Effect of senescence on morphological, functional and oxidative features of fresh and cryopreserved canine sperm

Abstract

The present research aimed to compare the hormonal profile, sperm quality and freezability of young and senile dogs. Dogs were assigned into Young Group (n = 11) and Senile Group (n = 11), additionally divided into Fresh Semen Group and Cryopreserved Semen Group. Males were evaluated for libido score and blood estrogen and testosterone assay. Sperm morphofunctional evaluations were performed based on Computer Assisted Sperm Analysis, morphology, mitochondrial activity, mitochondrial membrane potential, plasma and acrosomal membrane integrity, and DNA fragmentation. Sperm oxidative features were: protein oxidation, lipid peroxidation and production of advanced glycation end-products. Young dogs had higher libido score, sperm velocity average pathway, linearity of motility and mitochondrial activity index and lower percentage of major defects, total defects and proximal cytoplasmic droplet, despite the lack of difference between hormone profile of aged dogs. Fresh semen of senile dogs had increased percentage of spermatozoa with high mitochondrial membrane potential compared to young dogs and to cryopreserved sperm. Cryopreserved semen of young dogs had higher acrosomal membrane integrity compared to the Senile Group. In conclusion, sperm of aged dogs have reduced quality, signaled by higher morphological defects, ultimately altering sperm mitochondrial function and sperm kinetics. Furthermore, spermatozoa from senile dogs are more sensible to cryoinjury.

Keywords:

• Sperm mitochondrial potential
• acrosome integrity
• sperm cytoplasmic droplet
• aging
• dogs

Introduction

Animal longevity has increased significantly in the past years due to the remarkable advance in several areas of veterinary medicine. Hence, increasingly number of senile dogs is being used as breeders. On the other hand, the odds of senility are relatively unknown and insufficiently researched, hindering the reproductive approach to the geriatric patient [1]. Thus, it became challenging to understand reproductive features of senile animals, as for example characterize sperm functional changes. Therefore, the study of reproductive senescence in dogs is necessary not only to guarantee the reproductive success of elderly dogs, but as a model of studies for the reproductive aging man [2].

In animal reproduction, a decrease in fertilizing capacity and changes in seminal quality has been observed according to aging. Senescence has an apparent effect on testicular function in humans and there are evidences that spermatogenesis is incomplete in senile dogs [3]. In men, decreased sperm quality due to pathological or natural causes of aging has also been observed, setting an important factor in male infertility [4]. In senile dogs, in turn, lower percentage of normal spermatozoa in the ejaculate has been described previously [5].

The effect of men senescence has been studied in assisted reproduction technologies [1], attesting that the rate of births from in vitro fertilization and the rate of embryo implantation by intracytoplasmic sperm injection decrease significantly according to aging, more importantly in non-obstructive azoospermic men [6–8]. Nevertheless, the effect of male reproductive senescence remains controversial, especially regarding assisted reproduction [9]. For seminal cryopreservation, the consequences of aging are not well known, despite being considered an important step for assisted reproduction. Therefore, researches related to sperm cryopreservation in elderly dogs can provide, as experimental model, information for understanding the effects of reproductive senescence in assisted reproduction technologies.

Several theories related to biological aging have been developed and the common consensus is the description of a progressive loss of functionality according to aging, with consequent increase in disease susceptibility and incidence of several disorders [10]. Regardless of genetic influence, random (stochastic) factors can also be determinant for longevity, suggesting that the functional loss in aging results from the occasional accumulation of lesions, associated with environmental effects, causing progressive physiological decline [10]. Among the stochastic factors, there are different biochemical theories of the temporal influence, including the glycation process (i.e. Maillard reaction) and its subproducts (Advanced Glycation End-products—AGEs) [11] and oxidative stress. Therefore, experiments are required in order to clarify the influence of such processes on the reproductive aging in the canine species.

In light of the foregoing, the study on sperm parameters of senile dogs may contribute to the knowledge of the mechanisms involved in reproductive aging physiology and pathogenesis. Thus, the evaluation of sperm morphofunctional and oxidative features were selected for the study of the aging process in dogs. Moreover, the increase in knowledge on sperm cryopreservation of aged dogs can contribute to create alternatives for the maintenance of reproductive potential during senescence. Therefore, we aimed to compare the hormonal profile, sperm quality and freezability of young and senile dogs.

Materials and methods

This project was carried out in compliance with the standards established by the Bioethics Committee of the School of Veterinary Medicine and Animal Science—University of São Paulo, under protocol number 7045270115.

Animal and experimental groups

Twenty-two dogs were assigned into two experimental groups according to age: Young Group (n = 11) and Senile Group (n = 11). Each group was additionally divided into Fresh Semen Group and Cryopreserved Semen Group. Because the body mass index may influence sperm quality [12], the body weight of each dog was taken into account to classify the experimental groups, i.e. small dogs were considered senile with more than 8 years, medium dogs with more than 7 years old and large dogs with more than 6 years [13]. Young dogs, regardless of their size, were considered between 1 and 5 years of age.

Estrogen and testosterone serum assay

To evaluate serum concentration of testosterone and estrogen, dogs were submitted to an endocrine stimulation test [14] by an intramuscular injection of 0.2 μg kg⁻¹ of GnRH (Prorelin^®—Buserelin 4 μg mL⁻¹) during the morning period. After 1 h of the GnRH treatment, a single blood sample was collected by venous puncture into vacuum tubes without an anticoagulant. The tubes were then centrifuged for 10 min at 1500 × g, and serum was separated in aliquots of 0.5 mL and stored at −20 °C until hormone analysis.

Hormonal assays were performed with the use of commercial radioimmunoassay kits for quantitative measurement of testosterone and estrogen (Beckman Coulter^®), previously validated for dogs [15,16]. For estradiol, the intra-assay coefficients were 7.56% (high intra-assay) and 3.53% (low intra-assay) and, for testosterone, the low intra-assay coefficient was 4.52%. All reactions were performed in duplicate.

Semen evaluation and cryopreservation

A single semen sample was collected from each dog by digital manipulation of the penis, and an aliquot of the sperm-rich fraction was immediately analyzed (fresh semen), whereas the remainder sample was subjected to cryopreservation (cryopreserved sperm).

To analyze male libido, we exposed dogs to vaginal swabs of females in heat (proestrus or estrus) during semen collection. Thus, libido was scored on an arbitrary scale of 0 to 3, being 0: total failure of penile erection and sexual disinterest; 1: transient sexual interest without erection during manipulation of the penis and prepuce; 2: constant sexual interest and partial erection of the glans penis during prepuce manipulation; 3: Rapid erection and pelvic movements during prepuce manipulation. All dogs had no previous experience with semen collection by digital manipulation, therefore, libido subjective score was not biased by a reproductive conditioning.

Immediately after collection, the sperm fraction was evaluated for volume, color, aspect and sperm concentration. Subsequently, sperm morphological and functional analysis was performed. The third fraction of the ejaculate, corresponding to the prostatic fluid, was collected into plastic microtubes and maintained at −20 °C until analysis.

A single sperm sample from each dog was submitted to the one step cryopreservation protocol, using a 5% glycerol-based Tris-yolk-citric acid extender, according to previously described methodology [17]. We employed a single freezing-thawing cycle per dog. After at least 1 week, samples were thawed at 37 °C for 30 s and immediately evaluated.

Sperm motility evaluation was performed using the Computer Assisted Sperm Analysis (CASA, Hamilton-Thorne Ivos 12.3), with 10 µL of each seminal sample deposited between glass slide and cover slip, previously heated at 37 °C. Seven fields of view were randomly selected and evaluated according to the different motile patterns [18]: velocity average pathway (VAP; μm s⁻¹), curvilinear velocity (VCL; μm s⁻¹), velocity straight line (VSL; μm s⁻¹), amplitude of lateral head displacement (ALH; μM), beat cross-frequency (BCF; Hz), straightness (STR; %), linearity (LIN; %), percentage of motility and progressive spermatozoa. In addition, sperm population was classified into four groups based on speed: rapid, medium, slow and static (%).

For sperm morphology evaluation, we used the wet preparation technique assessed in samples fixed in 10% formol saline solution under phase-contrast microscopy at 1000× magnification [19], which classified spermatozoa, expressed as a percentage, into normal, with minor or major defects, as well as the specific detailing of the presence of proximal or distal cytoplasmic droplets.

To evaluate the integrity of the sperm plasma membrane, eosin/nigrosin staining was performed, allowing differentiation of cells with changes in plasma membrane permeability [20]. In brief, 5 μL of semen and 5 μL of the previously prepared eosin–nigrosin stain were placed in a prewarmed glass slide. The sperm smear was evaluated under light microscopy by counting 200 cells at 1000× magnification. The Fast-Green/Rose-Bengal staining was used to evaluate the acrosomal membrane integrity of spermatozoa [21]. Briefly, 5 μL of semen was mixed with 5 μL of fast green/rose bengal stain in a warmed glass slide. Smears were evaluated under light microscopy at 1000× magnification. For the analysis of sperm DNA integrity, the technique of toluidine blue staining was used [22]. Smears were evaluated under light microscopy at 1000× magnification. Damaged sperm (DNA fragmentation) were stained blue, while intact sperm (DNA integrity) were stainless.

Aiming to evaluate mitochondrial sperm activity, the cytochemistry technique of 3,3'-diaminobenzidine (DAB) was used, which provides an index of sperm mitochondrial activity into four classes: high sperm mitochondrial activity with 100% of the mid‐piece stained indicating full mitochondrial activity; medium sperm mitochondrial activity with more than 50% of the mid‐piece stained; low sperm mitochondrial activity with <50% of the mid‐piece stained; and absence of sperm mitochondrial activity with no staining in the mid‐piece [23].

The flow cytometry technique (BD FACSCalibur, Becton Dickinson, East Rutherford, NJ, USA) with sperm staining JC-1 fluorescent probe (5,5′,6,6′-tetrachloro1,1′,3,3′-tetramethylbenzimidazolylcarbocyanine iodide) was used to evaluate the mitochondrial membrane potential. About 1 μL of the JC-1 probe (50 μg mL⁻¹) was added to 188,000 diluted spermatozoa in 37.5 μL TALP. Samples were incubated at 37 °C for 10 min and then 300 μL of TALP were added and then sample was was analyzed by flow cytometry. The sperm population was selected by red fluorescence and then evaluated in the dot plot for yellow fluorescence. Sperm were separated into two populations: high mitochondrial membrane potential and low mitochondrial membrane potential. In spermatozoa with high mitochondrial potential, JC-1 formed complexes known as J-aggregates, with intense red fluorescence. For sperm with low mitochondrial membrane potential, JC-1 remained in the monomer form, which appears only as green fluorescence. Samples without dye (white) were used for proper separation of the spermatozoa in the graph, preventing considering other particles.

Evaluation of sperm and seminal plasma oxidative profile

The evaluation of sperm lipid oxidative stress susceptibility was performed by TBARS (Thiobarbituric Acid Reactive Substances) assay [24], by submitting sperm to a challenge with a reactive oxygen species (ROS) generating system. In brief, 0.5 mL of the previously prepared sperm suspension containing 1 × 106/mL of Tyrode’s albumin lactate pyruvate (TALP) was incubated with ferrous sulfate (125 μL, 4 mM) and sodium ascorbate (125 μL, 20 mM) for 1.5 h at 37 °C. Subsequently, 10% trichloroacetic acid at 4 °C was added, and the mixture was centrifuged (18,000 × g, 15 min) to promote the precipitation of proteins. Analysis was performed by mixing 500 μL of the previously prepared supernatant with 500 μL of 1% (TBA 1% diluted in 0.05 M sodium hydroxide) in a water bath kept at 90 °C to 100 °C for 15 min and then immediately cooled in an ice bath (0 °C) to interrupt the chemical reaction. The TBARS were quantified spectrophotometrically at a wavelength of 532 nm (Ultrospec 3300 Pro, Amersham Biosciences), as previously described. The lipid-peroxidation index was described as nanograms of TBARS/10⁶sperm.

To evaluate the protein oxidation of canine spermatozoa and seminal plasma, the protocols described previously [25,26] were employed, using 100 μL of seminal plasma or 20 million spermatozoa diluted in PBS. Trichloroacetic acid (100 μL of 20% TCA) was added and the mixture was then centrifuged for 10 min at 20,817 ×g and the supernatant discarded. Subsequently, 500 μL of 2,4-dinitrophenylhydrazine (10 mM) and 500 μL of 2 M HCl were added to the semen sample and blank sample, respectively. The mixtures were kept at 37° C for 1 h shaking every 15 min. Then 500 μL of 20% TCA was added, centrifuged for 5 min at 20,817 × g and the supernatant discarded. Subsequently, 1 mL of ethyl acetate/ethanol (1:1) was added and the mixture was centrifuged at 20,817 × g after 10 min. The pellet was resuspended in 2 mL of 1 M NaOH and the maximum absorbance generated was analyzed at 380 nm wavelength spectrophotometer (Ultrospec 3300 pro^®).

The advanced glycation end-products (AGEs) assay followed the protocol proposed by Schmitt, Schmitt [27], in which seminal plasma samples (2 mL) were placed in glass cuvettes and subjected to the analysis of a spectrofluorometer Fluorolog 3 (Jobin Ivon^®) excited at 330 nm wavelength. Results were expressed in wavelength (nm), directly proportional to the AGEs concentration.

Statistical analysis

Data were evaluated according to a factorial 2X2, with the following factors: age (young vs. senile) and semen processing (fresh vs. cryopreserved) by SAS System for Windows (SAS, 2000). Data were tested for normality of the residues (normal distribution) and homogeneity of variances. Whenever one of these assumptions was not accepted, data were transformed. If normality was not obtained, the NPAR1WAY procedure for non-parametric analysis of variance was used. For parametric data, the Least Significant Differences (LSD) test was used for comparison between groups. Additionally, response variables were submitted to Pearson and Spearman correlation tests for parametric and non-parametric variables, respectively.

A probability value of p < .05 was considered statistically significant. Results are reported as untransformed means ± SEM.

Results

Statistical interaction was detected between the age group of the dogs (Young vs. Senile) and semen processing (Fresh vs. Cryopreserved) only for the variables: acrosomal membrane integrity, high mitochondrial membrane potential, low mitochondrial membrane potential and sperm oxidation of protein.

No differences between groups were verified for estrogen assay (3.58 ± 0.79 pg mL⁻¹—Young Group and 6.60 ± 1.54 pg mL⁻¹—Senile Group) and testosterone assay (1.85 ± 0.26 ng mL⁻¹—Young Group and 2.79 ± 0.47 ng mL⁻¹—Senile Group). Moreover, we did not observe differences between groups for the following variables: ejaculate volume and aspect, sperm concentration, protein oxidation and advanced glycation end-products (AGEs) analyzed exclusively in the seminal plasma.

Young dogs presented higher libido score (2.91 ± 0.09) compared to senile dogs (2.09 ± 0.25). The semen of young dogs presented higher velocity average pathway (VAP) and linearity of motility compared to aged dogs, regardless of seminal processing (Figure 1A). Mitochondrial activity index was higher in the semen of young dogs in comparison to senile dogs (Figure 1B).

Figure 1. (A) Sperm linear motility (%), velocity average pathway—VAP (μm s⁻¹) and (B) mitochondrial activity index (%) in the Young and Senile groups. *Difference between groups (p < .05).

The fresh semen of senile dogs had increased percentage of spermatozoa with high mitochondrial membrane potential compared to young dogs (Figure 2A) and to cryopreserved semen (Figure 2B).

Figure 2. Sperm high mitochondrial membrane potential (%) in (A) fresh semen of the Young and Senile Groups and (B) in fresh and cryopreserved semen of the Senile Group. *Difference between groups (p < .05).

Cryopreserved semen of the Young Group presented higher acrosomal membrane integrity compared to the Senile Group (Figure 3). For both groups, fresh semen had higher acrosomal membrane integrity compared to post-thaw sperm (Figure 3). Considering the sperm morphofunctional analysis, young dogs had lower percentage of major defects, total defects and proximal cytoplasmic droplet (Figure 4).

Figure 3. Acrosome membrane integrity (%) in fresh and cryopreserved semen of the Young and Senile groups. *Difference between semen processing (p < .05). ⁺Difference between groups (p < .05).

Figure 4. Sperm defects (%) in the Young and Senile groups. *Difference between groups (p < .05).

For the Young Group, protein oxidation of the seminal plasma negatively correlated with linearity of motility (r = −0.63 and p = .04) and the amplitude of lateral head displacement of the spermatozoa—ALH (r = −0.6 and p = .05). Estrogen concentration positively correlated with sperm protein oxidation (r = 0.91 and p = .0001) and negatively with sperm velocity average pathway—VAP (r = −0.73 and p = .01).

For the senile group, protein oxidation positively correlated with the concentration of advanced glycation end-products—AGEs (r = 0.81 and p = .01), both in seminal plasma. Testosterone concentration negatively correlated with the percentage of minor sperm defects (r = −0.64 and p = .03) and with the concentration of the advanced glycation end-products in the seminal plasma (r = −0.77 and p = .02).

Discussion

In the present work we aimed to identify differences on hormonal, morphofunctional, and oxidative profile of fresh and cryopreserved sperm of young and senile dogs. Aged dogs presented adequate synthesis of testosterone and estrogen in response to exogenous stimulation with GnRH, thus demonstrating minimum influence of age on gonadal steroidogenesis. On the other hand, senile dogs presented lower libido score, although sexual interest and erectile function is directly related to testosterone concentration [28]. Therefore, in spite of the gonadal effectiveness in producing or converting testosterone and estrogen, it is not possible to rule out the existence of an imbalance in steroidogenic activity along the circadian cycle in elderly dogs. In fact, La Vignera et al. [29] showed that reduced number of high amplitude LH pulses in elderly men may be related to infertility, demonstrating that the levels of serum testosterone does not stand alone in male fertility. Furthermore, the imbalance in the expression of estrogen and androgen receptors at male reproductive tissue level appears to cause sperm dysfunction [30]. Hence, aged dogs may present an alteration in hormonal receptors at target organs, reducing the hormone response even under adequate serum concentration. For such statement, further studies on the quantification of tissue receptors for the main steroidogenic hormones are necessary.

In senile animals, we observed a negative correlation between testosterone concentration and the percentage of minor sperm defects and the concentration of advanced glycation end-products (AGEs). This result reinforces the importance of adequate circulating levels of testosterone for the maintenance of spermatogenesis and sperm maturation, especially in senescent dogs. Conversely, in young dogs, estrogen concentration positively correlated with sperm protein oxidation and negatively correlated with sperm velocity average pathway (VAP), indicating a possible detrimental effect of high estrogen concentration. Despite the fundamental role of estrogen in male reproductive function, the imbalance between hormone levels (estrogen and testosterone) is considered an important cause of spermatogenesis failure [30], even in young animals. In fact, the enzyme cytochrome P450 aromatase, responsible for the conversion of androgens into estrogen, is a content of the sperm cytoplasmic droplet and intermediate piece [30]. Therefore, we infer that the cytoplasmic droplet is responsible for the increase in sperm estrogenic concentration, and both factors lead to seminal alterations, such as oxidative stress (protein oxidation) and kinetic alteration of the spermatozoa.

In aged men, a reduction in seminal quality has been described, especially for sperm motility, morphology [31], semen volume, progressive motility, concentration, and total sperm count, negatively affecting reproduction capacity [32]. The overall results of our experiment also allow such observation for the canine species, as we detected an increase in sperm defects (mainly proximal cytoplasmic droplet) in aged dogs. In fact, dogs produce ejaculate with lower percentage of normal spermatozoa during the progression of senility [5], which is related to a failure in spermatogenesis or sperm maturation [33]. Therefore, we can affirm that senile dogs present alterations in sperm production, decreasing seminal quality by increased structural sperm defects.

Interestingly, young dogs had lower sperm mitochondrial membrane potential, although with a higher mitochondrial activity index compared to the Senile Group. Both evaluation parameters refer to mitochondrial function, however, the mitochondrial membrane potential is related to sperm energetic status, characterizing cellular metabolism [34]. Whenever mitochondrial membrane potential is high, it may be caused by a low production of ATP, leading to a compensatory mitochondrial mechanism in an attempt to increase energy production [35]. In men, sperm motility is independent of mitochondrial membrane potential [36]. Similarly, we noticed that semen of young dogs present higher velocity average pathway (VAP) and greater linearity of motility, despite lower mitochondrial membrane potential, compared to senile dogs. Thus, senility may interfere with sperm mitochondrial activity and, as a consequence, with sperm motility pattern, notwithstanding higher cellular metabolism. In addition, senile dogs had higher percentage of spermatozoa with cytoplasmic droplet, which we assume to be responsible for reduced mitochondrial activity and, conversely, increased mitochondrial membrane potential. Mitochondria is the main source of energy for sperm motility and homeostasis, through oxidative phosphorylation [37]. Therefore, the presence of a sperm midpiece proximal droplet can hinder oxidative phosphorylation by the mitochondria and suppress the action of metabolic enzymes responsible for catalyzing anaerobic glycolysis [38].

In the present study, we verified that cryopreserved sperm of senile dogs had increased acrosomal membrane damage compared to young dogs, demonstrating that senility sensitizes sperm acrosome to cryoinjury. Cryopreservation process causes acrosomal membrane changes, triggering early sperm capacitation [39,40]. Therefore, the effect of senility on sperm is represented by changes in the acrosomal membrane, especially on cryopreserved semen, significantly reducing its fertilizing potential.

Interestingly, we have not found differences on sperm oxidative stress between young and senile dogs, although oxidative stress has been suggested to be the main cause of sperm DNA damage in aged men [9,41,42]. However, dog senescence is associated with reduced quality of epididymal semen [43]. Therefore, we suggest that the reproductive consequence of canine senility is more related to molecular changes at spermatogenesis and sperm maturation levels.

In conclusion, despite the lack of steroidogenic hormone (testosterone and estrogen) imbalance and sperm oxidative stress, spermatozoa of aged dogs have reduced quality, signaled by higher morphological defects, mostly proximal cytoplasmic droplets, ultimately altering sperm mitochondrial function and sperm kinetics. Furthermore, spermatozoa from senile dogs are more sensible to cryoinjury, mainly in regards to acrosomal membrane damage, attesting the need to freeze canine semen ideally at reproductive maturity age. Hence, further studies should be performed with the main goal of comparing hormone expression (LH, estrogen and testosterone) at testicular and epidydimal level of young and senile dogs.

Acknowledgements

The authors acknowledge Prof. Marcilio Nichi, Nicolle Gilda Teixeira Queiroz-Hazarbassanov, Prof. Cristina de Oliveira Massoco, Prof. Maria Helena Bellini, Camilla Mota, Prof. Mayra Elena Assumpção, Priscila Viau, Prof. Claudio Alvarenga and Prof. Ricardo Pereira for their valuable methodological support.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo, (2015/05419–5).

Notes on contributors

Maíra Morales Brito

All authors carried out the experiment. Maíra M. Brito, Daniel S. R. Angrimani and Camila I. Vannucchi analyzed the data. All authors drafted the article.

Daniel de Souza Ramos Angrimani

All authors carried out the experiment. Maíra M. Brito, Daniel S. R. Angrimani and Camila I. Vannucchi analyzed the data. All authors drafted the article.

Giulia Kiyomi Vechiato Kawai

All authors carried out the experiment. Maíra M. Brito, Daniel S. R. Angrimani and Camila I. Vannucchi analyzed the data. All authors drafted the article.

Camila Infantosi Vannucchi

All authors carried out the experiment. Maíra M. Brito, Daniel S. R. Angrimani and Camila I. Vannucchi analyzed the data. All authors drafted the article.