1. Introduction
Semen cryopreservation is an effective method to preserve the genetic resources of different aviary breeds [
1]. This process is currently the only way for the ex situ in vitro storage of avian reproductive cells as the high yolk concentrations in avian eggs do not allow cryopreservation of oocytes [
2,
3]. In this way, one of the fundamental tools for the in vitro conservation of endangered breeds and high genetic value individuals is the development of germplasm banks, which are also a guarantee against the risk of epizootic diseases such as Avian Influenza [
1].
However, the freezing–thawing process causes osmotic changes and the formation of intracellular ice crystals resulting from the imbalance between antioxidant and pro-oxidant defenses which, in turn, increase the proportion of reactive oxygen species (ROS) [
4]. As a consequence, sperm membrane integrity and permeability are compromised and this process ultimately affects fertilization capacity and sperm viability [
5]. The high vulnerability of rooster spermatozoa to membrane peroxidation is due to the large amounts of polyunsaturated fatty acids in the membrane compared to mammals, especially docosatetraenoic acid (22:4n - 6) and arachidonic acid (20:4n - 6) [
6,
7].
To counteract this effect, avian seminal plasma contains several antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, as well as other antioxidant compounds such as vitamins C and E, pyruvate, glutathione, and carnitine [
8].
However, during cryopreservation, lipid peroxidation increases, which implies an attenuation of the aforementioned endogenous system, and the protection provided may be insufficient. In this regard, the addition of exogenous antioxidants is of interest, as there are a large number of antioxidant compounds that differ in their mechanisms of action, toxicity, and effectiveness [
9,
10].
Various plant extracts have antioxidant properties due to their high content of polyphenols, flavonoids, carotenes, gallic acid, tannins, and essential oils, which have major advantages over synthetic antioxidants as they generate fewer residues and cause less cytotoxicity [
11].
Specifically, hydroxytyrosol (3,4-dyhydroxyphenylethanol, HT) is a phenolic compound derived from olive oil (
Olea europea), which has antioxidant effects by reducing low-density lipoprotein oxidation, protecting against H
2O
2 cytotoxicity, and minimizing lactate dehydrogenase activity [
12,
13,
14,
15]. Specifically, this substance is obtained from alperujo olive pulp, which is a by-product generated in the two-phase system used in olive oil extraction [
16]. Given that the sustainable production of olive oil is currently a matter of general interest, and even more so in the region of Andalusia (southern Spain) where this sector is very strong, this extraction system represents a step forward in the industrial phase since as through this system, the pollutant load of the wastewater is considerably reduced [
17]. Therefore, the search for alternative uses for this by-product such as its use as an antioxidant would contribute to the optimization of this agricultural process [
18].
The effect of an antioxidant can be measured across multiple semen quality variables. The most important parameters are sperm motility, viability, morphology, and concentration [
19], and the use of statistical tools plays a pivotal role in determining which variables have the most discriminant power. Through discriminant canonical analysis (DCA), all the variables that are deemed appropriate can be considered simultaneously when distinguishing different populations [
20]. DCA is a dimension-reduction technique that is derived from canonical correlation and principal component analysis [
21]. As for principal component analysis (PCA), this statistical tool allows the development of a predictive model from a set of data showing the existing relationships between two or more variables [
22]. However, while PCA aims to explain the maximum amount of variance, the discriminant functions are generated to maximize the difference between groups. Once discriminant functions have been computed, their structure could be examined to identify the different variables that contribute most to the discrimination between the semen-freezing treatments [
23]. However, the complexity of the application of DCA produces considerable confusion among some researchers concerning its assumption, statistical properties, and data requirements [
24].
Alternatively, the chi-squared automatic interaction detection (CHAID) decision tree (DT) method is one of the most used data mining models. This method, belonging to the category of non-parametric methods, allows classification, prediction, regression, estimation, description, visualization, and dimensionality reduction [
25]. However, the use of CHAID DT presents certain methodological problems that should be solved. Model parameters should be specified before or during the application of the given algorithm. However, the main observed problems belong to different processes such as the rules for heterogenous populations splitting into smaller homogenous groups, the stopping criteria of the recursive process, and tree pruning [
26].
Therefore, the present work aimed to determine the antioxidant effect of different HT concentrations on the post-thaw quality of Utrerana endangered avian breed semen using N-methylacetamide (NMA) as a cryoprotective agent. For this, DCA was used to determine the differential clustering patterns of different sperm-freezing treatments according to the antioxidant concentrations and quantify the contribution of semen quality-related traits to each cryopreservation treatment.
4. Discussion
Seminal cryopreservation is an effective method for the conservation of animal genetic resources. However, there are some limitations in avian species because of the high incidences of cryoinjuries, which can lead to lethal and sublethal damage to spermatozoa [
2,
41,
42]. Due to the characteristics of avian spermatozoa, they are highly susceptible to oxidative stress. This leads to DNA damage and decreased motility in the spermatozoa and thus, reduced fertility [
42]. The high production of ROS causes lipid peroxidation of sperm membranes [
43]. To neutralize this effect, the addition of antioxidants to the extenders is helpful.
In this respect, HT is an important phenolic compound present in the fruit of the olive (
Olea europea), which has been isolated from alperujo olive pulp. This substance consists of a simple phenol that possesses a high antioxidant property and performs functions such as reducing the oxidation of low-density lipoproteins, minimizing lactate dehydrogenase activity, and protecting against H
2O
2 cytotoxicity [
12]. Moreover, HT has anti-inflammatory and antibacterial properties [
44]. Although there are studies that have evaluated the efficacy of adding HT in other species such as rats [
45], humans [
46], pigs [
47], rams [
48], and goats [
49], there is no evidence of its testing in roosters. For this reason, studies testing the effect of adding this type of antioxidant to poultry semen extenders could play a pivotal role in the development of poultry reproduction techniques.
However, the preliminary results obtained through descriptive statistics in the present study suggested that there is no improvement in viability, acrosome integrity, glutathione, ROS, LPO, HOST, morphology, motility, and kinematic parameters after the freezing–thawing process in samples with supplementation of different HT concentrations (
Table 1 and
Table 2). Conversely, an improvement of viability, acrosome integrity, HOST, TM, PM, VAP, VSL, VCL, and ALH variables is suggested when comparing the fresh sperm samples with the frozen–thawed samples.
These results were also observed by Arando Arbulu et al. [
49], who reported that there was no improvement in goat frozen semen quality when HT and another minor simple phenol such as 3,4-dihydroxyphenylglycol (DHPG) were added to the extender for semen freezing. However, according to results obtained in other species, HT addition to rat semen extender causes an improvement in sperm viability and motility parameters in fresh epididymal sperm [
45].
An increase in TM has been also reported in rams when a moderate concentration of HT was added to the extender [
50]. In this same species, Arando et al. [
48] observed that supplementation of semen with HT improves LPO values of frozen–thawed samples as well as in the case of DHPG and the mixture of both. Similarly, Li et al. [
51] have reported that the addition of 120 μmol/L of HT during boar semen storage at 17 °C has a positive effect on semen quality.
Even though no significant quality-related effects were observed in the present study, other authors have reported an improvement in the quality of semen from roosters when different levels of olive oil were added to the semen diluent during storage [
52]. In particular, these improvements were noticed in viability, acrosome integrity, and motility parameters. Also, Kacel and Iguer-Ouada [
53] have suggested a positive impact on the viability and motility of rooster semen when the olive oil is supplemented orally. The inclusion of olive oil in the rooster diet has also been reported to produce a decrease in the morphological injuries of the spermatozoa tail [
54]. Moreover, the addition of olive oil to the diet improves gonadal activity, reduces oxidative stress and lipid peroxidation, and promotes nitric oxide signaling, thereby preserving semen quality [
55]. In the same way, Eslami, et al. [
56] suggest that the enrichment of semen with another derivate of the olive such as oleic acid during cooled storage would decrease the negative effects of the lipid peroxidation in rooster seminal plasma and spermatozoa. However, further studies in frozen–thawed semen samples are needed to evaluate the effect of this type of antioxidant on rooster semen quality.
The addition to extender of melatonin [
57], quercitin [
43], L-carnitine [
58], glutamine [
59], and zinc [
60], among others, has shown beneficial effects in HOST variable values obtained in frozen–thawed semen. Moreover, some of these antioxidants also had a positive effect on the motility and viability variables in frozen–thawed semen.
The use of general descriptive statistics is important to provide an overview of the problem and, on this basis, to make decisions on how to address the problem. However, the development of DCA is decisive in differentiating which variables of those studied have a greater weight when evaluating the effect of supplementation of the cryopreservation extender with HT on post-thawing semen quality. After multicollinearity analysis, some variables that could present redundancy problems were eliminated (
Table 3).
The sperm viability variable was discarded due to redundancy problems. Previous authors have reported a high relationship between viability as measured by flow cytometry using SYBR-14 and PI and sperm motility using the CASA system when working with stallion sperm [
61,
62]. For this, measurement protocols must be followed correctly and thoroughly as sometimes, a decline in TM may be reversible and may represent to a certain extent a physiologic rather than a solely pathologic change [
62]. Sperm viability can also show redundancy problems with the morphology variable. There is a relationship between both variables in goat and bovine species since spermatozoa can be observed to be smaller after cryopreservation. In the case of goats, when thawing semen samples, spermatozoa show a smaller head length, width, area, and perimeter, which could also be related to the damage suffered by the sperm membrane after the osmotic differences that occur in the cryopreservation process. This leads to a reduction in the volume of the spermatozoa by changing the permeability of the membrane and thus, the cell content in the dead spermatozoa [
63,
64].
The acrosome integrity parameter was also eliminated in the multicollinearity test. This could be due to redundancy problems with the HOST variable. Semen manipulation, during incubation, refrigeration, and cryopreservation processes produces deterioration and changes in the acrosome and membrane of spermatozoa [
65]. These changes, therefore, could produce changes in sperm motility and sperm survivability [
66]. Thus, acrosome integrity during freezing can be highly correlated with most sperm-quality-related variables. In any case, the effect that an antioxidant may have on the acrosome will depend on the tested animal species and the basic composition of the cryopreservation extender supplemented [
67,
68]. Therefore, further studies in this field for avian species are necessary.
Finally, most of the motility-related variables were discarded since TM can synthesize and capture the variability in all these variables. Specifically, a strong correlation of VCL and VAP parameters with TM has been observed in rooster semen [
69]. Additionally, several kinematic parameters are usually strongly correlated with each other [
70]. Previous studies have related kinematic parameters with sperm morphology. However, the lack of standardization in measurements of motility-related traits of spermatozoa may lead to confusion about the relationship between sperm motility and sperm morphology [
70,
71].
After multicollinearity analysis, of the variables that remain, TM showed the highest discriminant power (
Table 6). This parameter is indicative of sperm functionality and only those spermatozoa with adequate motility are able to move up the female’s oviduct to reach the sperm storage tubules and subsequently, reach the fertilization zone [
72]. Sperm motility after a freeze–thaw cycle in poultry is reduced by 30 to 60% [
73]. Therefore, the use of TM, which is a rapid technique, provides a wealth of information on semen status. Moreover, the economic benefit of purchasing a CASA system is viable since this tool allows an increase in the efficiency of insemination dose production and higher fertility levels [
74]. Therefore, this system is becoming necessary in the daily operation of a poultry breeding center.
HOST showed a high discriminant power. This test can be used to assess the damage in the spermatozoa plasma membrane of spermatozoa due to the loss of permeability that occurs during the cryopreservation process [
75]. In this sense, the state of the plasma membrane could predict the fertilizing capacity of spermatozoa as it is considered a key element in the survival of spermatozoa in the female reproductive tract by acting as a selective barrier between the intracellular and extracellular environment [
76]. However, during the cryopreservation process, spermatozoa are exposed to an increasingly hypertonic medium. Therefore, an imbalance in sperm osmotic exchange occurs. The rupture of the plasma membrane supports the HOST parameter as highly variable and therefore has a high discriminating power [
77].
Finally, ALH was ranked third according to its discriminant power. This parameter corresponds to the maximum width of sperm head oscillation during movement, which facilitates sperm penetration into the zona pellucida of the mature oocyte [
78]. A relationship between this parameter and the total number of piglets born has been observed in porcine species [
79]. In roosters, good results were obtained for the ALH parameter when the straws were frozen at 3 cm above the level of liquid nitrogen, resulting in values that did not differ from those obtained in fresh samples [
80]. Thus, this could be considered the kinematic parameter of greatest interest, since it could act as a reliable predictor of the success of the in vitro fertilization process [
81].
Lastly, in order to observe the differences between the sperm-quality-related traits between the different freezing treatments used in the present study, the CHAID DT was used (
Figure 4). This statistical tool showed a large discriminant power of the ROS variable between the different cryopreserved treatments. In
Figure 4, it can be observed that the highest ROS values are observed in samples without the presence or with the lowest tested concentrations of HT. When ROS production is excessive, the generated mechanical damage, which is unrelated to plasma membrane peroxidation, may not be repairable by the addition of exogenous antioxidants [
82].