sexually active boars.
Citation: Keywords: spermatozoa, nutrition, fat-soluble vitamin, CASA, motility
Calderón-Calderón J, Sevilla F,
Roldan ERS, Barquero V and
Valverde A (2022) Influence of INTRODUCTION
Fat-Soluble Vitamin Intramuscular
Supplementation on Kinematic and Swine artificial insemination (AI) is performed using semen preserved in extender (1). Semen
Morphometric Sperm Parameters of samples to be employed are subjected to several quality tests in order to maximize the production
Boar Ejaculates.
Front. Vet. Sci. 9:908763. of doses for AI. Subjective semen evaluations have been replaced with objective analysis in effort
doi: 10.3389/fvets.2022.908763 to improve the assessment of fertilizing potential (2). Objective assessments provide the precision
Frontiers in Veterinary Science | www.frontiersin.org 1 July 2022 | Volume 9 | Article 908763
Calderón-Calderón et al. Fat-Soluble Vitamin Supplementation in Boars
and accuracy required to obtain reliability in the estimation of vitamins, such as vitamin E, impacts directly ATP concentration
quality variables (3) and would contribute to reducing technician (25), sperm production, and quality of ejaculates through
mistakes (4). To this end, computer-assisted semen analysis the swimming patterns and morphometric characteristics (26).
(CASA)representsavaluableresource(5). Because of the important role of vitamins, the present study
CASA technology has different modules for analyses, such was undertaken to examine the effect of season dietary
as CASA-Mot (motility and kinematics) and CASA-Morph supplementationoffat-solublevitaminsonsemenquality,paying
(morphometry) (6). Using CASA-Mot, spermatozoa can be particular attention to sperm morphology and kinematics.
classified according to their velocity as rapid, medium, slow,
and static and, moreover allow for a detailed analysis of MATERIALSANDMETHODS
kinematic variables (7). CASA-Morph assesses morphology by
using individual dimensions (length, width, area, perimeter) and Theuseandcareofanimalsinexperimentaltreatmentscomplied
shape (ellipticity, rugosity, elongation, and regularity) of the with the Costa Rica Institute of Technology animal welfare
spermhead(8). guidelines. Ethical approval has been given by the Committee of
Seminal production depends on multiple factors, such as Centro de Investigación y Desarrollo en Agricultura Sostenible
geneticimprovement,reproductiontechniques,health,nutrition, para el Trópico Húmedo at the Costa Rica Institute of
andhandling(9).Inboars,nutrition is of the utmost importance Technology (CIDASTH-ITCR) according to Section 08/2020,
because an inadequate balance in the diet affects the libido and article 1.0, DAGSC-100-2020.
sperm quality (10). For this reason, supplementation must take
into account season (11), age of sire (10), and weight and activity Animals and Location
of sire (12) in order to supply the necessary mineral, vitamin, The experiment was conducted at Agropecuaria Los Sagitarios
and protein requirements (13). Restrictions or deficiencies in S.A. commercial pig farm (Alajuela, Costa Rica) during 2020 in
some nutrients (14) entail a nutritional imbalance that could theNorthwestofCostaRica(RíoCuarto,10◦20′32′′ N,84◦12′55′′
influence the libido and the seminal quality of the ejaculate of W, Alajuela, Costa Rica). In this area, the height of the dry
the reproductive boar (10). season is from November to April and the rainy season is from
Reactiveoxygenspecies(ROS)arebelievedtobeimportantfor MaytoOctober.Sevensexuallymatureboarsfromacommercial
the normal sperm function, including processes underlying cell terminal sire line (SL: Duroc × Pietrain) at 32.2 ± 9.8 months
viability and preparation for fertilization (such as capacitation, of age at the beginning of the experiment were used as semen
hyperactivation, and the acrosome reaction) (15). However, donors in this study. Breeding boars were housed individually in
sperm are susceptible to peroxidative damage due to an well-ventilated pens with an average temperature of 25.8 ± 2.7◦C
imbalance between ROS production and the capacity of duringthetimeoftheexperiment.Datacollectionwasperformed
antioxidant systems (15). The increase in ROS causes damage to for 32 weeks, from January 4 to August 24th 2020, with the
the mitochondria; therefore, sperm with defective mitochondria first 2 weeks before initiating the trial allowing for adaptation to
would produce ATP inefficiently (16). In addition, an excess the diets.
of ROS can also generate errors during sperm production
(spermatogenesis) leading to a premature release of sperm from Diets
the germinal epithelium (17). Antioxidants can be classified The animals were fed with a standard breeder mixture,
into enzymatic and non-enzymatic (18). Enzymes, which are containing maize, soybean meal, mineral mixture, and common
responsible for protecting sperm in the epididymis, include salt, as ingredients to fulfill the nutrient requirements (27). Diets
glutathione peroxidase (GPx), phospholipid hydroperoxide were mixed completely, and males were fed as a total mixed
glutathione peroxidase (PHGPx), superoxide dismutase (SOD), ration 2 times daily at 0,700 and 1,300h; they consumed 2.5kg
glutathione reductase (GR) and catalase (CAT) (19). The main per day, and were provided with water ad libitum (Table 1).
non-enzymaticantioxidants are vitamins A, C, and E (19).
Vitamin A is known to be necessary for the normal process Treatments
of spermatogenesis (20), with retinoic acid being an alternative The animals were supplemented intramuscularly with a
R
metabolite of vitamin A; it controls the differentiation of commercial product (Vigantol E , Bayer) that provided fat-
spermatogonia and adhesion characteristics of spermatids (14). soluble vitamins. The fat-soluble vitamin supplementations were
Vitamins E and C are the most important non-enzymatic carried out monthly throughouttheexperiment.Theexperiment
antioxidants in nutritional supplementation (21). Vitamin E consisted of two treatments based on supplementation with
includes a group of fat-soluble compounds, tocopherols, and fat-soluble vitamins (A, D3, E) and a control treatment (24).
tocotrienols, that act as antioxidants against oxidative stress The experimental treatments included a control (COD) without
(16, 22). This is because vitamin E captures free radicals, fat-soluble vitamin supplementation. Treatment of FVD1
stabilizing the sperm membrane with the formation of less was based on the supply of 2,500,000 International Unit (IU)
harmful complexes (23). High supplementation with vitamin vitamin A, 375,000 IU vitamin D3, and 250mg vitamin E for
D (2,000–4,000 Ul·Kg−1), is positively associated with seminal every 400kg of weight (27). The treatment FVD½ consisted of
quality (24). supplementation of 50% of FVD1 (1,250,000 IU, 187,500 IU, and
Seminal quality has been linked to the presence of vitamins 125mg of vitamins A, D3, and E, respectively, for every 400kg
as supplementation (24). On the other hand, deficiency of of weight) (27). The assignments were completely random: two
Frontiers in Veterinary Science | www.frontiersin.org 2 July 2022 | Volume 9 | Article 908763
Calderón-Calderón et al. Fat-Soluble Vitamin Supplementation in Boars
TABLE1|Ingredients and chemical composition of diets. Switzerland) was used for slide staining, following the
manufacturer’s instructions. All slides were analyzed in a
Ingredients Min/Max Percentage (%) double-blind scheme.
Salt Min. 0.4 AssessmentofSpermMorphometryby
Salt Max. 0.5 CASA-Morph
Dry mater Max. 88.0
R
Crude protein (% of DM) Min. 16.0 Sperm head morphometry was analyzed using the ISAS v1
Crude fat (% of DM) Min. 2.0 (Integrated Semen Analysis System, Proiser R C D, Valencia,
Crude fiber (% of DM) Max. 7.0 Spain). The equipment consisted of a UB203 microscope
Min premixa 1.2 (UOP/Proiser R C D) equipped with a bright-field 100×
Chemicalcomposition objective and a 3.3 × photo-ocular. A digital video camera
Ash (% of DM) Max. 7.0 (Proiser 782m, Proiser R C D) was mounted on the microscope
Phosphorous (% of DM) Min. 0.7 to capture the images and transmit them to the computer.
Calcium (% of DM) Min. 0.8 The array size of the video frame grabber was 746 × 578 ×
Calcium (% of DM) Max. 1.0 8 bit, providing a resolution of the analyzed images of 0.084
Digestible energy (Mcal/kg DM) Min. 3.3 µm/pixel in both axes, and 256 gray levels (31). The resolution
of the images was 0.08µm per pixel in both the horizontal
NFE – 56.0 and vertical axes. The sperm heads were captured randomly
Min, minimum; Max, maximum; N.F.E, Nitrogen free extract [NFE% D 100 − (Moisture % in different fields with CASA-Morph, and only those that
CCrudeprotein % C Crude fat C Crude fiber % C Ash %)]. DM, Dry matter. aContained overlapped with background particles or other cells to interfere
3 2
195.0 g/kg calcium, 21.0 g/kg magnesium, 1.0 × 10 mg/kg cobalt, 3.0 × 10 mg/kg with the subsequent image processing were rejected as described
2 3 3
copper, 1.2 × 10 mg/kg iodine, 3.0 × 10 mg/kg iron, 2.2 × 10 mg/kg manganese,
3 by Barquero et al. (32). An initial erroneous definition of the
3.0 × 10 mg/kg zinc, 1.1 mg/kg selenium.
spermheadboundarywascorrectedbyvaryingtheanalysisfactor
of the CASA-Morph system. However, when it was not possible
boars in the COD group, two boars in the FVD1 grouped, and to obtain a correct boundary, the sperm head was deleted from
three boars in the FVD½ group. the analysis.
SemenCollectionandEvaluation AssessmentofSpermKinematicsby
Ejaculates were collected in the morning, 1 time per week, CASA-Mot
R
using the “gloved-hand” technique (28) and immediately placed For motility analysis, ISAS D4C20 disposable counting
in a water bath at 37◦C at the farm laboratory. In all cases, chambers (Proiser R C D, S.L., Paterna, Spain) were used after
◦
the sperm-rich fractions were collected and diluted with a being pre-warmed to 37 C. A volume of 2.7 µl of the diluted
commercial extender (Zoosperm ND5; Import-Vet, Barcelona, samples was distributed along the counting chamber fields by
Spain) using the procedure described by Barquero et al. (29). capillarity to fill it completely. Analyses were conducted using
R
Insemination doses contained a concentration of 3.7 ± 1.3 × the CASA-Mot system ISAS v1 (Integrated Semen Analysis
109 spermatozoa. From each boar, 8.6 ± 4.9 ejaculates were System,ProiserRCD,Paterna,Spain)fittedwithavideo-camera
obtained. From the treatments evaluated COD, FVD1, and (Proiser 782M, Proiser R C D), with 25 frames acquired per field
FVD½,11, 20, and 27 ejaculates were used, respectively. Semen at a frame rate of 50Hz and final resolution of 768 × 576 pixels
samples from each ejaculate were evaluated for total motility, as described Soler (33). The camera was attached to a microscope
progressiveness, and morphology, and only ejaculates with at UB203 (UOP/Proiser R C D) with a 1× eyepiece and a 10×
least 75% morphologically normal spermatozoa were used. negative-phase contrast objective (AN 0.25) and an integrated
The concentration was measured with Spermacue (Minitube, heated stage maintained at a constant temperature of 37.0 ±
0.5◦C. The CASA settings used were a particle area between 10
GmbH, Tiefenbach, Germany) following established protocols 2
◦ and 80 µm and a connectivity of 11µm according to Valverde
(30). Samples were stored at 17 C and were transported to (34). The percentage of total motile cells and progressive motility
◦
the laboratory under the same refrigerated conditions (17 C) (%) corresponded to spermatozoa swimming forward quickly
used for commercial distribution according to Barquero et al. in a straight line. The following parameters defined progressive
R
(29). Semen samples (1ml) were placed in an Eppendorf tube motility: straightness (STR, straightness index) ≥45% and
◦
(Sigma-Aldrich, St. Louis, MO, USA) and remained at 37 C for −1
30minbeforeevaluations. average path velocity (VAP) ≥25 µm·s , defined as the average
velocity over the smoothed cell path.
SamplePreparation for Morphometric ComputerizedKinematicsAnalysis
Analysis TheCASA-Motvariablesassessedinthisstudyincluded:straight-
−1
Ejaculates from each group were assessed in duplicate for line velocity (VSL, µm·s ), corresponding to the straight
morphometric analysis. A volume of 10 µl of each sample line from the beginning to the end of the track; curvilinear
−1
was mixed and smeared on a glass slide and subsequently air- velocity (VCL, µm·s ), measured over the actual point-to-
R
dried. The Diff-Quik kit (Medion Diagnostics, Düdingen, point track followed by the cell; average path velocity (VAP,
Frontiers in Veterinary Science | www.frontiersin.org 3 July 2022 | Volume 9 | Article 908763
Calderón-Calderón et al. Fat-Soluble Vitamin Supplementation in Boars
−1 TABLE2|Overall changes in seminal characteristics (mean ± SEM) of boar
µm·s ) the average velocity over the smoothed cell path; the
amplitude of lateral head displacement (ALH, µm), defined as ejaculates during the experiment.
the maximum of the measured width of the head oscillation Variable Season P-value
as the sperm swims; beat-cross frequency (BCF, Hz), defined
as the frequency with which the actual track crosses the Dry Rainy
smoothed track in either direction; motility (%), defined as the
percentage of total motile cells; and progressive motility (%), Semenvolume(ml) 267.42 ± 18.76 251.20 ± 19.51 ns
Total motility (%) 50.67 ± 0.77a 73.77 ± 0.83b **
corresponding to spermatozoa swimming rapidly forward in a a b
straight line as describe Soler (35). Three progression ratios, Progressive motility (%) 45.43 ± 0.82 67.63 ± 0.87 **
expressed as percentages, were calculated from the velocity Fast spermatozoa (%) 35.83 ± 1.34a 46.64 ± 0.85b **
measurements described above: linearity of forward progression Average speed spermatozoa (%) 16.70 ± 0.49a 13.91 ± 0.31b **
(LIN D VSL/VCL·100), straightness (STR D VSL/VAP·100), and Slow speed spermatozoa (%) 2.43 ± 0.19a 3.53 ± 0.12b **
wobble (WOB D VAP/VCL·100) (36). The CASA analyses were Static spermatozoa (%) 45.04 ± 1.24a 35.93 ± 0.78b **
6 −1
performed in seven microscope fields on a total of at least 600 Spermconcentration (×10 ·ml ) 267.35 ± 24.79 248.44 ± 22.85 ns
cells per sample. Normal sperm (%) 86.34 ± 3.29a 79.56 ± 2.99b **
Semendoses 13.92 ± 0.48a 12.97 ± 0.37b *
ComputerizedMorphometricAnalysis Abnormal sperm (%) 13.66 ± 3.29a 20.44 ± 3.00b **
Images from about 200 spermatozoa from each sample were nD58ejaculates.SEM,standarderrorofthemean.a,bLeastsquaremeansinarowwith
captured and analyzed, to obtain eight morphometric variable differing letters differ significantly (P < 0.05); ns: not significant. *P < 0.05; **P < 0.001.
values. Following the criteria of Boersma (37), the sperm heads
were measured on each slide for four primary parameters of
2
head size [length (L, µm), width (W, µm), area (A, µm ), and average, slow, and static spermatozoa), sperm concentration,
perimeter (P, µm)] and four derived dimensionless parameters normal and abnormal sperm (%), and semen doses. A normal
−1 2 −1
of head shape {ellipticity (L·W ), rugosity [4πA·(P ) ], distribution with an identity link function was assumed for
−1 −1
elongation [(L – W)·(L C W) ], and regularity [πLW·(4A) ]}. all response variables. ANOVA was further applied to evaluate
R
Data from each individual sperm cell were saved in an Excel statistical differences between treatments for all kinematic and
file(MicrosoftCorporation,Redmond,Washington,USA)bythe morphometricvariables.Otherfixedfactorswithpotentialeffects
software for further analysis. on sperm quality were also added to the model such as season
AssessmentofMorphologyofSperm and treatment × season interaction. A random residual effect
Variables was also added to the model to account for correlations between
A single technician carried out the assessments of sperm different ejaculates obtained from the same boar. The threshold
morphology.Spermwereclassifiedashavingnormalorabnormal for significance was defined as p < 0.05. Pairwise comparisons
morphologic features following WHO strict criteria (38). A total between season and treatment means were performed by the
of 200 sperm were analyzed per slide; 100 sperm from each of Tukey–Kramer test. Results were presented as mean ± standard
twodifferentlocationsontheslidewereassessed.Ifthedifference deviationofthemean.AlldatawereanalyzedusingtheIBMSPSS
betweenthepercentageofnormalsperminthetwoareaswas5% package,version23.0forWindows(SPSSInc.,Chicago,IL,USA).
or less, then the mean value was calculated (6). A subsample of
eachejaculate was used to prepare one slide per sample analyzed. RESULTS
Atotal of 10 µl aliquot was placed on a glass slide and covered
R
SemenCharacteristics
with a coverslip, and immediately brought to the Trumorph
R
system(ProiserRCD,SL,Paterna,España).Trumorph
exerted There was an effect of season on seminal variables analyzed (p
a constant force of 20 kiloponds (kp) uniformly distributed on <0.05): motility, swimming patterns, morphology, and semen
◦ productiondoses.Intherainyseason,aboarejaculatehadgreater
the surface of the coverslip, with a temperature of 65 C. For
assessment of the sample, a microscope with a 1× eyepiece and motility (total and progressive) and proportion of spermatozoa
a 40× negative-phase contrast objective was employed. Sperm withfastmovement.However,inthisseasonadecreasednumber
morphology was examined to categorize normal cells, proximal of semen doses was obtained. There were no differences (p
and distal cytoplasmic droplets, or flagellum defects such as > 0.05) between dry or rainy seasons for semen volume and
folded or coiled tails (39). spermconcentration(Table2).Therewasaninteractionbetween
treatment × season (p < 0.05). In the rainy season, FVD1
Statistical Analysis treatment resulted in higher total and progressive motilities than
Anormalprobabilityplotwasusedtoassessnormaldistribution. in the dry season (Figure 1). However, it was pointed a higher
The data obtained for the analysis of all sperm variables were semen volume and number of doses produced in the dry season
assessed for homoscedasticity by using the Levene test. Further, with the 100% fat-soluble vitamin supplementation treatment
sperm variables were analyzed using the Generalized Linear (FVD1)(Figure2).
Mixed Models (GLMM). The response variables were semen There was an effect (p < 0.05) of fat-soluble vitamin
volume, total and progressive motility, swimming patterns (fast, supplementation on seminal characteristics. The boars
Frontiers in Veterinary Science | www.frontiersin.org 4 July 2022 | Volume 9 | Article 908763
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