Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation
Contents
Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer-assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.
1 INTRODUCTION
Buffalo spermatozoa are more vulnerable to cryopreservation than cattle sperm, a fact that interferes directly with fertility potential (Ahmad, Anzar, Shahab, Ahmad, & Andrabi, 2003). This feature can be attributed to specific biochemical factors, such as differences in the lipid composition of plasmalemma (Andrabi, 2009) and in the seminal plasma proteins between these species (Singh et al., 2014). Additionally, more marked negative effects on seminal plasma of buffaloes can be related to low concentrations of natural antioxidants, which can be reduced even further during dilution and cryopreservation (Kumar, Jagan Mohanarao, Arvind, & Atreja, 2011). The removal of seminal plasma has become an option to improve the quality of semen in different species, including buffaloes (Goyal, Tuli, Georgie, & Chand, 1996), cattle (Bergeron, Crête, Brindle, & Manjunath, 2004), sheep (Luna et al., 2015) and horses (Kareskoski & Katila, 2008). In stallions, seminal plasma is removed routinely by centrifugation because of the large volume of seminal plasma characteristic of these animals. However, some authors report deleterious effects of centrifugation on sperm quality, especially sperm motility, due to the damage caused by the mechanical stress (Macpherson et al., 2001).
One alternative to centrifugation is the removal of seminal plasma by filtration using the SpermFilter® (Ceafepe Tecnologia Veterinária Ind., Sorocaba, São Paulo, Brazil). In horses, the use of filtration has yielded total motility, sperm morphology and sperm recovery rates similar to those of centrifugation, resulting as an option in laboratories or farms that do not possess a centrifuge (Roach et al., 2016). In cattle, the filtration device provided better results of freezability and in vitro embryo production and has become an efficient option in insemination centres (Campanholi et al., 2017).
Besides the controversial effects of plasma removal, this method has been few studied in buffaloes, in which ejaculates are submitted to cryopreservation. In addition, filtration (SpermFilter®) has not been reported in this species and its effect on buffalo spermatozoa is unknown. Therefore, this study tested the hypothesis that the removal of seminal plasma by filtration improves the quality of cryopreserved buffalo semen, causing less damage to spermatozoa than centrifugation. For this purpose, the centrifugation and filtration techniques were compared to a group not submitted to seminal plasma removal by evaluating the functional and kinetic characteristics of spermatozoa.
2 MATERIALS AND METHODS
The study was approved by the Ethics Committee on Animal Use of the Faculty of Agrarian and Veterinary Sciences, UNESP, Jaboticabal, (Protocol No. 12072/14).
2.1 Collection and processing of fresh semen
The samples were collected from February to April 2015 (winter in the Amazon region) at the Biotechnology Center of Animal Reproduction (CEBRAN/UFPA), in Castanhal, Pará, Brazil (1°18′18″ S and 47°56′36″ W). The region is characterized by a tropical humid climate with a mean temperature of 26°C, annual rainfall of 2,300–2,800 mm and monthly rainfall of 67–399 mm (Alvares, Stape, Sentelhas, Gonçalves, & Sparovek, 2013).
Semen from seven Murrah buffalo bulls (Bubalus bubalis) aged between 4 and 6 years and maintained under uniform nutritional conditions were collected once a week for 2 months with an artificial vagina. Five to eight ejaculates were obtained per bull, totalling 141 ejaculates. Immediately after collection (maximum two min), semen samples were evaluated in the laboratory nearby 25 m to the collection centre. Samples used in this trial had the following criteria (CBRA, 2013): concentration: >300 million/ml, sperm vigour: >3, sperm motility: >70% and morphologically normal spermatozoa above 70%. Each ejaculate was diluted 1:3 (semen:extender) in egg yolk and glycerol extender (Botu-Bov®, Botupharma, Botucatu, Brazil), and the samples were divided into three groups: control group (with seminal plasma) and two groups submitted to seminal plasma removal by centrifugation (CE) and filtration (F).
For the control samples, semen was diluted conventionally at a concentration of 30 × 106 spermatozoa/dose in Botu-Bov® extender. For samples of the CE group, the semen was transferred to 15-ml Falcon tubes and centrifuged (Damon IEC HN-SII LabCentrifuge, Minnesota, USA) at 600 g for 10 min at room temperature (Della'Aqua, Papa, Alvarenga, & Zahn, 2001). Seminal plasma was removed by discarding the supernatant, and the pellet was resuspended in Botu-Bov® at the same concentration as the control group. For samples of group F, the semen was filtered using SpermFilter® and Botu-Bov® extender. For this purpose, the semen was added to the membrane of the filter and gentle rotational movements were performed on a Petri dish for approximately 5 min. The spermatozoa retained on the filter were resuspended in Botu-Bov® diluent at the same concentration as the control group, according to the technique described by Ramires Neto et al. (2013).
2.2 Cryopreservation of semen
The diluted semen of the three groups was packaged into 0.25-ml sterile straws at room temperature. The straws were cooled in a MiniTub® refrigerator (Minitub do Brasil, Porto Alegre, Brazil) at rate of 3°C/min until stabilization at 5°C for 4 hr. After this period, the straws were arranged on screens and maintained in an isothermal box filled with 2.5 cm liquid nitrogen at 3.5 cm above the liquid nitrogen vapour for 20 min, following a freezing curve of 20°C/min until the straws had reached approximately-153°C (Graham, 1996). The straws were then directly immersed in liquid nitrogen, placed in racks, identified and stored in a cryogenic container at 196°C until the time of post-thaw analysis.
For the subsequent analyses, the samples of the three groups were thawed in a water bath at 37°C for 30 s using a pool of two straws for analysis.
2.3 Computer-assisted sperm analysis (CASA)
The sperm samples were analysed by CASA in a Hamilton Thorne apparatus (Version 14, Hamilton Thorne Biosciences, Beverly, MA, USA). Briefly, 10 μl semen of each group was placed in a Makler Counting Chamber® (Sefi-Medical Instruments Ltd., Haifa, Israel), previously heated to 38°C. Sperm motility was analysed with the Animal Motility software in five automatically chosen fields in a standardized manner. The following sperm motility parameters were evaluated: percentage of total motility (TM), percentage of progressive motility (PM), percentage of rapid motility (RAP), average path velocity (VAP, μm/s), straight-line velocity (VSL, μm/s), curvilinear velocity (VCL, μm/s), amplitude of lateral head displacement (ALH, μm), beat cross-frequency (BCF, Hz), straightness (STR, %) and linearity (LIN, %).
The equipment was set up for bovine pattern as follows: frames per second: 60 Hz; minimum cell size: 6 pixels; contrast for the cell: 60 pixels; contrast of immobile cells: 60 pixels; minimum VAP mean = <40 μm/s; minimum VAP for progressive cells = <30 μm/s; minimum VSL for slow cells = <20 μm/s; STR: 60%; magnification: 1.89; temperature: 38°C.
2.4 Sperm function tests
After cryopreservation, four sperm function tests were performed. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and the quantity of reactive oxygen species (ROS) were evaluated by flow cytometry (BD LSR Fortessa, Becton Dickinson, Mountain View, CA, USA), equipped with blue (488 nm, 100 mW), red (640 nm, 40 mW) and violet (405 nm, 100 mW) lasers. In each sample, 10,000 sperm cells were analysed at rate of 800 events/s. Hoechst 33342 dye (100 mg/ml) was added to ensure that cellular debris and other particles were excluded from the analysis. Data were analysed with the bd facsdiva software 6.1.
Lipid peroxidation was estimated in a spectrophotometer (UV-vis Ultrospec 3300 Pro Spectrophotometer, Biochrom Ltd., Cambridge, UK) at a wavelength of 532 nm.
2.4.1 Evaluation of plasmalemma and acrosomal membrane integrity
All chemical reagents, unless otherwise mentioned, were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Propidium iodide (PI; P4170), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA, L0770) and Hoechst 33342 (H342; 14533) were used as probes. For analysis, 50 μl H342 (100 μg/ml), 20 μl PI (50 μg/ml) and 0.25 μl FITC-PSA (2 μg/ml) were added to 200 μl of a thawed semen sample diluted in TALP medium to a concentration of 10 × 106 sperm/ml. The mixture was homogenized, incubated for 15 min at 37°C and analysed by flow cytometry (Freitas-Dell'Aqua et al., 2012).
The percentage of four subpopulations of spermatozoa was estimated in this analysis according to positive or negative reactivity of the probes: damaged plasmalemma membrane and intact acrosome (DPMIA), damaged plasmalemma and acrosome membranes (DPAM), intact plasmalemma and acrosome membranes (IPAM), and intact plasmalemma membrane and damaged acrosome (IPMDA).
2.4.2 Evaluation of mitochondrial membrane potential
5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide, (JC-1) and H342 were used as probes for the determination of mitochondrial membrane potential. Briefly, 10 μl JC-1 and 50 μl H342 were added to 300 μl of a thawed semen sample diluted in TALP to a concentration of 10 × 106 spermatozoa/ml. The mixture was homogenized, incubated for 15 min at 37°C protected from light and analysed by flow cytometry (Evenson & Ballachey, 1986). Two subpopulations were identified: high and low mitochondrial membrane potential.
2.4.3 Quantification of reactive oxygen species
The5-(-6)-carboxy-2,7-dichlorodihydrofluoresceindiacetate (DCFDA; D6883, Molecular Probes Inc., Eugene, OR, USA) probe was used to quantify ROS according to Guthrie and Welch (2006). For this purpose, 0.5 μl DCFDA (20 μM), 50 μl PI (50 μg/ml) and 50 μl H342 (100 μg/ml) were added to 500 μl of a thawed semen sample diluted in TALP to a concentration of 1 × 106 spermatozoa/ml. The samples were homogenized and incubated for 60 min at 25°C protected from light. Two subpopulations were observed: ROS-positive total cells and ROS-positive intact cells.
2.5 Evaluation of induced lipid peroxidation
After thawing the straws, 1600 μl PBS was added to each 200 μl semen sample and the mixture was centrifuged (Eppendorf 5804R Centrifuge, Hamburg, Germany) at 800 g for 10 min. The supernatant was discarded and 1600 μl PBS was again added, followed by centrifugation for complete removal of the extender. Next, 50 μl iron sulphate (4 mM) and 50 μl vitamin C (20 mM) were added and the mixture was incubated for 90 min at 37°C. Proteins were precipitated by adding 600 μl of an ice-cold solution of 10% trichloroacetic acid (T 9159) and centrifugation at 18,000 g for 10 min, and 800 μl of the supernatant was removed for the assay.
At the time of the assay, 500 μl 1% TBA (T 5500) dissolved in 0.05 N sodium hydroxide (NaOH; S 8045) was added and aliquots containing this mixture were incubated in a boiling water bath (100°C) for 15 min to permit the reaction of lipid peroxidation. The reaction was stopped by cooling the samples on ice (0°C). TBARS were quantified in a spectrophotometer, and the results were compared to a standard curve prepared with malondialdehyde (10,838-3) (Nichi et al., 2006).
2.6 Statistical analysis
Sperm variables obtained by CASA (MT, MP, RAP, VAP, VSL, VCL, ALH, BCF, STR and LIN), flow cytometry (DPMIA, DPAM, IPAM, IPMDA, HMP, LMP, ROS + IC, ROS + TC) and TBARS assay (TBARS) were previously tested for normality and homoscedasticity. When data were not normal, transformation was used. Data were analysed by ANOVA, using PROC GLM (SAS System for Windows 9.3, 2000). Fixed effect of treatment and random effect of bull were included in the model, and comparisons between treatments were made by Tukey test. Results were expressed as the mean ± standard error of means (SEM). Differences were considered when p ≤ .05, and tendency when .05 < p < .10.
3 RESULTS
Table 1 shows the sperm variables evaluated by CASA. Treatment effect was non-significant (p > .05) for MT, MP, RAP, STR or LIN variables. However, VAP, VSL, VCL and ALH were higher (p < .05) in group CE than in group F, while the control group (with seminal plasma) did not differ from both. BCF was higher in group F and control (p < .01) compared to group CE.
| Variable | Group | p value | ||
|---|---|---|---|---|
| Control (n = 47) | Centrifugation (n = 47) | Filtration (n = 47) | ||
| TM (%) | 69.57 ± 2.03 | 71.92 ± 2.01 | 66.44 ± 2.01 | .16 |
| PM (%) | 59.97 ± 1.83 | 61.13 ± 1.83 | 57.09 ± 1.85 | .28 |
| RAP (%) | 67.17 ± 2.04 | 69.28 ± 2.04 | 63.42 ± 2.06 | .12 |
| VAP (μm/s) | 93.15 ± 1.15ab | 96.65 ± 1.15a | 90.73 ± 1.17b | <.01 |
| VSL (μm/s) | 78.76 ± 0.91ab | 80.33 ± 0.91a | 76.87 ± 0.92b | .02 |
| VCL (μm/s) | 137.39 ± 2.19ab | 144.01 ± 2.21a | 133.35 ± 2.22b | <.01 |
| ALH (μm) | 5.64 ± 0.10ab | 5.96 ± 0.10a | 5.60 ± 0.10b | .02 |
| BCF (Hz) | 17.80 ± 0.30a | 16.67 ± 0.30b | 18.59 ± 0.31a | <.01 |
| STR (%) | 85.25 ± 0.50 | 83.72 ± 0.50 | 85.16 ± 0.51 | .06 |
| LIN (%) | 61.52 ± 0.67 | 59.87 ± 0.67 | 61.56 ± 0.68 | .13 |
- Results are reported as the mean ± standard error.
- Means in the same row followed by different superscript letters differ from one another (p < .05).
- TM, total motility; PM, progressive motility; RAP, rapidly moving spermatozoa; VAP, average path velocity; VSL, straight-line velocity; VCL, curvilinear velocity; ALH, amplitude of lateral head displacement; BCF: beat cross-frequency; STR, straightness; LIN, linearity.
Concerning sperm function after thawing, the percentage of cells with integrity of both membranes (IPAM) was not affected by any treatment (p = 0.19). Besides that, filtration reduced the percentage of sperm cells with plasmalemma damage and intact acrosome (DPMIA) compared to control group, but increased the percentage of cells with both membranes damaged (DPAM). For these two variables, CE group did not differ from F or control groups. On the other hand, centrifugation increased the percentage of sperm with damaged acrosome even with intact plasmalemma (IPMDA) compared to F group. In this case, both groups did not differ from control group (Table 2).
| Variable | Group | p value | ||
|---|---|---|---|---|
| Control (n = 47) | Centrifugation (n = 47) | Filtration (n = 47) | ||
| DPMIA (%) | 47.88 ± 1.99a | 41.30 ± 1.96ab | 40.45 ± 2.01b | .07 |
| DPAM (%) | 15.23 ± 1.06b | 17.43 ± 1.04ab | 22.18 ± 1.07a | <.01 |
| IPAM (%) | 33.74 ± 2.09 | 37.91 ± 2.06 | 32.02 ± 2.11 | .19 |
| IPMDA (%) | 0.34 ± 0.06ab | 0.77 ± 0.05a | 0.28 ± 0.06b | .03 |
| HMP (%) | 35.46 ± 2.23 | 39.51 ± 2.20 | 33.42 ± 2.26 | .14 |
| LMP (%) | 61.63 ± 2.51 | 57.90 ± 2.47 | 61.53 ± 2.54 | .70 |
| ROS + IC (%) | 21.83 ± 4.60b | 56.68 ± 4.53a | 20.96 ± 4.7b | <.01 |
| ROS + TC (%) | 17.94 ± 3.46b | 35.50 ± 3.41a | 16.07 ± 3.50b | <.01 |
| TBARS (ng/ml) | 342.41 ± 29.60 | 342.68 ± 361.16 | 290.09 ± 22.11 | .60 |
- Results are reported as the mean ± standard error.
- Means in the same row followed by different superscript letters differ from one another (p < .05); 0.05 < p < .10 indicates a tendency.
- DPMIA, damaged plasma membrane and intact acrosome; DPAM: damaged plasma and acrosome membranes; IPAM, intact plasma and acrosome membranes; IPMDA, intact plasma membrane and damaged acrosome; HMP, high mitochondrial membrane potential; LMP, low mitochondrial membrane potential; ROS + IC, intact cells producing ROS; ROS + TC, total cells producing ROS.
The removal of seminal plasma by either centrifugation or filtration did not affect mitochondrial membrane potential compared to the control group (p > .05; Table 2). However, centrifugation produced larger amounts (p < .01) of ROS when compared to F group and control group. Nevertheless, neither centrifugation nor filtration increased the degree of lipid peroxidation, as demonstrated by the lack of a difference in the concentration of TBARS between the all groups (p = .60; Table 2).
4 DISCUSSION
From our knowledge, the present study is the first to compare the effects of seminal plasma removal by filtration and centrifugation in buffaloes in sperm kinetics and function, determined by membrane integrity, mitochondrial potential and lipid peroxidation.
Our results of sperm kinetics were satisfactory, and better than others described for buffalo species (Gonçalves et al., 2014; Mandal, Nagpaul, & Gupta, 2003). In our study, motility parameters were not influenced by plasma removal, inversely to reports in equine (Love et al., 2005) and buffaloes (Ahmad et al., 2003), in which a greater sperm motility was found when seminal plasma was absent. Nevertheless, we found greater velocity parameters when removal of seminal plasma was carried out by centrifugation compared to filtration, and we speculate that this effect could be a result of the own method of plasma removal, once is known that centrifugation can hyperactivate spermatozoa (Centola, Herko, Andolina, & Weisensel, 1998). Although high VCL and ALH are positively correlated with fertilization rate (Verstegen, Iguer-Ouada, & Onclin, 2002), we believe that, in our study, this capacity could be at least partially compromised because centrifugation reduced BCF, a feature associated to sperm vigour (Contri, Valorz, Faustini, Wegher, & Carluccio, 2010).
Subpopulations according to membrane integrity were affected differently between treatments. Major percentage of sperm cells (>40%) was found in subpopulation with only plasmalemma damaged, and control enhanced this problem compared to F group, suggesting that components of the seminal plasma in contact with the membrane may increase permeability and thus render vulnerability to cryopreservation (Thérien, Moreau, & Manjunath, 1999).
When both (plasmatic and acrosomal) membranes were evaluated, filtration resulted in greater damage, in accordance to a similar study in cattle (Campanholi et al., 2017). As this method of filtration was developed and efficacy has been demonstrated in stallions (Alvarenga et al., 2012), we infer that differences in seminal characteristics between buffaloes and stallions may have contributed to the results obtained in the present study. However, the reasons for this effect remain unknown.
Finally, acrosomal damage not altering plasmalemma integrity was more evident in semen samples submitted to centrifugation, indicating an anticipation of acrosome reaction. In a study on human sperm, acrosomal responsiveness was found to be increased when seminal plasma was removed (Cross, 1993).
The removal of seminal plasma did not interfere with the mitochondrial membrane potential. In the present study, the percentage of cells with a high mitochondrial membrane potential was lower than that of cells with a low mitochondrial membrane potential after cryopreservation, in agreement with other reports on buffaloes (Kadirvel, Periasamy, & Kumar, 2012; Mohan & Atreja, 2014).
As expected, centrifugation increased production of ROS, corroborating the findings of Guimarães et al. (2014), who centrifugated bovine semen samples on a continuous Percoll density gradient. Similar results have been reported for human semen which requires the use of centrifugation to select faster spermatozoa, but an increase in ROS levels is observed after the procedure (Shekarriz, DeWire, Thomas, & Agarwal, 1995).
Despite the observation of higher ROS production due the centrifugation, no increase in lipid peroxidation after cryopreservation was detected in groups submitted to seminal plasma removal, regardless of the technique used (centrifugation or filtration). This increase in ROS may have been caused by the mechanical force of centrifugation rather than by the absence of seminal plasma. In addition, the increase in ROS levels in the CE group was not sufficient to cause injuries to the sperm cell membrane, as demonstrated by the TBARS results that did not differ between groups.
In conclusion, the hypothesis that the removal of seminal plasma causes less damage to spermatozoa and improves the quality of buffalo semen was not confirmed, as this procedure did not improve sperm kinetics, did not reduce the percentage of damage to plasmalemma and acrosomal membrane and did not permit an increase in mitochondrial membrane potential. Similarly, we could not confirm the hypothesis that filtration is less damaging to cells than centrifugation. Although the filtration method has advantages over centrifugation by reducing the production of ROS, its application to buffalo semen is not justified because the results are similar to those obtained with the method using seminal plasma.
ACKNOWLEDGEMENTS
We also thank Botupharma (especially Dr. José Antônio Della'Aqua Júnior) for providing the extender and SpermFilter®; Central de Biotecnologia de Reprodução Animal (CEBRAN); Instituto de Zootecnia for CASA analysis (FAPESP Grant: 2012/05555-8) and for the statistical analysis; Departamento de Radiologia e Reprodução Animal, FMVZ–Unesp, Botucatu, for permitting the use of the flow cytometer, and Departamento de Reprodução Animal, FMVZ-USP, for execution of the TBARS assay.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
Albuquerque conducted the entire experiment, drafted and submitted the manuscript; Morais, Reis and Miranda participated in the first stage of the experiment (semen collection); Ribeiro and Monteiro performed CASA analysis; Paz was responsible for statistical analysis; Nichi and Kawai performed TBARS assay; Della'Aqua and Papa worked with flow cytometry, Viana and Miranda edited the manuscript; Gimenes and Monteiro were involved in the experimental design, analysis of data and edited the manuscript.
