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

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 6 different replicates) were divided into 4 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.

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 2-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).

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

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 2 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 2 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 2 mo (April and May 2022).

For the development of this study, a total of 16 Utrerana breed roosters aged between 1 and 3 yr 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 wk. However, this cycle may extend until 156 wk 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 (≥3 × 10⁹ 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 min, 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 4,700 g (Comteifa, S.L., Barcelona, Spain) with subsequent ultrafiltration of the liquid phase at 3,000 Da, the liquid phase above 3,000 Da being concentrated at 2L 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 3,000 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 1 h at a temperature reduction rate of 0.3°C/min. The pool was divided into 4 aliquots which were diluted with a diluent containing different concentrations of Pectoliv-80A to obtain 4 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 2 parts as the dilution was carried out in 2 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 min, 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 × 10⁶ spz/straw. Thirty 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) 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

The reactive oxygen species (ROS) were measured with the commercial kit DCFH-DA (Sigma-Aldrich, St. Louis, MO). For this, 1000 µL of semen (4×10⁶ 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 2,600 rpm for 5 min, the supernatant was removed and 1,000 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 (20×10⁶ spz/mL) and 10 µL of C11-BODIPY^581/591 (10 µM) were incubated in a cytometer tube for 30 min at 37°C in the dark. The sample was then centrifuged at 2,600 rpm for 5 min, the supernatant was removed and 1,000 µL of cytometer fluid was added for reading. Sperm that emitted at the green wavelength (FL1) were considered positive for C11-BODIPY^581/591.

Acrosome Integrity

For the assessment of acrosome integrity, the CyFlow Cube 6 Cytometer was also used. Thus, 300 µL of semen (20×10⁶ spz/mL) together with 15 µL of fluorescein isothiocyanate-conjugated peanut agglutinin (PNA-FITC, 100 µg/mL; Sigma-Aldrich) and 30 μL of propidium iodide (PI; 6 µM) were deposited in a cytometer tube. Incubation was carried out in the dark for 5 min 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 5,000 (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, 6 explanatory variables were included; 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) 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:

$$VIF=\frac{1}{1-R^2},$$

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:

$$D_{ij}^2=\left({\overline{\mathrm{Y}}}_i-{\overline{\mathrm{Y}}}_j\right)CO{V}^{-1}\left({\overline{\mathrm{Y}}}_i-{\overline{\mathrm{Y}}}_j\right),$$

where $D_{ij}^2$: distance between population i and j; COV⁻¹: inverse of the covariance matrix of measured variable x; ${\overline{\mathrm{Y}}}_i$ and ${\overline{\mathrm{Y}}}_j$: 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) 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.

Table 2.

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

Treatment	Variable	Mean	Std. Deviation	Minimum	Maximum
Control	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
	LPO (MFI)	1007.67	56.18	956.00	1115.00
	Acrosome integrity (%)	22.80	8.90	14.14	38.78
T1	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
	LPO (MFI)	1062.33	97.90	940.00	1164.00
	Acrosome integrity (%)	23.42	6.11	18.18	33.33
T2	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
	LPO (MFI)	1022.17	86.15	889.00	1108.00
	Acrosome integrity (%)	30.46	15.77	13.13	52.53
T3	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
	LPO (MFI)	978.50	84.86	897.00	1138.00
	Acrosome integrity (%)	23.40	13.63	10.11	40.00

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.

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 3 discriminant functions revealed after discriminant analyses, 2 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).

Table 3.

Summary of the results of Pillai's trace of equality of covariance matrices of discriminant canonical functions.

Trace	F (Observed value)	F (Critical value)	DF1	DF2	P-value	Alpha
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.

Figure 1.

Canonical variable functions and percentages of self-explained and cumulative variance.

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 2 traits that contributed significantly (P < 0.05) to the discriminant ability of significant discriminant functions.

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 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.

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

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 2 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.

Figure 2.

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

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).

Figure 3.

Cladogram constructed from Mahalanobis distances across different treatments.

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 2 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 2 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 to 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 nonradicals (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 2 variables that provided a large amount of information and showed a great discriminant power when we differentiate between the 4 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.