jagomart
digital resources
picture1_Supplementary Nutrition Pdf 146142 | Roldan Ers Influence


 130x       Filetype PDF       File size 0.69 MB       Source: digital.csic.es


File: Supplementary Nutrition Pdf 146142 | Roldan Ers Influence
originalresearch published 12 july 2022 doi 10 3389 fvets 2022 908763 inuence of fat soluble vitamin intramuscular supplementation on kinematic and morphometric sperm parameters of boar ejaculates josue calderon calderon1 ...

icon picture PDF Filetype PDF | Posted on 11 Jan 2023 | 2 years ago
Partial capture of text on file.
                                                                                                                                                            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
The words contained in this file might help you see if this file matches what you are looking for:

...Originalresearch published july doi fvets inuence of fat soluble vitamin intramuscular supplementation on kinematic and morphometric sperm parameters boar ejaculates josue calderon francisco sevilla eduardo r s roldan vinicio barquero andanthonyvalverde animal reproduction laboratory school agronomy costa rica institute technology alajuela department biodiversity evolutionary biology national museum natural sciences spanish research council csic madrid spain faculty agri food alfredo volio mata experimental station university cartago ejaculate quality can be regarded as multifactorial with nutrition being a factor that could directly the present study aimed to evaluate seminal associated seasonal boars seven sexually mature were randomly allotted one three groups fed supplementary diets for weeks control treatment cod without vitamins containing edited by administered intramuscularly which was based elisabeth pinart d e fvd girona reviewedby semen collected at day francesca mercati int...

no reviews yet
Please Login to review.