Evaluation of Microscopic,Flow Cytometric,and Oxidative Parameters of the Frozen–Thawed Bull Sperm in a Freezing Extender Containing Myo-Inositol

Abstract

Introduction:

This research was conducted to assess the effect of myo-inositol (MYO) in the freezing extender on the semen quality and oxidative stress parameters of frozen–thawed bull sperm.

Materials and Methods:

Semen samples were obtained from four bulls (n = 24, six ejaculates per bull), twice a week, and diluted into four equal aliquots in freezing extenders containing different concentrations of MYO (0, 2, 3, and 4 mg/mL). After a freezing/thawing process, velocity parameters, plasma membrane integrity, apoptosis status, malondialdehyde level, and oxidative stress parameters were assessed.

Results:

Supplementation of freezing extender with 3 mg/mL MYO resulted in higher rapid motility (62.22% ± 2.63%), progressive motility (77.45% ± 2.65%), viability (78% ± 0.91%), plasma membrane integrity (86 ± 0.85), catalase (20.03 ± 0.39 U/mL) activity, and lower significance of lipid peroxidation (3.60 ± 0.15 nmol/dL) than those of the control group (p < 0.05). A significantly lower percentage of normal morphology and intact acrosomes were observed for frozen–thawed semen in the extender supplemented with 4 mg/mL MYO than those of the control group (p < 0.05). Freezing of the sperm in the extender containing 3 mg/mL of MYO leads to a higher percentage of live cells (38.3 ± 2.76). Beat-cross-frequency, amplitude of lateral head displacement, linearity, total antioxidant capacity, total peroxidase activity, early apoptotic status, and superoxide dismutase activities were not affected by MYO levels in the extenders (p > 0.05).

Conclusion:

The findings of this study suggest that the supplementation of the freezing extender with 3 mg/mL MYO resulted in a higher quality of frozen–thawed bull sperm.

Introduction

Semen cryopreservation has accelerated the breed improvement via an artificial insemination technique.¹ Sperm cryopreservation induces cellular damages due to cold shock, enhanced oxidative stress, intracellular ice crystal formation, sperm membrane alteration, and DNA damage, as well as changes in the sperm functionality and metabolism,² resulting in the reduction of the sperm quality and fertilizing capacity of spermatozoa.³

The membranous structures such as the plasma membrane, acrosome, and mitochondrial membranes are highly sensitive to the freeze–thawing process.⁴ The high levels of polyunsaturated phospholipids in the plasma membrane of the spermatozoa are also considered an important factor for the sperm susceptible to lipidperoxidation,⁵ which leads to cellular damage during the freezing process.⁶ Oxidative stress is due to the excessive production of reactive oxygen species (ROS) accompanied by the failure of the antioxidant protection system.⁷ As a consequence, the freezing–thawing process leads to a decrease in sperm motility, activation of the intrinsic apoptosis pathway, loss of mitochondrial membrane potential (MMP), deterioration of plasma membrane functionality, and DNA fragmentation.⁸ Development of antioxidant formulation is becoming extremely important in decreasing the harmful effect of ROS on frozen sperm.^9,10 Therefore, a wide range of vitamins, sugars, enzymes, and amino acids have been added to semen extenders, aiming to increasing the quality of frozen–thawed sperm.^11,12

Myo-inositol (MYO), synthesized from glucose-6-phosphate, is the most biologically active form of inositol.¹³ Bull testicular sperm can synthesize lipids from inositol, which play a critical role in generating crucial molecules for sperm survival in the epididymis.¹⁴ Cell signaling, cell cytogenesis, chromatin dynamics and remodeling, regulation of ion channel opening, gene expression, and epigenome control are all regulated by MYO and its derivatives.¹⁵ Several studies have proven that MYO plays an important role in antioxidant properties that increase sperm quality in both normozoospermic men and patients with poor sperm parameters.^16–18 Supplementing ram sperm extender with MYO protects the enzyme from damage caused by freezing or lipopolysaccharides and preserves acrosome integrity.^19,20 MYO also increases the quality of canine sperm, such as percentage sperm motility, plasma membrane integrity, and chromatin integrity.²¹ It has been reported that treating equine sperm with MYO improves the sperm motility and intactness of the plasma membrane.²² It also improves sperm motion characteristics such as amplitude of lateral head displacement (ALH).¹¹ Furthermore, it enhances sperm mitochondrial activities by stimulating oxidation and adenosine triphosphate production, consequently condensing chromatin and avoiding apoptosis.¹⁸ The addition of MYO to sperm freezing medium improves the human sperm parameters and protects them against DNA fragmentation and apoptosis-like changes.¹³ Infertile women undergoing ovulation induction for in vitro fertilization (IVF) or intracytoplasmic sperm injection have a higher clinical pregnancy rate while using MYO supplements.²³ As a result, it has been determined that this antioxidant is an excellent alternative for enhancing the semen extender and producing high-quality semen following the freeze–thawing process.

The objective of this study was to investigate the effects of different concentrations of MYO on the motion kinematics, oxidative stress parameters, apoptosis-like change, and acrosomal status in Holstein bull spermatozoa.

Materials and Methods

Ethics statement

In this study, bull semen was provided by the Iranian Nahadehaye Dami Jahed (NDJ) Company (Karaj, Iran). All experimental procedures were approved by the Animal Ethics Committee of the University of Tabriz, Iran (IR.TABRIZU.REC.1399.043).

Animals and semen collection

Twenty-four ejaculates were collected from four mature Holstein bulls (3–5 years old) twice a week for 3 weeks (two ejaculates/week/bull = six replicates/bull) using an artificial vagina. Immediately after collection, the samples were kept at 35°C until further analysis. Only semen samples with progressive motility (PM) ≥70%, and concentration ≥1.0 × 10⁹ spermatozoa/mL were enrolled in this study. We pooled the semen to eliminate individual differences and balance the sperm contribution of each bull.²⁴

Experimental design

Sperm concentrations of pooled bull ejaculates were determined using a calibrated photometer (IMV^® Technologies, L'Aigle, France) and dilution was carried out with 250 mmol/L Tris (Tris-hydroxymethyl amino-methane), 90 mmol/L citric acid, 70 mmo/L fructose, 100 IU/mL penicillin G, 100 μg/mL streptomycin, glycerol 7% (v/v), and egg yolk 20% (v/v) to obtain the final sperm concentration of 25 × 10⁶ spermatozoa/mL. The pH and osmolality were set at 6.8 and 300 mOsm/kg, respectively.²⁵ After dilution, the semen samples were divided into four equal aliquots and were added with one of the four different concentrations of MYO (0, 2, 3, and 4 mg/mL). The semen extended samples were loaded into 0.5 mL straws (IMV Technologies), equilibrated for 4 hours at 4°C, and then frozen using a programmable freezer (Digit Cool^®; IMV Technologies) (4°C to −10°C at 5°C/min; −10°C to −100°C at 40°C/min; and −100°C to −140°C at 20°C/min). After 1 week, the frozen samples were thawed for 30 seconds at 37°C in a water bath for microscopic evaluation.

Evaluation of sperm motility parameters

In this experiment, each sample was diluted to 25 × 10⁶ spermatozoa/mL with phosphate-buffered saline buffer, then a 5 μL specimen was put onto a chamber slide (previously warmed to 37°C) established for bull sperm; 38°C, 4; 20 μm height; Leja^® slide) of the CASA system (AndroVision^®, minitube), and sperm motion parameters were assessed.²⁶ Sperm total motility (TM; %), PM (%), curvilinear velocity (VCL; μm/s), straight linear velocity (VSL; μm/s), average path velocity (VAP; %), beat-cross-frequency (BCF; Hz), ALH (μm), straightness (STR; %), linearity (LIN; %), and wobble (WOB; %) were evaluated in at least six fields by counting 400 sperm for each sample.²⁷

Assessment of sperm viability

Sperm viability was assessed with modification of the eosin–nigrosin stain solution [1% (w/v) eosin B and 5% (w/v) nigrosin in 3% trisodium citrate dehydrate solution].²⁸ For this purpose, approximately equal volumes of semen and stain (5 + 5 μL) were mixed, and smeared using a second slide. At least 200 spermatozoa were counted using a light microscope (Scope.A1 ZEISS). Pink-stained samples were counted as dead sperm and unstained were counted as live sperm.

Sperm morphology

For evaluation of sperm abnormalities, 10 μL of frozen–thawed semen was pipetted into 1 mL Hancock's solution.²⁹ Hancock's solution comprised 62.1 mL formalin (37%), 150 mL sodium chloride (NACL), 150 mL buffer solution, and 500 mL double-distilled water. Sperm defects were classified as head, neck, midpiece, and tail defects. At least 200 spermatozoa were assessed under brightfield and oil immersion using a phase-contrast microscope (magnification: × 1000).

Sperm plasma membrane integrity

The functional integrity of sperm plasma membrane was evaluated by the hypoosmotic swelling test (HOST). HOST is based on the sperm membrane's resilience to stressful situations in a hypoosmotic solution. In brief, 30 μL of thawed semen and 300 μL of a hypoosmotic solution (13.5 g fructose and 7.35 g sodium citrate dissolved in 1 L water; osmolality of 100 mOsm/kg) were mixed. This mixture was incubated at 37°C for 60 minutes, then 10 μL of the mixture was transferred onto a prewarmed slide, mounted with a coverslip, and was promptly assessed ( × 400 magnifications) under a phase-contrast microscope. Finally, at least 200 sperm with curled and nonswollen tails were evaluated.

Acrosomal integrity

Acrosomal integrity was evaluated according to Thys et al.³⁰ using a 500 μL sperm suspension in a microtube. After centrifugation, the sperm pellet was dissolved in 100 μL of ethanol (96%) and kept at room temperature for 30 minutes, after which 10 μL of sperm suspension was kept on a glass slide to allow slow evaporation of the ethanol. Then, 30 μL of fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (50 μL/mL) was added to the sperm. The glass slide was incubated for 20 minutes at room temperature, and then dipped 10 times in distilled water. Afterward, prepared slides were allowed to dry and were mounted with glycerol. We observed and recorded at least 200 sperm per slide by a fluorescent microscope (BX51; Olympus, Tokyo, Japan) at × 400 magnification. Sperm with green fluorescence in the head area were considered to be intact, whereas those with no fluorescence in the head area were considered to have a damaged acrosome. Each of the four treatments was replicated three times.

Phosphatidylserine translocation assay by flow cytometry

To assess the externalization of phosphatidylserine (PS) as an indicator of apoptosis, sperm were rinsed in calcium (Ca) buffer and readjusted to a concentration of 1 × 10⁶ cells/mL.³¹ Subsequently, 10 μL of Annexin V-FITC (0.01 mg/mL) was added to a 100 μL sperm suspension and incubated for 20 minutes on ice. Then 10 μL of propidium iodide (PI) was added to the suspension and incubated on ice for 10 minutes. For each sample, 10,000 events were examined by flow cytometry (Becton-Dickinson, San Khosoz, CA). Four different sperm were identified: viable or nonapoptotic cells that are negative for Annexin-V and exclude PI staining (A⁻/PI⁻), early apoptotic cells that bind Annexin V but exclude PI (A⁺/PI⁻), late apoptotic cells that bind both Annexin-V and PI (A⁺/PI⁺), and necrotic cells that exclude Annexin-V and bind PI (A⁻/PI⁺). Green fluorescence was measured by a 530/30 nm (FL1) bandpass filter, and propidium (red florescence) was measured by a 585/42 nm (FL2) bandpass filter. Each of the four treatments was replicated three times.

Oxidative stress parameters

The ferric reducing/antioxidant power (FRAP) analysis was performed to determine total antioxidant capacity (TAC).³² In a cuvette, 10 μL of frozen–thawed semen was combined with 1.0 mL of FRAP reagent [300 mmol/L acetate buffer; 10 mmol/L 2,4,6-tri-(2-pyridyl)-1,3,5-triazine, 98.00%; and 20 mmol/L FeCl₃·6H₂O were mixed in a 10:1:1 ratio], and the absorbance was measured after 4 minutes at 593 nm with a Unico UV/2100 PC spectrophotometer (China). The concentration of malondialdehyde (MDA) level, as an indicator of lipid peroxidation in the samples, was assayed using the thiobarbituric acid (TBA) assay.³³ In brief, 0.2 mL of the frozen–thawed semen was combined with 1 mL cold trichloroacetic acid 20% (w/v) to precipitate the protein. The precipitate was centrifuged, and 1 mL of the supernatant was mixed with 1 mL of TBA solution (0.67%). The solution was vortexed and incubated in a boiling water bath (100°C, 10 minutes). After cooling, the absorbance of the supernatant was read using a spectrophotometer (Unico UV/2100 PC spectrophotometer, China) at 532 nm. The MDA concentration was expressed as nmol/dL. Catalase (CAT) activity was determined using the Goth method.³⁴ To this end, 0.1 mL of frozen–thawed semen was incubated with 1 mL of substrate (hydrogen peroxide) in sodium/potassium phosphate buffer (60 mmol L⁻¹, pH 7.4) at 37°C for 2 minutes. The enzymatic process was stopped by adding 1.0 mL of ammonium molybdate (32.4 mmol L⁻¹), and hydrogen peroxide was measured at 405 nm using a spectrophotometer. The CAT activity was recorded in U/mL. Superoxide dismutase (SOD) activity was assayed by the oxidation of pyrogallol.³⁵ Auto-oxidation of pyrogallol was measured for 4 minutes at 420 nm. The amount of enzymes that caused 50 percent inhibition in auto-oxidation of pyrogallol was defined as one unit of SOD activity (U/mL). Total peroxidase activity (TPA) was determined by the procedure previously described.³⁶ The assay system comprised 100 mM citrate buffer (pH 5), 50 μL o-dianisidine (2.5 mg/mL), and 20 μL of semen samples in a final volume of 1 mL. The reaction was initiated by the addition of 50 μL of freshly prepared 3.5 mM H₂O₂ in the assay mixture. One unit of peroxidase is defined as the amount of the enzyme that catalyzes the conversion of one micromole of o-dianisidine per minute, under the indicated conditions.

Statistical analysis

Each experiment was replicated three times. The normal distribution of quantitative variables was assessed by the Shapiro–Wilk test, and data analysis was performed by the generalized linear model procedure of SAS 9.2 (SAS Institute Inc., Cary, NC). Mean separation was performed using the Duncan's multiple rang test at p ≤ 0.05. Data were expressed as the mean ± standard error of the mean.

Results

Effect of MYO on sperm characteristics and kinematic parameters

Table 1 shows the effects of different concentrations of MYO on the semen characteristics and motion parameters of bull sperm after freeze–thawing. Supplementation of freezing extender with 3 mg/mL MYO produced the most significant percentage of rapid sperm motility (62.22% ± 2.63%, p ≤ 0.01), progressive sperm motility (77.45% ± 2.65%, p ≤ 0.05), and viability (78% ± 0.91%, p ≤ 0.05) compared with the control group. There was no significant difference in sperm TM among the treatment groups and control groups (p ≥ 0.05). As shown in Table 1, supplementation of the freezing extender with 3 mg/mL MYO increased VSL (48.5 ± 1.69 μm/s), VAP (58.70 ± 1.76 μm/s), VCL (111.5 ± 3.84 μm/s), and STR (0.82% ± 0.004%) compared with the control group (p ≤ 0.05). The ALH, BCF, LIN, and WOB were not affected by different concentrations of MYO in the semen extender (p ≥ 0.05).

Table 1.

Effect of Different Concentration of Myo-Inositol on Post-Thaw Characteristics and Kinematic Properties of Cryopreserved Bull Sperm (Mean ± Standard Error of the Mean)

Variables	Concentrations of MYO (mg/mL)
Variables	0	2	3	4	p
PM (%)	72.10 ± 0.47^b	75.70 ± 1.14^a,b	77.45 ± 2.65^a	71.56 ± 0.99^b	0.05
TM (%)	76.76 ± 2.15	82.27 ± 1.09	82.77 ± 2.41	79.25 ± 1.25	0.12
RM (%)	52.19 ± 1.05^b	56.74 ± 1.56^a,b	62.22 ± 2.63^a	54.90 ± 1.96^b	0.01
VCL (μm/s)	93.62 ± 3.20^b	101.8 ± 5.34^a,b	111.5 ± 3.84^a	102.5 ± 3.27^a,b	0.05
VSL (μm/s)	38.6 ± 1.76^b	42.0 ± 2.61^b	48.5 ± 1.69^a	42.3 ± 1.60^b	0.02
VAP (μm/s)	49.29 ± 1.75^b	52.69 ± 2.93^a,b	58.70 ± 1.76^a	53.10 ± 1.93^a,b	0.05
BCF (Hz)	11.24 ± 0.56^b	11.52 ± 0.38^a,b	12.83 ± 0.47^a	11.79 ± 0.37^a,b	0.12
ALH (μm)	1.22 ± 0.03	1.26 ± 0.03	1.35 ± 0.04	1.03 ± 0.26	0.42
STR (%)	0.78 ± 0.007^b	0.79 ± 0.010^b	0.82 ± 0.004^a	0.79 ± 0.011^b	0.02
LIN (%)	0.410 ± 0.008	0.412 ± 0.008	0.435 ± 0.028	0.412 ± 0.008	0.11
WOB (%)	0.527 ± 0.007	0.517 ± 0.004	0.525 ± 0.002	0.517 ± 0.006	0.49
Viability (%)	75 ± 0.40^b	76 ± 0.85^a,b	78 ± 0.91^a	77 ± 0.75^a	0.05

a,b

Different letters within the same row show significant differences (p < 0.05).

ALH, amplitude of lateral head displacement; BCF, beat-cross-frequency; LIN, linearity; MYO, myo-inositol; PM, progressive motility; RM, rapid motility; STR, straightness; TM, total motility; VAP, average path velocity; VCL, curvilinear velocity; VSL, straight-line velocity; WOB, wobble.

Effect of MYO on sperm morphology

Data related to sperm morphology are presented in Table 2. Supplementation of the freezing extender with a high concentration of MYO (4 mg/mL) reduced the percentage of normal morphology compared with those of other groups (p ≤ 0.05). The highest percentage of midpiece defects (2.75 ± 0.62) was recorded in the high dosage of MYO (4 mg/mL) when compared with the other groups (p ≤ 0.05). No significant difference was found among treatment groups in percentages of the sperm with excess residual cytoplasm, and head and tail defects.

Table 2.

Effect of Supplementation of the Semen Extender with Myo-Inositol on Frozen–Thawed Bull Sperm Morphology

Variables	Concentrations of MYO (mg/mL)
Variables	0	2	3	4
Normal forms	86.25 ± 2.59^a	82.5 ± 2.66^a	82.25 ± 3.52^a	78.5 ± 1.19^b
Head defect	2.25 ± 0.25	3.25 ± 1.25	4.5 ± 1.04	4.75 ± 1.10
Midpiece defect	00 ± 0.0^a	0.75 ± 0.47^a	0.5 ± 0.28^a	2.75 ± 0.62^b
Tail defect	2.5 ± 0.86	4 ± 1.08	2.5 ± 1.04	2.75 ± 0.62
Excess residual cytoplasm	4.75 ± 1.25	5.25 ± 1.65	5 ± 1.47	5.25 ± 1.31

a,b

Different letters within the same row indicate significant differences (p < 0.05).

Effect of MYO on sperm plasma membrane and acrosomal integrity

The results showed that plasma membrane functionality was higher (p ≤ 0.05) in 3 mg/mL MYO compared with the control group (Fig. 1A). There were no statistically significant differences between 3 and 4 mg/mL MYO. On the contrary, as shown in Figure 2B, the greater levels of MYO (4 mg/mL) led to lower percentages of intact acrosomes in comparison with other concentrations and control groups (p ≤ 0.05). Comparison of different concentrations of MYO showed that 2 and 3 mg/mL MYO produced a high percentage of intact acrosomes compared with 4 mg/mL MYO (p ≤ 0.05).

FIG. 1.

(A) Effect of supplementation of the freezing extender with MYO on PMI and (B) acrosomal integrity in post-thawed bull sperm.^a–cDifferent letters indicate significant difference (p < 0.05) between different groups. Values are expressed as the mean ± SEM. MYO, myo-inositol; PMI, plasma member integrity; SEM, standard error of the mean.

FIG. 2.

Flow cytometry dot plot for apoptosis with Annexin-V and PI (FL1: Annexin-V fluorescence, FL2: PI fluorescence) in thawed sperm supplemented with different concentrations of MYO [2 mg/mL (A); 3 mg/mL (B); and 4 mg/mL MYO (C)], and control group without MYO (D). Events in the lower-left quadrant (Q4) represent negative for Annexin-V and PI (A⁻/PI⁻) (viable sperm), and events in the lower-right quadrant (Q2) represent early apoptotic spermatozoa (A⁺/PI⁻). Spermatozoa in the upper-right quadrants represent (Q2) late apoptotic cells (A⁺/PI⁺); spermatozoa in the upper upper-left quadrants (Q1) represent necrotic cells (A⁻/PI⁺). FITC, fluorescein isothiocyanate; PI, propidium iodide.

Effect of MYO on sperm apoptosis

As shown in Table 3 and Figure 2, supplementation of the freezing extender with 3 mg/mL MYO increased the percentage of viable cells compared with the control group (p < 0.05), while supplementation of the freezing extender with 4 mg/mL MYO increased the percentage of late apoptotic cells compared with the control group (p ≤ 0.05). No significant differences were observed in the early apoptotic cells among different concentrations of MYO and control group. Supplementation of the freezing extender with different concentrations of MYO decreased the percentage of necrotic cells compared with the control (p < 0.05).

Table 3.

Flow Cytometric Evaluation of Apoptosis Statues in Frozen–Thawed Bull Sperm Supplemented with Myo-Inositol

Variables	Concentrations of MYO (mg/mL)
Variables	0	2	3	4
Live cells (%)	21.39 ± 2.03^a	31.26 ± 4.19^b	38.3 ± 2.76^b,c	21.53 ± 1.06^a,d
Early apoptotic (%)	2.92 ± 1.38	2.07 ± 0.04	5.9 ± 1.14	3.7 ± 1.7
Late apoptotic (%)	33.55 ± 1.25^a	39.76 ± 3.49^a	37.10 ± 6.48^a	47.70 ± 0.085^b
Necrotic (%)	42.16 ± 0.44^a	26.66 ± 7.51^b	15.88 ± 4.92^b	27.06 ± 1.79^b

The data are expressed as the mean ± standard error of the mean.

a–d

Different rows indicate significant differences (p < 0.05) between different groups.

Analysis of MDA levels and antioxidant activities

According to Table 4, supplementation of freezing extender with different concentrations of MYO had no effect (p > 0.05) on the levels of TAC, SOD, and TPA after the freeze–thaw process. Supplementation of freezing extender with 3 mg/mL MYO produced the most significant levels of CAT compared with the control group (p < 0.05; Table 4). MDA formation was lower (3.60 ± 0.15 nmol/dL) in semen samples extended in the presence of 3 mg/mL MYO, compared with the control group (p ≤ 0.001).

Table 4.

Effect of Different Concentrations of Myo-Inositol on Sperm Biochemical Parameters During Cryopreservation of Bull Semen (Mean ± Standard Error of the Mean)

Variables	Concentrations of MYO (mg/mL)
Variables	0	2	3	4
TAC (μmole/mL)	1.23 ± 0.67	1.26 ± 0.066	1.19 ± 0.044	1.11 ± 0.043
SOD (U/mL)	1.95 ± 0.04	1.93 ± 0.039	1.92 ± 0.032	1.85 ± 0.05
TPA (U/mL)	42.85 ± 6.18	51.50 ± 5.26	35.03 ± 2.57	46.11 ± 5.40
CAT (U/mL)	8.08 ± 1.42^c	14.88 ± 2.20^b	20.03 ± 0.39^a	13.57 ± 2.66^b,c
MDA (nmol/dL)	5.10 ± 0.20^a	3.99 ± 0.17^b	3.60 ± 0.15^b	4.10 ± 0.27^b

a–c

Different letters within the same row indicate significant differences (p < 0.05).

CAT, catalase; MDA, malondialdehyde; SOD, superoxide dismutase; TAC, total antioxidant capacity; TPA, total peroxidase activity.

Discussion

The freeze–thaw procedure induces cold shock, ice crystal formation, osmotic pressure disturbance, membrane alteration, and oxidative stress in sperm.⁶ We observed the beneficial effects of MYO on the quality of frozen–thawed bull sperm. In an earlier study, a positive influence of MYO on the motility of post-thaw ram sperm was reported.³⁷ Similarly, MYO addition to human sperm before freezing increased the total and progressive sperm motility in infertile patients with oligoasthenoteratozoospermia (OAT).^38–40 This indicated that supplementing human semen cryoprotectant with MYO improves the TM and PM after thawing. Previous studies showed that MYO is mainly produced by Sertoli cells and is involved in several processes including the regulation of motility, capacitation, and acrosome reaction of sperm cells.^16–18 In addition, it causes the activation of phospholipase C and Ca channels, thus affecting the sperm motility through increased Ca ions.⁴¹ A possible explanation for this might be that MYO improved human sperm motility through improvement of the sperm mitochondrial function.⁴²

Our results revealed that a supplementation semen extender of 3 mg/mL concentration resulted in improved sperm viability following a freeze–thawing process. We assumed that the improved sperm viability rate by MYO may be due to the increased membrane resistance to deterioration against ice crystals. Interestingly, a previous report showed that treatment with MYO had no effect on the human sperm viability.⁴³ In addition, a study indicated that it had no effect on the post-thaw ram semen quality when inositol was included in the tris base extender.⁴⁴ This inconsistency may be due to the species of animals, the extender composition, and the amount of the doses of MYO used in these studies.

A previous study showed that the addition of MYO to equine semen cooling extenders had no effect, except ALH, which was higher in MYO compared with the control group.¹¹ Our results showed that the supplementation of semen extender with 3 mg/mL MYO improved sperm kinematic variables, including VSL, VCL, VAP, BCF, and STR compared with the control group. These velocity parameters are correlated with sperm fertilization ability, IVF, and pregnancy rate.¹² Therefore, it is possible that a portion of the beneficial effects of MYO on the motion parameters of frozen–thawed samples would be related to the integrity of the plasma membrane. MYO may also protect the sperm against ice formation in intracellular spaces, forming an outside coating layer, or penetrating the cell membrane to minimize recrystallization. However, in the group supplemented with 4 mg/mL MYO, sperm PM, acrosome integrity, and curvilinear distance were considerably minimized, probably due to the higher amount of antioxidants added to the freezing extender, increase in osmotic pressure, decrease in physiological concentration of ROS, and enhancement of the fluidity of the plasma membrane, making sperm more susceptible to membrane damage.¹³ However, more experiments are required to determine the exact molecular mechanism by which high levels of antioxidants induce adverse effects on the cell.

A previous study showed that sperm morphology is probably the most relevant parameter of the traditional semen evaluation and can be used as a valid biomarker of functional deficiencies, providing information about the chances of conception.⁴⁵ In patients with OAT, it was shown that MYO reduced DNA fragmentation and improved the morphology of the sperm membrane.⁴⁶ Based on our findings, midpiece defects were lower in the extender containing 3 mg/mL MYO. For men with OAT, it was found that midpiece defects or abnormal organization of sperm mitochondrial sheaths causes serious motility alterations and lower fertilization potential.⁴⁷ The current study showed that higher concentrations of MYO exerted deleterious effects on the normal morphology, resulting in excess residual cytoplasm and abnormal morphology in post-thaw sperm. It has been shown that ROS production was positively correlated with the proportion of sperm with amorphous heads, damaged acrosomes, midpiece defects, cytoplasmic droplets, and tail defects.⁴⁸ In this study, we did not measure the MMP, however, it was reported that ROS cause damage to the mitochondrial membranes, and on the contrary, it increases impairment of sperm function, which includes sperm motility, viability, and morphology.^49,50

It is well-known that plasma membrane disruption due to ROS results in increased membrane permeability and uncontrolled intracellular ion concentration.⁵¹ MYO has previously been reported to have protective effects on the plasma membrane of canine frozen sperm.²¹ We recorded a higher percentage of spermatozoa with intact membranes at 3 mg/mL MYO, suggesting that it plays a role of protective agent for plasma membrane stabilization during the freezing process. Two hypotheses have been proposed to explain the beneficial effect of antioxidants on sperm: improved fluidity of the sperm membrane and chemically converted peroxyl radicals to hydroperoxides that are not toxic for sperm.⁹ Acrosomal integrity is important for sperm fertility since an intact acrosome is needed for the incidence of acrosome reactions and sperm penetration into the oocyte.¹¹ In the current study, low concentrations of MYO (2 mg/mL) led to an increase in the percentage of intact acrosomes, including the freezing extender (control). Therefore, the structural and functional integrity of sperm supplements of exogenous antioxidants is essential to provide protection against native-produced ROS in the freezing–thawing process.

We used Annexin ⁻V/PI to increase the detection accuracy of PS translocation as a sign of apoptosis in sperm. The externalization of PS increased during the sperm freezing.¹⁰ We assumed that the addition of MYO to the extender could minimize apoptotic markers such as PS, due to the stabilizing effect of MYO on the sperm plasma membrane. In this regard, MYO can prevent ROS-induced apoptosis by improving mitochondrial function and reducing DNA fragmentation.^13,52 However, our results showed that adding different concentrations of MYO to the freezing extender did not increase the number of viable cells as determined by flow cytometry. Similarly, previous findings showed that addition of MYO to the sperm of normozoospermic did not have an impact on PS externalization and chromatic compactness.⁴⁰

MDA, a key product of polyunsaturated fatty acid peroxidation in the cell, is utilized as an index of oxidative damage. It has been shown that the rate of lipid peroxidation increases in cryopreserved mammalian spermatozoa.⁵³ We found that the supplementation of 3 mg/mL MYO decreased the MDA production in comparison with the control group. In line with our results, supplementation of human sperm with MYO before freezing led to decreased MDA levels.¹³ Interestingly, supplementation of semen extender with 4 mg/mL of MYO was found to result in a higher level of MDA compared with other different concentrations of MYO. It seems that a high concentration of MYO can lead to loss of membrane fluidity and integrity, making sperm more susceptible to lipid peroxidation during the freezing and thawing process.¹³

The results of the present study confirmed that extended supplementation with MYO had no effect on the level of some antioxidant enzymatic activities such as TAC, SOD, and TPA. In line with our results, Kulaksiz et al.³⁷ mentioned that supplementation of ram semen extender with MYO had no effect on levels and activities of SOD. On the contrary, our results showed that supplementation of semen extender with 3 mg/mL MYO can improve CAT activity in sperm after a freeze–thawing process. It has been shown that higher levels of CAT in the semen extender decreased the production of ROS and protected the human spermatozoa against damage from freezing.⁵⁴ This approach improved the chance of natural pregnancy and outcomes of IVF.⁵⁵ These results suggested that low concentrations of MYO may be necessary to improve intracellular antioxidant levels during bovine sperm cryopreservation.

Conclusions

The results revealed that the supplementation of the freezing extender with a lower concentration of MYO resulted in a higher quality of frozen–thawed bull sperm. A high (4 mg/mL) concentration of MYO does not have a good cryoprotective effect. Further research is needed to determine the effect of MYO on the fertility rate following artificial insemination.

Footnotes

Acknowledgments

The authors gratefully acknowledge the financial support from the University of Tabriz. They also thank the staff at the Research Center of Iranian Nahadehaye Dami Jahed (NDJ) Company (Karaj, Iran) for providing use of the bull and facilities utilized in this study.

Authors' Contributions

A.M.: Methodology, data curation, and investigation. R.A.: Conceptualization, investigation, methodology, writing—original draft, project administration, and formal analysis. H.T-N.: Biochemical analysis and methodology. M.R.: Sperm analysis, methodology, and supervision. R-J.J.: Writing—reviewing and editing.

Author Confirmation Statement

Dr. Mohammadi, Dr. Asadpour, Dr. Tayefi-Nasrabadi, Dr. Rahbar, and Dr. Jafari-Joozani are from the University of Tabriz (Tabriz, Iran), where research and education are the primary functions.

Author Disclosure Statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Funding Information

No funding was received for this article.

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