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Evaluation of the effect of the addition of an olive oil-derived

antioxidant (Pectoliv-80A) in the extender for cryopreservation of
rooster sperm through the use of a discriminant statistical tool

Esther Dı́az Ruiz , Antonio González Ariza ,

José Manuel León Jurado , Ander Arando Arbulu ,
Juan Fernández-Bolaños Guzmán , Alejandra Bermúdez Oria ,
Juan Vicente Delgado Bermejo , Francisco Javier Navas González

PII: S0032-5791(24)00209-8
DOI: https://doi.org/10.1016/j.psj.2024.103630
Reference: PSJ 103630

To appear in: Poultry Science

Received date: 5 January 2024

Accepted date: 4 March 2024

Please cite this article as: Esther Dı́az Ruiz , Antonio González Ariza , José Manuel León Jurado ,
Ander Arando Arbulu , Juan Fernández-Bolaños Guzmán , Alejandra Bermúdez Oria ,
Juan Vicente Delgado Bermejo , Francisco Javier Navas González , Evaluation of the effect of
the addition of an olive oil-derived antioxidant (Pectoliv-80A) in the extender for cryopreservation
of rooster sperm through the use of a discriminant statistical tool, Poultry Science (2024), doi:
https://doi.org/10.1016/j.psj.2024.103630

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 EFFECT OF PECTOLIV 80-A IN CRYOPRESERVED SPERM

Evaluation of the effect of the addition of an olive oil-derived antioxidant (Pectoliv-

80A) in the extender for cryopreservation of rooster sperm through the use of a

discriminant statistical tool

Esther Díaz Ruiz*, Antonio González Ariza†,1, José Manuel León Jurado†, Ander
Arando Arbulu*, Juan Fernández-Bolaños Guzmán‡, Alejandra Bermúdez Oria‡, Juan
Vicente Delgado Bermejo*, Francisco Javier Navas González*

*Department of Genetics, Faculty of Veterinary Sciences, University of Córdoba,

Córdoba, 14071, Spain.
†
Agropecuary Provincial Centre, Diputación of Córdoba, Córdoba, 14071, Spain.
‡
Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, (CSIC), Sevilla,
41013, Spain.

1
Corresponding author: Antonio González Ariza; angoarvet@outlook.es; Tel.: +34-679-
305-661
ABSTRACT

During the poultry sperm cryopreservation process, an excess of reactive oxygen

species is generated resulting in oxidative stress which harms the quality of avian

spermatozoa. To counteract this effect, the addition of exogenous antioxidants, such as

Pectoliv-80A (a by-product of olive oil), to the cryopreservation diluent is interesting.

For this purpose, 16 roosters belonging to the Utrerana avian breed were used. Six

semen pools (from the six different replicates) were divided into four aliquots

corresponding to different concentrations of Pectoliv-80A that were tested (0, 300, 400,

and 500 μg/mL), and the cryopreservation process was carried out. To evaluate post-

thawing semen quality, different parameters such as motility, membrane functionality,

reactive oxygen species production, lipid peroxidation, and acrosome integrity were

studied. A discriminant canonical analysis was used to determine both the differences

between the Pectoliv-80A concentration groups and the discriminant power of the

aforementioned parameter used for semen evaluation. Total motility and membrane

functionality were reported to be the most discriminant variables for differentiating the

different antioxidant enrichment groups and concluded that concentrations of 300

μg/mL showed the most desirable quality of post-thawing semen. The present study

could lead to the optimization of both cryopreservation and quality evaluation

techniques of the sperm of rooster species, that support the conservation program of

endangered local breeds.

KEYWORDS

Genetic resources; antioxidant; rooster; semen quality assessment; discriminant

canonical analysis
 INTRODUCTION

Sperm cryopreservation is a fundamental tool within assisted reproduction

techniques in avian species, for the conservation of germplasm of both endangered

breeds and genetically valuable animals for the improvement of production. This

conservation method is also a more cost-effective conservation strategy than keeping

genetic resources in vivo (Silversides et al., 2012; Woelders, 2021; Zong et al., 2023).

Therefore, governments should prioritize in vitro conservation programs by

implementing strategies to develop different aspects of germplasm banks, which will

increase productivity and facilitate the preservation and exchange of genetic material

between countries (Paiva et al., 2016).

The conservation of genetic diversity allows genotypes to be more sustainable by

adapting appropriately to environmental changes, such as climatic or market changes,

new management practices, and the emergence of diseases (Tisdell, 2003). Furthermore,

in rural areas, local livestock breeds are essential to maintain the population in these

areas, and the disappearance of these resources would lead to the migration of these

people to urban centers, endangering these communities and, therefore, the food supply

(Cardellino, 2003).

However, during the cryopreservation process, oxidative stress is generated due to

an imbalance between the production of reactive oxygen species (ROS) and the

antioxidant capacity of the endogenous system present in the avian seminal plasma

(Akhtar et al., 2022). This phenomenon is even more accentuated in the avian species

due to the characteristic morphology of avian spermatozoa, as the head is very narrow,

with a low amount of cytoplasm, and the tail is very long, which hinders motility,

causing damage at the mitochondrial level and in the midpiece of the spermatozoa

(Mohammad et al., 2021). These morphological characteristics coupled with the fact
that avian spermatozoa contain large amounts of polyunsaturated fatty acids in the

plasma membrane make them more susceptible to oxidative stress leading to DNA

damage and reduced fertility (Masoudi et al., 2018; Mehaisen et al., 2020). Although

minimum levels of ROS are necessary for the fertilization process to develop properly,

when these levels are exceeded in excess, sperm functionality is altered (Guthrie and

Welch, 2012). The main ROS-producing organelles are the mitochondria and the cell

membranes of spermatozoa (Brouwers and Gadella, 2003; Agarwal et al., 2005). When

there is an excess of ROS production, it is usually the result of electron leakage from

uncoupled oxidative phosphorylation of mitochondria, which results in sperm cells

decreasing their mitochondrial membrane potential, impairing mitochondrial ATP

production and thus sperm motility (Guthrie and Welch, 2007).

In this sense, the addition of exogenous antioxidants in the cryopreservation

extender may be beneficial to mitigate the excessive production of ROS and the

resulting peroxidation which allows preserving the characteristics of rooster sperm

leading to a higher fertilizing capacity after processing (Khiabani et al., 2017; Al-

Mutary, 2021). In fact, there are many antioxidants that have been tested in roosters

after their addition to the diluent, such as vitamins C and E (Amini et al., 2015),

quercetin (Appiah et al., 2020), L-carnitine (Fattah et al., 2017), and glutamine

(Khiabani et al., 2017), among others.

Alperujo is a by-product generated in the olive oil industry that is obtained when the

two-phase continuous extraction system is applied, being a source of substances with

high added value such as mineral celluloses, hemicelluloses, pectins, gums, tannins, and

polyphenols. Some of these substances have antioxidant effects, such as hydroxytyrosol

(3,4-dyhydroxyphenylethanol, HT), a phenolic compound that reduces oxidation of

low-density lipoproteins, protects against H2O2 cytotoxicity and minimizes lactate

dehydrogenase activity (Fernandez-Bolanos et al., 2008; Rubio-Senent et al., 2012;

Bermúdez-Oria et al., 2019; Bermúdez-Oria et al., 2020). This substance has been tested

in roosters (Díaz Ruiz et al., 2023a), and its addition in the diluent has resulted in an

improvement of the post-thawing semen quality, as well as in other animal species

(Hamden et al., 2010; Arando et al., 2019; Zhang et al., 2020b; Arando Arbulu et al.,

2021). In addition to HT, there is another minor phenolic compound, 3,4-

dihydroxyphenylglycol, which has also been tested for its antioxidant effect in roosters

(Díaz Ruíz et al., 2023b), sheep (Arando et al., 2019), goats (Arando Arbulu et al.,

2021) and dogs (Shakouri et al., 2021).

As mentioned above, when the alperujo is thermally treated and the solid and liquid

phases are separated, other types of compounds such as pectic polysaccharides are

obtained in the liquid phase (Rubio-Senent et al., 2012). This is the case of Pectoliv-

80A, which possesses a high antioxidant property that is directly related to its

antiproliferative capacity, which would be able to reduce the production of ROS due to

its free radical scavenging capacity (Bermúdez-Oria et al., 2019). Pectoliv-80A is a type

of "modified pectin" and is considered to have been treated with pH adjustment by

alkali or acid, heat, or enzyme, whose biological activity is attributed to its galactan side

chains (Maxwell et al., 2012). Its antioxidant action could be related to its high content

of phenolic compounds. The complete chemical composition of this substance is shown

in Table 1 (Bermúdez-Oria et al., 2019).

For the evaluation of semen quality, some routine techniques are normally used

more frequently on thawed rooster sperm, such as the assessment of morphology,

membrane functionality (hypo-osmotic swelling test; HOST), and mass motility, which

are inexpensive methods that do not require specialized equipment. (Łukaszewicz et al.,

2008). (Bucak et al., 2009). (Van de Hoek et al., 2022).

 However, in addition to these routine techniques, there are more specialized

techniques such as the CASA system and flow cytometry, which are used as poultry

sperm quality evaluation methods (Svoradová et al., 2019). (Boe-Hansen and Satake,

2019). (Bréque et al., 2003; Słowińska et al., 2018; Mehaisen et al., 2020; Rezaie et al.,

2021).

In any case, the use of statistical tools allows the prediction of the semen quality

parameters that provide the most information on the avian species according to the

study in question. This is the case of discriminant canonical analysis (DCA), a

procedure that allows the development of a predictive model from a set of data showing

the existing relationships between two or more variables, which has proven to be

effective in revealing patterns of data clustering (Adeyemi and Oseni, 2018). The DCA

determines linear functions of quantitative variables that distinguish at most two or

more classification groups while maintaining the minimum possible variation between

these groups (Cruz-Castillo et al., 1994). By means of this tool, it will be possible to

take into account all the variables considered appropriate simultaneously when

distinguishing between study groups, so that we can determine the variables that

contribute most to the discrimination between semen freezing treatments (Yeater et al.,

2004). In fact, in the area of avian reproduction, this analytical technique has previously

been successfully applied ( González Ariza et al., 2022a; Díaz Ruiz et al., 2023a). DCA

has also been used in the field of poultry production with the objectives of increasing

broiler performance (Rosario et al., 2008), classifying eggs of different genotypes of

local breeds of hens according to egg quality traits (González Ariza et al., 2021a;

González Ariza et al., 2022b), and performing biometrical characterizing of local

genotypes of hens (González Ariza et al., 2021b).

 In short, the objective of the present study is to evaluate whether the addition of

Pectoliv-80A in the cryopreservation extender at different concentrations has any effect

on post-thawing rooster semen quality by determining by means of a DCA which are

the seminal quality variables with the greatest discriminatory power when evaluating

this effect. The aim is to improve the technique of seminal cryopreservation concerning

the use of antioxidants, which will be an advantage for the conservation of avian genetic

resources.

MATERIAL AND METHODS

Ethical Approval

Protocols and animals used in the present research were managed following the

prescriptions and regulations of the European Union (2010/63/EU) in its transposition to

Spanish law (RD 53/2013). The data analyzed in this study were not obtained by

experimental procedures but were obtained in the daily framework of an avian

reproduction center and a center for the conservation of native breeds (both in the

Agropecuary Provincial Center; Diputación de Córdoba). Thus, the present study is not

covered by the legislation on the protection of animals used for scientific purposes and

is outside the scope of evaluation of the Ethics Committee of the University of Córdoba.

Animals and semen collection

The present study took place in the facilities of the Agropecuary Provincial Center

(Diputación de Córdoba), in the south of Spain (37◦54’50.9"N-4◦42’40.4"W) for two

months (April and May 2022).

For the development of this study, a total of 16 Utrerana breed roosters aged

between 1 and 3 years were used. The selection of the sampled individuals was

performed considering the age when the used genotype is reared in its common
production system. Contextually, the typical egg production cycle in commercial strains

lasts about 72 weeks. However, this cycle may extend until 156 weeks in around a third

of the Utrerana breed population (González Ariza et al., 2022b).

The animals were handled according to the prescriptions and regulations of the

European Union (2010/63/EU) in its transposition to Spanish law (RD 53/2013). The

roosters were housed in individual cages of suitable dimensions and received a

commercial diet based on 15.20% crude protein, 4.60% crude fat and oils, 3.20% crude

fiber, 14.00% crude ash, 4.10% calcium, 0.66% phosphorus, 0.19% sodium, 0.31%

methionine, and 0.72% lysine, as well as water ad libitum.

During the study period, semen was collected once a week per animal. A total of 6

replicates were used in the study. Studies using more than 10 roosters and 5 or more

replicates have been considered to be of the highest research quality when evaluating

the antioxidant enrichment of rooster semen extenders (Leão et al., 2021).

The technique used for sperm collection was the abdominal massage technique

described by Burrows and Quinn (1937). Each week, once the semen was collected, all

ejaculates meeting the following minimum quality criteria were pooled: ejaculate

volume (≥0.2 mL), sperm concentration in the ejaculate (≥3x109 spz/mL), and mass

motility (≥3 points out of 5). To determine ejaculate mass motility, a drop of semen

sample was placed on a slide without coverslip, examined under a compound

microscope (100×) and scored on a scale of 1 to 5 (Mussa et al., 2023). Ejaculates

contaminated by blood, urine, or feces were discarded.

Isolation of Pectoliv-80A from olive oil waste

Hydrothermal treatment of alperujo was carried out using a prototype steam

treatment reactor, designed by the Department of Food Phytochemistry of the Instituto

de la Grasa (Seville, Spain). Fresh alperujo samples were treated with saturated steam at

a temperature of 80°C for 60 minutes, which was injected directly to increase the

contact with the alperujo. After steam injection, the pressure was reduced in a controlled

manner until atmospheric pressure was reached. To separate the solid and liquid phases

of the alperujo, the samples were centrifuged at 4700 g (Comteifa, S.L., Barcelona,

Spain) with subsequent ultrafiltration of the liquid phase at 3000 Da, the liquid phase

above 3000 Da being concentrated at 2 L in a rotary evaporator and precipitated with

70% EtOH. Finally, the residue that remained insoluble in alcohol was freeze-dried and

part of it was hydrolyzed with 1 M NaOH for 1 h, with subsequent neutralization with 1

M HCl. To remove the salts from the resulting liquid, it was ultrafiltered at 3000 Da and

then lyophilized. The extract obtained is known as Pectoliv-80A (Bermúdez-Oria et al.,

2019).

Experimental design

Once the pool was made with ejaculates that met minimum quality criteria, it

was refrigerated at 5°C in a programmable cooler (Cell incubator SH-020S, Welson,

Korea) for one hour at a temperature reduction rate of 0.3°C/min. The pool was divided

into four aliquots which were diluted with a diluent containing different concentrations

of Pectoliv-80A to obtain four different treatments whose final concentrations of the

antioxidant are: control (0 μg/mL), T1 (300 μg/mL), T2 (400 μg/mL) and T3 (500

μg/mL). This amount of antioxidants was divided into two parts as the dilution was

carried out in two steps. The first one was carried out with a diluent (Sasaki et al., 2010)

whose composition was: 0.2 g D (+)-glucose, 3.8 g D (+)-trehalose dihydrate, 1.2 g L-

glutamic acid monosodium salt, 0.3 g potassium acetate, 0. 08 g magnesium acetate

tetrahydrate, 0.05 g sodium citrate tri-basic dihydrate, 0.4 g BES, 0.4 g Bis-Tris and

0.001 g gentamicin sulfate (pH= 6.8, osmolarity= 360 mOsm). After 30 minutes, a
second dilution was performed with the same diluent containing 18% N-

methylacetamide (NMA; final concentration 9%) as a cryoprotectant. Finally, the

samples were packed in 0.25 mL straws with a final concentration of 250 x 106

spz/straw. 30 minutes after the second dilution, straws were placed in nitrogen vapor at

a height of 4 cm for 30 min. In the end, the straws were immersed in liquid nitrogen (-

196 °C) until use. For thawing, straws were immersed in a water bath at 5 °C for 100 s

(Sasaki et al., 2010).

Sperm quality assessment

Sperm motility.

Sperm motility was assessed using a Computer Assisted Sperm Analyser

(CASA) IVOS 12.3 (Hamilton-Thorne Bioscience, MA, USA) by diluting a sperm

sample with FA of the above diluent to a concentration of 50 × 106 spz/mL and

depositing 5 µL in a Life Optic chamber. To be considered spermatozoa, the cells had to

reach an area between 2 and 60 μm2, and to be categorized as progressive motile they

had to have average apth velocity (VAP) >50 μm/s and straightness (STR) >70%.

Membrane functionality (HOST).

For the study of plasma membrane functionality, HOST was performed

(Jeyendran et al., 1984). For this, 25 µL of sperm were diluted in 500 µL of

hypoosmotic solution (1 g sodium citrate and 100 mL double distilled water; 100 mOs-

mol/kg) and incubated at 37 °C for 30 min. The dilution was then fixed in 2%

glutaraldehyde and observed under a phase contrast microscope (×400 magnification).

A total of 200 spermatozoa were analyzed and considered intact when the spermatozoa

were coiled.

Reactive Oxygen Species (ROS).

 The reactive oxygen species (ROS) were measured with the commercial kit

DCFH-DA (Sigma-Aldrich, St. Louis, MO, USA). For this, 1000 µL of semen (4 x 106

spz/mL) and 1 µL of DCFH-DA (25 µM) were mixed and incubated at 25°C for 30 min.

After this time, the sample was centrifuged at 2600 rpm for 5 min, the supernatant was

removed and 1000 µL of cytometer fluid was added for evaluation.

Lipid Peroxidation (LPO).

The lipid peroxidation (LPO) was assessed using a CyFlow® Cube 6 Cytometer

(Sysmex Europe GmbH, Norderstedt, Germany). This cytometer is composed of a 488

nm blue laser and a 638 nm red laser, with features of interchangeable optical filters.

First, 200 µL of semen (20 x 106 spz/mL) and 10 µL of C11-BODIPY581/591 (10 µM)

were incubated in a cytometer tube for 30 min at 37 °C in the dark. The sample was

then centrifuged at 2600 rpm for 5 min, the supernatant was removed and 1000 µL of

cytometer fluid was added for reading. Sperm that emitted at the green wavelength

(FL1) were considered positive for C11-BODIPY581/591.

Acrosome Integrity.

For the assessment of acrosome integrity, the CyFlow® Cube 6 Cytometer was

also used. Thus, 300 µL of semen (20 x 106 spz/mL) together with 15 µL of fluorescein

isothiocyanate-conjugated peanut agglutinin (PNA-FITC, 100 µg/mL; Sigma-Aldrich,

St. Louis, MO, USA) and 30 μL of propidium iodide (PI; 6 µM) were deposited in a

cytometer tube. Incubation was carried out in the dark for 5 minutes at room

temperature and 1200 µL of cytometer fluid was added for reading.

Statistical analysis

Overall Descriptive Statistics

 Data obtained in control, T1, T2, and T3 cryopreservation treatments were

summarized through the use of descriptive statistics techniques. Mean, standard

deviation, minimum, and maximum were obtained for the following variables: total

motility (TM), HOST, ROS, LPO, and acrosome integrity. To perform this analysis, the

descriptive statistics routine of the data description package of XLSTAT 2022 (Pearson

Edition; Addinsoft, Paris, France) was used.

Normality and Bayesian ANOVA tests

Shapiro-Francia W’ test was computed to discard violations of the normality

assumption. This test was chosen because the sample number used in the present study,

which ranged from 5 to 5000 (Royston, 1993). Both normally and non-normally

distributed variables were obtained (p > 0.05), Thus, a Bayesian ANOVA test was

chosen to analyze differences between the different cryopreservation treatments. The

results from the Bayesian ANOVA test reported medians in the TM (F = 5.384, pv =

0.007), PM (F = 3.503, pv = 0.034), and HOST (F = 4.034, pv = 0.021) variables that

significantly differ. By contrast, the ROS (F = 0.260, pv = 0.853), LPO (F = 1.067, pv =

0.385), and acrosome integrity (F = 0.576, pv = 0.638) variables did not report

significant differences. In this way, the presence of differences in some variables across

the different rooster cryopreservation treatments used in the present work justified the

subsequent election of DCA statistics.

DCA

For the DCA, six explanatory variables were included; total motility (TM),

progressive motility (PM), HOST, ROS, LPO, and acrosome integrity. The different

concentrations of Pectoliv-80A (control, T1, T2, and T3) were used as classification

criteria and variability between groups was measured.

 Sample size used in this study has been reported to be robust. Some authors have

reported a minimum sample size of at least 20 observations for every 4 or 5 predictors,

and the maximum number of independent variables to palliate possible distortion effects

should be n-2, where n is the sample size (Poulsen and French, 2008; Marín Navas et

al., 2021).

Strong and independent relationships across predictors were ensured by running

the multicollinearity analysis. The forward and backward stepwise selection methods

were used, with the same variables. However, the progressive selection method was

performed as it requires less time to perform than the backward selection method

(González Ariza et al., 2021b). To perform multicollinearity and DCA analyses, the

Discriminant Analysis routine of the Analyzing Data package of XLSTAT software

(Addinsoft Pearson Edition 2014, Addinsoft, Paris, France) was used.

Multicollinearity Preliminary Testing.

Before running a DCA, the multicollinearity assumption must be tested to ensure

that variance explanatory potential not to be overinflated due to variables’ redundancies.

The variance inflation factor (VIF) is the most common indicator used in detecting

multicollinearity. A recommended VIF value of 5 was used in this study (Rogerson,

2001). VIF was computed by using the following formula as a subroutine of the

Discriminant Analysis routine of the Analyzing Data package of XLSTAT software:

where R2: coefficient of determination of the regression equation.

DCA Model Reliability.

 The Pillai’s trace criterion is the only acceptable test to be used in cases of

unequal sample sizes. The assumption of equal covariance matrices was tested through

this method in the discriminant function analysis (Zhang et al., 2020a). Pillai’s trace

criterion was computed as a subroutine of the Discriminant Analysis routine of the

Analyzing Data package of XLSTAT software. The set of predictors considered in the

DCA can be considered statistically significant if a significance is ≤ 0.05. Pillai trace

criterion has been argued to be the most robust statistic for general protection against

deviations from normality and homogeneity of variance of multivariate residuals. The

larger the observed value of the Pillai trace, the greater the evidence that the set of

predictors has a statistically significant effect on the values of the response variable.

DCA Efficiency.

Wilks' Lambda test, also called Rao's approximation, has been used in the present

study to evaluate the variables’ contributions to the discriminant function. As Wilks's

lambda approaches 0, the variable's contribution to the discriminant function increases.

The functions can be used to explain group adscription if the significance value is ≤

0.05 (Anuthama et al., 2011).

Independent Factor Discriminant Potential Evaluation, Canonical Coefficients,

and Loading Interpretation and Spatial Representation.

Next, once the variables whose discriminant potential was based on the mean

differences between the different treatments were analyzed, the discriminant function

analysis was used to identify those whose discriminant potential could be based on their

ability to determine higher percentages of assignment of observation within its group.

The discriminant loading of the different variables is considered to be significatively

discriminating when values of ≥|0.40| are obtained. The inclusion of redundant variables
in the function was prevented using a stepwise procedure technique. Large absolute

values in the loadings of the standardized coefficients of each variable lead to a higher

discrimination capacity and a higher percentage of correct classification.

Data were standardized following procedures reported by Manly and Alberto

(2016). Then, Squared Mahalanobis distances and principal component analysis were

computed, using the following formula:

where : distance between population i and j; COV-1: inverse of the covariance

matrix of measured variable x; and : means of variable x in the ith and jth

populations, respectively.

A dendrogram was built through the conversion of the squared Mahalanobis

distance matrix into an Euclidean distance matrix. For this, the underweight pair-group

method arithmetic averages (UPGMA; Rovira i Virgili University, Tarragona, Spain),

and the Phylogeny procedure of MEGA X 10.0.5 (Institute of Molecular Evolutionary

Genetics, The Pennsylvania State University, State College, PA, USA) were used.

RESULTS

Descriptive Statistics

The mean, standard deviation, minimum, and maximum for each variable studied

according to the different treatments considered are shown in Table 2.

DCA Model Reliability and Efficiency

The PM variable was discarded from further analyses due to multicollinearity

problems (VIF values over 5). Significant Pillai's trace criterion determined the validity
of the DCA (Table 3). Of the three discriminant functions revealed after discriminant

analyses, two showed a significant discriminant ability (Table 4). The discriminatory

power of the function F1 was high (eigenvalue of 1.463) with 96.67% of the variance

being explained by F1 and F2 (Figure 1).

Independent Factor Discriminant Potential Evaluation, Canonical Coefficients,

and Loading Interpretation and Spatial Representation

The discriminating ability of the different variables studied is shown in Table 5.

The greater discriminating power of a variable in question is related to a high value of F

and consequently, lower values of Wilks' Lambda, which translates into a better

position in the rank. The present analysis revealed that TM and HOST were the only

two traits that contributed significantly (P<0.05) to the discriminant ability of

significant discriminant functions.

Table 6 reports discriminant canonical coefficient loadings for representative

variables across discriminant functions. Values of ≥|0.40| for the discriminant loading of

the standardized coefficients of a certain variable can be considered to be substantially

discriminating variables.

Spatial Representation

Figure 2 suggests clear differentiation across treatments. The relative position of

centroids was determined through the substitution of the mean value for observations in

each term of the first two discriminant functions (F1 and F2) to obtain x and y-axis

coordinates. The larger the distance between centroids, the better the predictive power

of the canonical discriminant function in classifying observations.

Mahalanobis distance represents the probability that an observation showing an

unknown background belongs to a particular group. It can be computed through the

relative distance of the problem observation to the centroid of its closest group. Then,

the hit ratio was calculated (Figure 3).

DISCUSSION

Animal genetic resources comprise all animal species, breeds, and strains that

have scientific, economic, and cultural value to humanity in terms of food and

agricultural production for the present and future (Toro et al., 2009). The conservation

of genetic resources of the different local poultry breeds is also important due to their

high adaptability to alternative systems, resistance to extreme climatic conditions, and

resistance to diseases, as well as a high ability to survive in rough terrain (Machebe and

Ezekwe, 2002). In this sense, sperm cryopreservation (ex situ in vitro conservation) is

an effective method to safeguard genetic biodiversity, as artificial insemination (AI)

with good-quality sperm allows the development of breeding programs (Tiwari et al.,

2022).

For this reason, the evaluation of the seminal quality is essential. During the

cryopreservation process, various cellular lesions are produced as a consequence of the

ROS production, among others, allowing the supplementation of the cryopreservation

diluent with exogenous antioxidants to mitigate oxidative and nitrosative stress (Tiwari

et al., 2022).

For the study of semen quality, there are various available techniques, ranging

from the more routine ones that evaluate morphology or mass motility to more

specialized ones such as those carried out using the CASA system or flow cytometry.

To determine which techniques provide the most information on post-thaw semen

quality when the cryopreservation extender is supplemented with the antioxidant

Pectoliv-80A, a DCA was carried out. Moreover, this statistical approach allows us to
know if the Pectoliv-80A addition to the rooster cryopreservation semen extender

improves the results obtained for the different studied variables and which

concentrations of Pectolive-80A offer the best post-thawing results.

After the multicollinearity analysis, the PM variable was eliminated since it

shows redundancy problems with other variables that remain, such as TM, since, as

demonstrated in humans, the TM variable predicts viability results more accurately than

PM (Palomar Rios et al., 2018). Furthermore, a very close relationship between the two

parameters has been shown in frozen-thawed rooster sperm (Blesbois et al., 2008).

Once the multicollinearity analysis was performed, TM and HOST turned out to

be the only variables that significantly differ within the different studied groups

(control, T1, T2, and T3) and with the high discriminant power among the remaining

variables. This finding proves that the evaluation of these two parameters is mandatory

to assess the effect of Pectoliv-80A supplementation in the cryopreservation extender on

post-thawing rooster semen quality. The results obtained by Salehi et al. (2020) also

suggest a positive relationship between TM, HOST, and sperm viability in roosters.

Motility after a freeze-thaw cycle in poultry sperm is reduced by 30-60%. Thus,

this trait measurement is of choice to assess the level of spermatozoa damage during the

cryopreservation procedure because ROS production impairs sperm motility (Cremades

et al., 2005; Long, 2006). Increasing knowledge about TM is interesting since this

parameter is readily identifiable and reflects several essential aspects of sperm

metabolism (Katila, 2001). Moreover, TM is one of the main parameters most closely

related to fertilization ability as only those spermatozoa with an adequate degree of

motility will be able to ascend through the hen's vagina to reach the fertilization zone

(Leão et al., 2021; Muvhali et al., 2022). In any case, some factors influence TM, such

as the thawing rate, which must be adequate to maintain sperm quality, with recent
studies suggesting that the use of high temperatures could be beneficial (Salih et al.,

2021).

On the other hand, HOST can be used to determine the functionality of the

sperm plasma membrane in roosters after the freeze-thaw cycle based on the resistance

of the membrane to hypo-osmotic stress conditions (Moghbeli et al., 2016; Lotfi et al.,

2017). The importance of assessing the integrity of the plasma membrane lies in the

intimate relationship between the viability and the fertilizing capacity of sperm. Thus,

HOST plays a pivotal role in spermatozoa survival in the female reproductive tract since

it acts as a selective barrier between the intracellular and the extracellular environment

(Vazquez et al., 1997; Gwathmey et al., 2006). The HOST evaluation technique is

simple and inexpensive, that, in humans, has also been used as a predictor of successful

in vitro fertilization and infertility diagnosis which could be extrapolated to avian

species (Check et al., 1989).

In any case, the Pectoliv-80A enrichment of rooster semen extenders improved

TM and HOST variables in post-thawing semen. This is due to its high antioxidant

activity given its high content of phenolic compounds (Bermúdez-Oria et al., 2019).

Specifically, Banihani (2017) concluded that olive oil supplementation maintains semen

quality in adequate ranges by improving gonadal function, reducing oxidative damage

and lipid peroxidation, and promoting nitric oxide signaling.

Moreover, results obtained in the present research determine which antioxidant

concentration is the most suitable to supplement the cryopreservation diluent.

Bayesian ANOVA reported significant differences between the different

treatments for the TM and HOST variables. These analyses show that the use of

Pectoliv-80A in the extender improves the post-thaw quality of rooster semen, with the
T1 treatment having the most favorable results. In the case of the TM parameter,

obtained values were higher when the cryopreservation extender was supplemented at a

concentration of 300 μg/mL of Pectoliv-80A (T1), which corresponds to the lowest

concentration of those studied. These results are in agreement with those obtained in

other studies testing antioxidants in rooster sperm, in which high concentrations of

antioxidants are detrimental to the quality of thawed sperm (Amini et al., 2015;

Masoudi et al., 2018). Furthermore, this effect has also been observed under refrigerated

conditions (Touazi et al., 2018) and even in other species such as sheep (Arando et al.,

2020). The fact that high concentrations of antioxidants could produce detrimental

effects on spermatozoa may be because increased plasma membrane fluidity occurs in

the presence of high amounts of antioxidants, causing sperm to become more

susceptible (Shoae and Zamiri, 2008). Furthermore, as Touazi et al. (2018) describe,

high concentrations of olive oil are detrimental to other organisms such as bacteria

where olive oil is able to penetrate the lipid structure of the cell wall causing membrane

destruction, so this phenomenon could be similar to what occurs in spermatozoa.

However, in the case of HOST, although major differences were reported

between the control samples and the rest, similar values were obtained in the different

treatments in which Pectoliv-80A was added to the extender (T1, T2, and T3). In any

case, values higher than 60% for the HOST variable have been reported to be

considered acceptable (Sanyal et al., 2023). Moreover, this method has been reported to

be less sensitive in avian sperm when compared with spermatozoa of mammalian

(Matson et al., 2009). However, results obtained in the present study indicate that

Pectoliv-80A concentrations between 300 and 500 μg/mL did not produce variations of

biochemical damage in membrane spermatozoa (Santiago-Moreno et al., 2009).

 By contrast, other variables such as ROS, LPO, and acrosome integrity were

considered in the present study and were not found to be significantly discriminant. As

mentioned above, there exist different types of techniques for the evaluation of semen

quality, the most commonly used in the avian species being the evaluation of sperm

viability, motility, morphology, and concentration (Tabatabaei, 2012).

A strong negative correlation has been found between oxidative status and

motility, in the bovine species (Gallo et al, 2021). Large amounts of polyunsaturated

fatty acids, which are highly susceptible to oxidation compose the spermatozoa plasma

membrane. LPO produces a decrease in membrane fluidity, that is detrimental to sperm

functions, such as motility (Aitken, 2020). On the other hand, a by-product of

mitochondrial activity is ROS. These are a large class of molecules that include non-

radicals (ozone, simple oxygen, lipid peroxides, and hydrogen peroxide), radicals

(hydroxyl ion, superoxide, nitric oxide, or peroxyl, among others), and other oxygen

derivatives. At physiological concentrations, ROS activates intracellular pathways

underlying some functions like spermatozoa maturation, capacitation, hyperactivation,

acrosomal reaction, and gamete fusion (Ferramosca and Zara, 2017). However, an

overproduction of ROS causes the antioxidant defense systems to be overwhelmed,

resulting in a state of oxidative stress. Spermatozoa have a reduced antioxidant capacity,

so they are particularly susceptible to oxidative stress (Aitken, 2020). However, sperm

motility is not only influenced by the oxidative status of the cell. For example, the

energy source for sperm motility is derived from the cell's mitochondrial activity. Thus,

TM combines the explainability derived from many other variables, and therefore, this

variable results in greater discriminating powerin the DCA.

Concerning acrosome integrity, previous work on rooster species has suggested

that the effect of antioxidants on the acrosome is not a reliable indicator of semen
quality after cryopreservation (Mehaisen et al., 2020). Thus, despite the great amount of

effort made by researchers in the search for an antioxidant that has a positive effect on

the integrity of the acrosome, to present, no clear action on this variable has been

observed, both in poultry (Partyka et al., 2013; Mehaisen et al., 2020; Partyka et al.,

2020) and in mammalian species (Bucak et al., 2007; Bucak et al., 2009; Sarıözkan et

al., 2009; Manee-In et al., 2014; Gibb et al., 2015).

Parameters such as motility, morphology, or HOST are the most commonly

used. Therefore, these techniques are the most standardized, provide the most

information at a global level, and are cheaper and simpler techniques that can be applied

easily in developing countries where resources, both in terms of equipment and

specialized personnel, are limited, which is accentuated in the field of production of

local poultry breeds (González Ariza et al., 2021c). However, the assessment of semen

quality by conventional techniques shows the disadvantage of being less statistically

reliable as subjectivity comes into play (Evenson et al., 2002).

The introduction of other more objective techniques such as the CASA system in

the early 1980s as well as the even earlier application of flow cytometry in the late

1960s for the evaluation of spermatozoa has been a breakthrough in this field. These

techniques are constantly being improved thanks to better optics and instruments,

however, in the avian species, as they have been applied later, they have yet to be

standardized to a greater extent (Boe-Hansen and Satake, 2019). In the case of flow

cytometry, it is a technique that is increasingly used in studies assessing semen quality

in some avian species as it allows the objective and accurate assessment of subcellular

changes in a large number of spermatozoa in terms of shape, size, and even some

functional characteristics in a short period (Słowińska et al., 2018). Specifically, this

tool can be used to evaluate the integrity of the acrosome, the activity of the
mitochondrial membrane, and ROS and DNA damage, among others. Nowadays, there

exists a wide variety of fluorescent probes that allow a better evaluation of the different

cells, since in the case of sperm it is common for there to be contamination by blood

cells, leukocytes, or bacteria. Thus a cellular distinction is very important. On the other

hand, the use of the CASA system, which is based on optical microscopy and 2D

videomicrography, allows measuring both, motility and different kinematic parameters

(Boe-Hansen and Satake, 2019).

Conclusively, in avian species, the semen quality evaluation is important to

detect subfertile individuals and to predict fertility after the application of cryopreserved

sperm by AI. The Pectoliv-80A enrichment at low concentrations (300 μg/mL) of the

rooster semen extender produces a significant improvement in the TM, PM, and HOST

traits in post-thawing semen. TM and HOST traits are the two variables that provided a

large amount of information and showed a great discriminant power when we

differentiate between the four different treatments (control, T1, T2, and T3) under study

in this work. Moreover, DCA has been proven and validated as an efficient statistical

tool to discriminate between different cryopreserved semen treatments and allows us to

optimize this type of study since this tool indicates which techniques provide us with

the most usable information from a scientific point of view. Lastly, the standardization

of some specialized techniques such as flow cytometry in the poultry species, could

provide us with other types of information, which would allow us to advance in the

knowledge generated in these new techniques of cryopreservation of genetic material.

ACKNOWLEDGMENTS

This work would not have been possible if it had not been for the assistance of the

Centro Agropecuario Provincial de la Diputación de Córdoba, the Instituto de la Grasa,

and the PAIDI AGR 218 research group.

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Table 1. Chemical composition (g/100 g) and glycosyl residue composition (% molar
ratio) of Pectoliv-80A.
Compound Quantity (mean ± SD)
Uronic acid 29.93 ± 0.92
Neutral sugar 30.22 ± 0.92
Phenol 10.93 ± 0.32
Rhamnose 5.82 ± 0.36
Arabinogalactan 23.11 ± 0.28
Xylose 2.12 ± 0.05
Mannose 1.82 ± 0.03
Galactose 13.36 ± 0.07
Glucose 3.70 ± 0.05

Table 2. Mean, standard deviation, minimum, and maximum of TM, HOST, ROS,
LPO, and acrosome integrity according to the different treatments considered.

Std.
Treatment Variable Mean Minimum Maximum
Deviation
TM (%) 37.83 11.92 17.00 49.00
HOST (%) 63.28 7.63 51.71 70.44
ROS (MFI) 1011.67 53.90 950.00 1091.00
Control
LPO (MFI) 1007.67 56.18 956.00 1115.00
Acrosome
22.80 8.90 14.14 38.78
integrity (%)
TM (%) 49.17 5.91 42.00 56.00
HOST (%) 71.78 2.59 68.66 75.00
ROS (MFI) 1024.33 132.40 819.00 1150.00
T1
LPO (MFI) 1062.33 97.90 940.00 1164.00
Acrosome
23.42 6.11 18.18 33.33
integrity (%)
TM (%) 39.67 6.87 32.00 49.00
HOST (%) 71.97 3.27 67.91 76.04
ROS (MFI) 1055.50 38.17 999.00 1100.00
T2
LPO (MFI) 1022.17 86.15 889.00 1108.00
Acrosome
30.46 15.77 13.13 52.53
integrity (%)
TM (%) 29.67 7.84 21.00 42.00
HOST (%) 72.16 6.06 65.85 82.63
ROS (MFI) 1043.50 115.32 881.00 1199.00
T3
LPO (MFI) 978.50 84.86 897.00 1138.00
Acrosome
23.40 13.63 10.11 40.00
integrity (%)
Control: 0 μg/mL Pectoliv-80A in semen extender; T1: 300 μg/mL Pectoliv-80A in
semen extender; T2: 400 μg/mL Pectoliv-80A in semen extender; T3: 500 μg/mL
Pectoliv-80A in semen extender.
Table 3. Summary of the results of Pillai’s trace of equality of covariance matrices of
discriminant canonical functions.
F F
Trace (Observed (Critical DF1 DF2 p-value Alpha
value) value)
1.151 2.242 1.856 15 54 0.016 0.05

Table 4. Canonical Discriminant analysis efficiency parameters to determine the

significance of each canonical discriminant function.

Test of Function(s) Wilks' Lambda Chi-square df Sig.

1 through 3 0.195 30.285 15 0,011
2 through 3 0.479 13.607 8 0,049
3 0.924 1.465 3 0,690
df: degrees of freedom.

Table 5. Results for the tests of equality of group means to test for difference in the
means across sample groups once redundant variables have been removed.

Variable Rank Wilks' Lambda F DF1 DF2 p-value

TM (%) 1 0.553 5.384 3 20 0.007
HOST (%) 2 0.623 4.034 3 20 0.021
LPO (MFI) 3 0.862 1.067 3 20 0.385
Acrosome integrity (%) 4 0.921 0.576 3 20 0.638
ROS (MFI) 5 0.962 0.260 3 20 0.853

Table 6. Discriminant loadings for each variable that determine the relative weight of
each trait on each discriminant canonical function.
F1 F2 F3
TM (%) -0.778 0.421 0.174
HOST (%) 0.291 0.818 -0.239
ROS (MFI) 0.170 0.170 0.288
LPO (MFI) -0.412 0.275 0.095
Acrosome integrity (%) 0.082 0.183 0.883
Figure 1. Canonical variable functions and percentages of self-explained and
cumulative variance.

Figure 2. Territorial map depicting the results of the canonical discriminant analysis on
the different treatments.
2

1.5

1
T1
0.5 T2

0 T3
F2

-0.5

-1

-1.5 CONTROL

-2

-2.5
-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3
F1
Figure 3. Cladogram constructed from Mahalanobis distances across different
treatments.

Conflicts of Interest

All the authors involved in this study (Esther Díaz Ruiz, Antonio González Ariza, José
Manuel León Jurado, Ander Arando Arbulu, Juan Fernández-Bolaños Guzmán, Alejandra
Bermúdez Oria, Juan Vicente Delgado Bermejo, and Francisco Javier Navas González)
declare no conflict of interest.