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ISSN: 2574 -1241
ISSN: 2574 -1241
Received: August 10, 2023; Published: August 18, 2023
*Corresponding author: Jose Adelson Alves Do Nascimento, Afya Faculdade De Ciências Médicas Garanhuns, Brazil
DOI: 10.26717/BJSTR.2023.52.008232
Sheep farming in the Northeast region of Brazil is an important component of the local economy. The objective of this work was to test the effect on seminal quality of ovine plant-derived antioxidants extracted from Abarema cochliacarpos, aiming to decrease reactive oxygen species (ROS) in cryopreserved ovine semen samples. Sheep semen was collected in triplicate, frozen with cryopreservation extender extract with addition of Abarema cochliacarpos oil, at the following concentrations: 0 (control), 2.5 mg; 1.25 mg; 0.67mg; 0.31 mg and 0.2 mg. The data obtained by the sinusoidal arc were submitted to analysis of variance. The results indicate that there were no significant differences between treatments for membrane integrity or mitochondrial potential, and in evaluating computer-assisted sperm analysis (CASA) between concentrations on the parameters used in the study. There was, however, a difference in the parameters of progressive motility, linearity, straightness, and frequency of cross-flagellar beating, with emphasis on the concentration of 1.25 mg of A. cochliacarpos, not indicating an adverse effect. The 1.25 mg concentration was the most effective in this experiment.
Keywords: Sheep Farming; Seminal Quality; Oil Extract; Cryopreservation
Abbreviations: ROS: Reactive Oxygen Species; AI: Artificial Insemination; CASA: Computer Assisted Sperm Analysis; ROS: Reactive Oxygen Species; CEUA: Committee for Ethics in the Use of Animals; PMI: Plasma Membrane Integrity; A. Cochliacarpos: Abarema Cochliacarpos; LABRAPE: Animal Reproduction Laboratory of Pernambuco; UFRPE: Federal Rural University of Pernambuco; UAG: Garanhuns Academic Unit; CBRA: Brazilian College of Animal Reproduction; PBS: Phosphate Buffered Saline Solution; CFDA: 6 Carboxyfluorescein; MMP: Mitochondrial Membrane Potential; DIC: Completely Randomized Design; ANOVA: Analysis of Variance; MP: Progressive Motility; PIM: Progressive Individual Motility; LIN: Linearity; STR: Straightness; BCF: Crossed Flagellar Beat Frequency; MT: Total motility; VCL: Track Speed; VSL: Progressive Speed; VAP: Route Speed; ALH: Amplitude of Lateral Displacement of the Head; WOB: Oscillation Index
One of the most used techniques to improve the productive potential of animals is artificial insemination (AI). It is among the oldest biotechnological techniques applied for animal genetic improvement, being the one that produced the greatest genetic gain due to the ease of its practical domain, making it the most widespread [1]. To perform AI, semen from tested animals may be used, handled fresh or stored under refrigeration and freezing. Semen, when frozen, suffers several structural, chemical, and physical damages to the spermatozoa, leading to a decrease in motility, viability, and fertility parameters [2]. These damages are linked to the process of cooling, freezing, and thawing [3]. The occurrence of this damage differs between species, due to variation in sperm size and shape, as well as in lipid composition. The alterations caused by this process are due to changes in temperature, oxidative injuries via reactive oxygen species, formation of ice crystals, DNA damage, alterations in the sperm membrane, osmotic stress, in addition to the toxicity of cryoprotectants [4]. To minimize oxidative damage, several studies such as Banday, et al. [5] used media with antioxidant substances, thus improving post-thawing sperm quality.
The addition of antioxidants based on substrates of plant origin is a viable alternative [6]. Abarema cochliacarpos, a plant species native to Brazil, has numerous medicinal properties. For example, Silva, et al. [7] reported the presence of catechins, which show evidence of antioxidant activity and ability to fight free radicals [8]. Therefore, the aim of this investigation was to test the effect on seminal quality of ovine plant-derived antioxidants extracted from A. cochliacarpos to decrease ROS in cryopreserved ovine semen samples. The results of the study will be valuable to improve biotechnologies aimed at sheep farming and its reproduction, in addition to increasing savings and reducing damage to sperm membranes.
Extraction and purification of Abarema cochliacarpos
The research was carried out in January 2018 at the Animal Reproduction Laboratory of Pernambuco (LABRAPE), located on the premises of the Federal Rural University of Pernambuco (UFRPE), Academic Unit Garanhuns (UAG). Sheep from rural properties in the municipality of Garanhuns, in the Mesoregion of Agreste Pernambucano, were used. The experiment was approved by the Committee for Ethics in the Use of Animals (CEUA), under license number 127/2017.
Two adult rams were selected after andrological examination. After being conditioned to collection with an artificial vagina, semen collections were performed on alternate days. During semen collection, ewes were used as dummies, not necessarily in estrus, which was manually contained. The semen was received in a silicone collection cone and graduated Falcon® tubes.
As the semen was collected, it was sent to the Animal Reproduction Laboratory of Pernambuco (LABRAPE) to carry out the spermogram, quantifying the following parameters: volume (mL), sperm concentration (x109 spermatozoa/mL), progressive individual motility (PIM , 0 -100%) and vigor (0-5), according to the criteria recommended by the Brazilian College of Animal Reproduction (CBRA, 2013). An aliquot of 2.5μL of semen diluted in 997.5 μL of phosphate buffered saline solution (PBS) was pipetted, thus obtaining a saline solution at a concentration of 1:400, based on the sperm count, the concentration was defined camera-assisted sperm.
The semen was diluted by the addition of the diluent medium, with the addition of A. cochliacarpos oil. The semen received pre-established concentrations of the extract. The oil obtained by aqueous extraction was subjected to several tests of antioxidant activity [7,8] and later, the analysis was performed together with ram semen where the concentrations were used: C1:0 (control); C2: 2.5; C3: 1.25; C4: 0.67; C5: 0.31; C6: 0.2 milligrams of extract.
After dilution the semen was packaged in 0.25 ml straws and subjected to freezing in the TK3000® machine (TK Freezing Technology LTDA, Uberaba, Brazil), using a quick-freezing curve (P3:S1) with a stabilization time of 1 hour according to previous studies in our laboratory [9]. Then, the freezing curve started (-20°C /min) until reaching -120°C. Then, the straws were immersed and stored in liquid nitrogen (-196°C) until later use.
After freezing the desired samples, sperm parameters were evaluated after thawing (37 °C for 30 seconds).
Analysis of sperm kinetics was performed using Computer-Assisted Sperm Analysis (CASA). An aliquot (3 μL) of the diluted sample was placed on a slide and covered with a coverslip (18 x 18 mm), both preheated (37°C), and evaluated under a phase contrast microscope (Eclipse 50i, Nikon, Japan, 100x). Images were captured by a video camera (Basler Vision Technologies TM A312FC, Germany). For each sample, five random fields were selected, with records of at least 500 spermatozoa. The analyzed variables were: Total motility (MT, %), Progressive motility (MP, %), Linearity (LIN, %), Straightness (STR, %); Track Speed (VCL, μm/s), Progressive Speed (VSL, μm/s); Velocity of travel (VAP, μm/s), Amplitude of lateral displacement of the head (ALH, μm/s); cross-flagellar beat frequency (BCF,%). CASA system values were measured with the following settings: temperature 37°C; magnification, 100x; number of images, 25; images per second, 25; head area, 20 to 70 μm2; PAV: slow 10 μ/s < medium 45 μ/s < fast 75 μ/s; progressiveness, 80% STR; circular, 50% LIN. Plasma membrane integrity (PMI) [10].
The following procedures were performed to analyze plasma membrane integrity (PMI). Aliquots (40 μL) of semen from each experimental group were distributed in microtubes (1.5 mL) for each evaluation, to which 5 μL of propidium iodide, 5 μL of CFDA (6 carboxyfluorescein) and 5 μL of of paraformaldehyde. Then, 5 μL of each sample were placed on a slide and covered with a coverslip (18 x 18 mm) and submitted to evaluation under a fluorescence microscope. For each sample, five random fields were selected, with a record of 200 spermatozoa, those that presented green color being classified as intact.
For mitochondrial membrane potential (MMP) analysis, 5 μL of JC-1 (Stock solution 5 mg/mL in DMSO; Working solution 153 μM in DMSO) was added to semen samples containing 40 μL, distributed in microtubes of 1 .5ml. Subsequently, the samples were incubated for 5 min at room temperature and evaluated. Cells with intermediate partsstained orange were classified as having high mitochondrial membrane potential. For each sample, five random fields were selected, with a record of 200 spermatozoa.
Completely randomized design (DIC), with six treatments and six replications (3 per animal). The treatments were composed by the concentrations: C1:0 (control); C2: 2.5; C3: 1.25; C4: 0.67; C5: 0.31; C6: 0.2 mg of the extract. The data obtained were submitted post-transformation by sinusoidal arc (arcsen P/100) to analysis of variance (ANOVA) by the “F” test, with the averages of the treatments compared with the Tukey test at 5% significance.
(Table 1) The results of five extracts (Table 2) show that there were no significant differences between concentrations after semen thawing in the following parameters: TM, VCL, VSL, VAP, WOB and ALH.
Table 1: Shows that the semen collected is healthy and complies with the standard contained in the Manual for Andrological Examination and Evaluation of Animal Semen. This material will be used as a basis for analysis and comparison with later results.
Note: mL: Milliliter.
Table 2: Mean values after thawing of ram semen with addition of vegetable extract, with evaluation in the CASA computerized system.
Note: Progressive motility (PM); Linearity (LIN); Straightness (STR); Trail speed (VCL); Progressive Speed (VSL); Speed of travel (VAP); Amplitude of lateral head displacement (ALH); Total Motility (MT); wobble index (WOB); Micromeres per second (μm/s).
However, there was a significant difference between treatments when the parameters of progressive motility (PM), linearity (LIN), straightness (STR) and cross-flagellar beat frequency (BCF) were evaluated. However, it was demonstrated that concentrations of less than 1.25 mg of the extract showed lower percentage values of the parameters evaluated in the CASA. This may lead to the conclusion that concentrations below 1.25 mg may not have the expected antioxidant action. However, a hypothesis that could explain the fact that values higher than this could compromise sperm quality after thawing is also not validated using higher concentrations of this extract in this study. This hypothesis corroborates Marsico [11] that different extracts at different concentrations can present different biochemical behaviors.
Thus, it seems reasonable to assume that the concentration of 1.25 mg is the most appropriate, as values above and below the referent do not provide an adequate response in ram semen. Therefore, sheep farmers would need to increase the type of thawing medium for semen, thus preserving some of its primordial characteristics and, consequently, ensuring a greater probability of success in the sheep fertilization process. In this experiment (Table 3), no significant differences were observed between treatments for impairment of plasma membrane integrity and mitochondrial membrane potential. This means that A. cochliacarpos does not have an adverse effect on PMI and MMP at different concentrations in the present study, as also observed by Santos [12] who also used a plant extract based on green propolis as an antioxidant in the cooling of equine semen. Still, there was a decrease in sperm motility. There is no evidence, as far as we know, about the effect of A. cochliacarpos extract, especially on the pattern of mitochondrial activity and state of apoptosis in mammalian spermatozoa.Table 3: Mean values of the PMI and MMP Integrity evaluation using fluorescent probes.
Note: Plasma Membrane (PMI); Mitochondrial Membrane Potential (MMP).
The study analyzed five extracts (Table 2) and concluded that there were no significant differences in post-thawing semen concentrations in relation to parameters such as TM, VCL, VSL, VAP, WOB and ALH. However, there was a significant difference between treatments when evaluating PM, LIN, STR and BCF. Concentrations lower than 1.25 mg of the extract resulted in lower percentage values in the CASA, suggesting a limited antioxidant action. The hypothesis that concentrations above 1.25 mg negatively affected sperm quality was not confirmed. Thus, it seems that the concentration of 1.25 mg is the most appropriate to maintain the characteristics of ram semen and increase the chances of successful fertilization. No significant differences were found in the effects on PMI and MMP with different concentrations of A. cochliacarpos, which indicates its safety in these respects. However, a decrease in sperm motility was observed. So far, there is no evidence of the effects of A. cochliacarpos extract on mitochondrial activity and apoptosis in mammalian spermatozoa.
Abstract
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