The effects of different levels of Nano selenium on the quality of frozen-thawed sperm in ram

ABSTRACT

The effects of Nano selenium as antioxidant was studied on ram semen. Samples were collected twice a week for 2 months from 4 rams. Extenders were supplemented with Nano selenium (1 and 2 µg/ml) and no antioxidants (control group). The frozen sperm samples were stored for 30 days. Sperm viability, total motility and progressive motility, plasma membrane integrity, abnormal sperms and the damage of acrosome membrane were measured on days 0, 15 and 30 of storage and level of Malondialdehyde on day 30. The results showed that 1 µg/ml Nano-Se significantly increased the percentage of viability, total and progressive motility, plasma membrane integrity (HOST) and decreased acrosome membrane damaged and abnormal sperms compared to 2 µg/ml Nano-Se and control groups (P < 0.01). Storage time of 30 days compared to 0 and 15 days decreased significantly viability, total and progressive motility and plasma membrane integrity (P < 0.01), and increased the percentage of sperms and acrosome membrane damage (P < 0.01). The amount of lipid peroxidation (nmol/ml) in Nano selenium 1 µg (0.508 ± 0.098) and 2 µg (0.878 ± 0.098) was less than the control group (1.17 ± 0.098, P < 0.01). It can be concluded that the addition of 1 µg/ml Nano-Se to extender improved the post-thawing quality and oxidative variables of ram semen during storage.

Highlight

• Nano Selenium reduced MDA after freeze-thawing.

• Nano Selenium improved motility rate after freeze-thawing.

• Nano Selenium improved plasma membrane integrity after freeze-thawing.

KEYWORDS:

• Antioxidant; oxidative stress
• nano selenium
• ram sperm

1. Introduction

The protocol of freezing-thawing process of semen in mammals has undergone a great evolution (Lebeuf et al. 2000). Freezing process caused ultrastructural, biochemical and functional changes in sperm membranes resulting in decreased viability after the freeze-thawing process (Meagher and Fitzgerald 2000). Freezing of ram sperm confront with problems such as decrease in viability and motility. Therefore, using an appropriate freezing environment that can protect sperm from damage and maintain the viability and motility after freeze-thawing is an important step in the application of artificial insemination in ram (Evans and Maxwel 1989). Almost all cells contain substances and enzymes that neutralize the toxic effects of various reactive oxygen species (ROS), but the amount of antioxidants in the sperm is lower than that of other cells and is more vulnerable to oxidative stress (Sreegith et al. 2005). Plasma membrane of mammalian sperm, fish and birds is full of unsaturated fatty acids and phospholipids. High amounts of unsaturated fatty acids with multiple bonds (PUFAs¹) cause sperm to become very sensitive to lipid peroxidation, which is positively correlated with male infertility (Surai et al. 1998).

Oxidation of fatty acids leads to the production of reactive oxygen species (ROS). These radicals are necessary in normal conditions for some activities and sperm physiological processes but excessive production of ROS in the sperm can reduce membrane fluidity, DNA fracture, damage to proteins, and ultimately reduced sperm motility and fertility (Peris et al. 2007). Protection of the balance between the production of ROS and the antioxidant system is essential to maintain the survival of the sperm, which is balanced by the storage and freezing of the sperm (Breque et al. 2003). In order to prevent increased sperm lipid oxidation and increase the sperm storage time in the laboratory, the use of natural antioxidants in the sperm diluent environment seems necessary. Selenium can be mentioned as the best natural and effective antioxidant in preventing the oxidation of spermatozoa. Selenium is one of the essential minerals of the body that has many physiological effects. For example, as one of the components of Selenoproteins in Glutathione Peroxidases (GPx) and Thioredoxin reductase (TrxR), both of which have different biological roles. Oxidative damage is one of the effective factors in male fertility that selenium plays an important role in protecting from these injuries and in maintaining male fertility. Selenium is a cofactor or activates the Glutathione Peroxidase enzyme, which is one of the strongest natural antioxidants, and catalyzes the decomposition of lipid peroxidases and hydrogen peroxide (Hill et al. 1996). In a study conducted on Guinea pigs, the long-term shortage of selenium in the diet of guinea pigs has been shown to reduce the concentration and sperm motility and the appearance of cytoplasmic droplets in the sperm (Liu et al. 1982). However, one of the limiting factors for selenium consumption is bioavailability (the rate of entry into blood circulation and tissues) and its toxicity (Tarze et al. 2007). Therefore, the use of other forms of selenium that has less toxic and more beneficial, is more appropriate. Selenium nanoparticles were appropriate with low toxicity compounds and bioavailability, which greatly increase the production of Selenoproteins in the body (Peng et al. 2007). Regarding the properties of Nano selenium in reducing oxidative damage, it seems that the addition of antioxidants to the sperm diluent environment would preserve the qualitative characteristics of ram semen after the freeze-thawing process.

The objective of the present study was to evaluate the effect of the addition of Nano selenium on the quality of semen during the freeze-thawing process.

2. Material and method

2.1. Animal and semen collection

This research was conducted on four Ghezel breed of 2–3-years-old rams with an average weight of 58 ± 2 kg. Semen samples were collected by an artificial vagina from each ram twice a week for 2 months. The rams were kept in an indoor area as a group and had free access to water and food and licking salts. The sperm samples after collecting were immediately transferred to the laboratory. Fresh sperms have been studied in terms of volume, concentration, total motility, viability and morphology, and only samples with a concentration of over 3 billion sperm and a progressive motility of over 70% were used for dilution.

2.2. Extender preparation and sperm dilution

An aliquot was diluted with Tris (2.71 g), citric acid (1 g), fructose (1.4 g), penicillin (100,000 IU) and streptomycin (100 mg) in 100 ml distilled water. Then 73 ml of this solution was mixed with glycerol (7 ml), egg yolk (20 ml). Two ml of the diluents were spilled into 3 sterile tubes and antioxidants (Nano selenium size was 30 nm) were added to each tube based on the experimental groups:

Control group: diluent based on Tris$\begin{array}{c}\mathrm{NS}1\mathrm{group}:1\mathrm{ml}\mathrm{diluent}\mathrm{based}\mathrm{on}\mathrm{Tris}+1\mu \mathrm{g}\mathrm{of}\mathrm{Nano}\mathrm{selenium}\\ \mathrm{NS}2\mathrm{group}:1\mathrm{ml}\mathrm{diluent}\mathrm{based}\mathrm{on}\mathrm{Tris}+2\mu \mathrm{g}\mathrm{of}\mathrm{Nano}\mathrm{selenium}\end{array}$

2.3. Cryopreservation procedure

After mixing antioxidants with diluent, sperm samples were added to each tube with ratio 1:10 (240 × 10⁶ progressive motile sperm). Sixteen French straws (0.25 ml) were drawn and stored in a refrigerator for 90 min to reach 5°C, and then were placed at 4 cm above the liquid nitrogen for 8–10 min and then immersed in the liquid nitrogen for storage(Hozyen et al. 2019). Samples were evaluated on days 0, 15 and 30 of the experiment. The evaluated traits included the total motility and progressive motility percent, the viability percent, sperm membrane integrity, morphological characteristics of the sperm, the damage of the acrosome membrane of the sperm at all times, also, measurement the Malondialdehyde of the samples were used to measure the amount of lipid peroxidation of the plasma membrane of sperm on day 30 of the experiment

2.4. Assessment of sperm quality

2.4.1. Analysis of standard semen parameters by CASA

Sperm motility and motion variables were estimated by computer-assisted sperm motility analysis (CASA, video test-sperm 3.1, St. Petersburg, Russia). For evaluation by the system thawing was carried out on days 0, 15 and 30. One straw of each treatment and replicates was thawed, and was assessed individually. Five fields, which were randomly selected by the computer, were analysed for each semen sample. The thawed semen sample was analyzed for the following eight motility variables: Total sperm motility (TM, %); progressive motility (PM, %); average path velocity (VAP, µm/s); straight line velocity (VSL, µm/s); curvilinear velocity (VCL, µm/s); amplitude of lateral head displacement (ALH, µm); straightness (STR, %); linearity (LIN, %); using a phase-contrast microscope (Labomed LX400; Labomed Inc., Culver City, CA, U.S.A.) (Mostafapor and Farrokhi Ardebili 2014).

2.4.2. Sperm viability parameters

Eosin-nigrosin colouring was used to evaluate the percentage of live and dead sperm. For this purpose, a drop of eosin-nigrosin colour, which had already reached 38°C, was placed on a slide and then 10 µl of the diluted sperm solution with sodium citrate 2.9% was added with a ratio of 1–100 and prepared with another slide. Viability was assessed by counting 200 cells at a magnification of ×400 under phase-contrast microscopy. Only sperm with strict exclusion of stain were counted as viable and sperm displaying partial or complete purple staining were considered nonviable (Zhanghaneh et al. 2013).

2.4.3. Abnormal sperms

To calculate the percentage of abnormal sperm, eosin-nigrosin colouring was used. Sperms were studied by using a phase-contrast optical microscope in five different fields (Ntemka et al. 2019).

2.4.4. Sperm plasma membrane integrity

Plasma membrane functionality of spermatozoa after freeze-thawing was assessed by hypo-osmotic swelling (HOS) test, according to the previously described method (Santiago-Moreno et al. 2009) with minor modifications. The HOS test predicts membrane integrity by determining the ability of the sperm membrane to maintain equilibrium between the sperm cell and its environment. The influx of the fluid due to hypo-osmotic stress causes the cell to expand and bulge, especially in the tail. This change can be readily observed with a phase-contrast microscope. HOS test involves incubating a portion of semen (10 µl containing 400 × 10⁴ sperm cells) and 100 mOsm sodium citrate solution (50 µl) at 38°C for 10 min. A sample was then prepared on a slide and the percentage of sperm cells with a swollen ‘bubble’ around the curled flagellum was determined by counting 200 cells/slide with the use of phase-contrast microscopy (1000× magnification).

2.4.5. Damage of the acrosome membrane

To determine the damage of acrosome membrane, the sample was diluted 1:100 with sodium citrate 2.9% at 37°C and stained with eosin-nigrosin. Then was observed with a magnification of 1250× under the microscope. Between the dead sperms, 20 sperms were counted and their acrosome were examined. Finally, the ratio of damage acrosome in dead sperm was calculated. The method was based on the (WHO 2010) guidelines.

2.4.6. Determination of sperm lipid peroxidation

Thiobarbituric acid test was used to determine the amount of lipid peroxidation of spermatozoa (Esterbauer and Cheeseman 1990). In this test, the level of Malondialdehyde or MDA (one of the final products of lipid peroxidation) is measured as an indicator of lipid peroxidation by the reaction of Thiobarbituric acid. In order to precipitate the proteins, 1 ml semen from each treated group was mixed with 2 ml of Trichloroacetic acid in a sterile tube on day 30 of experiment. Then, 1 ml the Hydroxytoluene-boiled solution or BHT² (2% in ethanol) and 1 ml of EDTA³ were added to the solution for preventing the happening lipid peroxidation during the experiment. Then the tubes were centrifuged for 15 min at 1200× rounds. After completion of the centrifuge, 1 ml from the top of the solution was mixed with 1 ml of Thiobarbituric acid (0.67%) in a micro tube and placed in 95°C water for 20 min. After cooling the samples, the absorbance of the samples was measured at 532 nm by a spectrophotometer.

2.5. Statistical analysis

Data were analysed using the Glm and Mixed procedures of SAS 9.2 software. For comparison of means, the least square means (LSM) were compared by Tukey–Kramer method. Differences with values of P < 0.01 were considered to be statistically significant. The resulted were presented as the LSM ± SEM. The model used in the present study was as follows: Y_ijk = µ + T_i + Ramj + Timek + eijk where Y_ijk is observation records, µ is the mean of population, T_i is fixed effect of treatment (Nano selenium, i = 1,2), Ram_j is the random effect of ram that was nested within treatment, Time_k is the effect of time(date)(k = 0,15,30) and eijk is the random residual error.

3. Results

The collected semen samples were initially characterized in terms of their volume, percentage of viability sperms, total motility and progressive motility percent, abnormal sperms percent, concentration and wave motion to assess their suitability for freezing (Table 1).

Table 1. Descriptive statistics of ram sperm parameters before dilution.

Variable	Count	Mean	Minimum	Maximum	Standard deviation	Coefficient of variation
Volume (ml)	24	1.54	1	2	0.51	23.01
Wave motion (1–5)	24	4.87	4	5	0.34	6.93
Total motility (%)	24	86.25	80	91	3.35	3.89
Progressive motility (%)	24	80.61	73.60	86.40	3.18	3.94
Viability sperms (%)	24	90.33	84	95	3.18	3.52
Abnormal sperms (%)	24	2.92	2	4	0.65	22.42
Concentrate (×10^9)	24	3.18	2.16	4.17	0.51	16.04

Descriptive statistics between evaluated traits after the sperm freeze-thawing process has been showed in Table 2. In the present study, the results of 30 days of storage and subsequent evaluation for characteristics such as the percentage of abnormal sperm, the percentage of live sperm, motility, acrosome membrane integrity, Hypo-osmotic swelling test (HOST) and Malondialdehyde levels has been investigated.

Table 2. Descriptive statistics of evaluated traits of frozen-thawed ram sperms.

Variable	Count	Mean	Minimum	Maximum	Standard deviation	Coefficient of variation
Total motility (%)	216	65.49	37	86	0.734	16.48
Progressive motility (%)	216	59.23	31.82	80.84	0.726	19
Viability sperms (%)	216	68.68	40	90	0.728	15.58
Abnormal sperms (%)	216	6.71	2	16	0.189	41.51
Acrosome membrane damage (%)	216	6.25	3	13	0.147	34.65
HOST test (%)	216	62.18	35	84	0.733	17.32
Malondialdehyde (nm/ml)	39	0.822	0.252	2.411	0.060	45.93

The results showed that the diluent containing different levels of Nano selenium compared to the control group significantly altered the sperm motility characteristics after freezing. According to Table 3, diluent containing 1 and 2 µg/ml Nano selenium, resulted in a significant increase in ram sperm motility than the control group 74.81 ± 0.486 and 65.44% ±0.486, respectively, after the freeze-thawing process (P < 0.01). Also, diluents containing 1 and 2 µg/ml Nano selenium significantly increased the progressive motility (PM), VAP, VSL, VCL, ALH, STR and LIN of sperm after freezing and thawing compare with the control group (P < 0.01). The highest percentage of PM was observed in day zero (67.49 ± 0.533) and the lowest was the day of 30 experiment (52.13 ± 0.533) (P < 0.01). Also, other parameters of CASA (VAP, VSL, VCL, ALH, STR and LIN) significantly decreased during storage time (P < 0.01). The results (Table 4) showed that the percentage of TM and PM has significantly decreased during storage time (P < 0.01). The diluents containing 1 and 2 µg/ml Nano-selenium had a significant increase in the viability of sperm with a mean of 77.92 ± 0.467 and 68.62 ± 0.467, respectively, than the control groups after the freezing process (P < 0.01). The effect of time of storage on the viability of frozen sperm showed that the viability of sperm after freezing and thawing on days 0, 15 and 30 was significantly decreased with a mean of 76.40 ± 0.467, 68.88 ± 0.467 and 60.77 ± 0.467, respectively, (P < 0.01). 1 and 2 µg/ml Nano selenium with mean 5.013 ± 0.188 and 6.56 ± 0.188, respectively, significantly improved the sperm morphology and reduction in the percentage of abnormal spermatozoa from the control group after freezing (P < 0.01). The results showed that duration storage had a negative and significant effect on the sperm morphology, so that the least percentage of sperm abnormality was related to the day zero (4.75 ± 0.188) and the highest percentage was related to day 30 of the experiment (8.91 ± 0.188) (P < 0.01). The results of this study showed that the percentage of Sperms with plasma membrane integrity (positive HOST) during the freeze-thawing process in diluents containing 1 and 2 µg of Nano selenium had a positive and significant increase compared to the control group (P < 0.01). The results of this experiment showed that the percentage of sperm membrane integrity (positive HOST) during the cooling process was significantly decreased in all treatments (P < 0.01). The least percentages of sperm membrane integrity were observed in day 30 and the highest percentage was observed in day of zero (P < 0.01).

Table 3. Effect of different levels of nano selenium on ram sperm after thawing process.

Trait	Treatment
	control	Nano selenium 1 µg	Nano selenium 2 µg
Total motility (%)	56.22 ± 0.486^c	74.81 ± 0.486^a	65.440.486^b
Progressive motility (%)	50.66 ± 0.533^c	68.56 ± 0.533^a	59.78 ± 0.533^b
(VAP, µm/s)	41.56 ± 2.09^c	52.36 ± 2.09^a	46.83 ± 2.09^b
(VSL, µm/s)	40.03 ± 1.65^c	58.09 ± 1.65^a	50.37 ± 1.65^b
(VCL, µm/s)	98.02 ± 4.37^c	149.93 ± 4.37^a	121.12 ± 4.37 ^b
(ALH, µm)	2.58 ± 0.16^c	4.87 ± 0.16^a	3.63 ± 0.16^b
(STR, %)	21.86 ± 0.019^c	38.42 ± 0.019^a	30.59 ± 0.019^b
(LIN, %)	0.33 ± 0.016^c	0.54 ±0.016^a	0.42 ± 0.016^b
viability (%)	59.47 ± 0.467^c	77.92 ± 0.467^a	68.62 ± 0.467^b
Abnormality (%)	8.56 ± 0.188^c	5.01 ± 0.188^a	6.56 ± 0.188^b
acrosome membrane damage (%)	7.63 ± 0.271^c	4.98 ± 0.271^a	6.15 ± 0.271^b
plasma membrane integrity (%)	52.93 ± 0.475^c	71.41 ± 0.475^a	62.22 ± 0.475^b
Malondialdehyde (nmol/ml)	1.17 ± 0.098^c	0.508 ± 0.098^a	0.878 ± 0.098^b

Note: Non-like letters in the row indicate a significant difference (P < 0.01).

Table 4. Effect of storage time on frozen ram sperm characteristic.

Trait	Time
	Day 0	Day 15	Day 30
Total motility (%)	73.36 ± 0.486^a	65.56 ± 0.486^b	57.55 ± 0.486^c
Progressive motility (%)	67.49 ± 0.532^a	59.38 ± 0.532^b	52.13 ± 0.532^c
VAP, (µm/s)	55.47 ± 1.89^a	50.63 ± 1.89^b	43.02 ± 1.89^c
VSL, (µm/s)	60.12 ± 1.73^a	51.42 ± 1.73^b	39.87 ± 1.73^c
VCL, (µm/s)	153.01 ± 4.89^a	141.20 ± 4.89^b	123.01 ± 4.89^c
ALH, (µm)	5.07 ± 0.19^a	4.12 ± 0.19^b	2.93 ± 0.19^c
STR, (%)	42.57 ± 0.02^a	30.86 ± 0.02^b	22.17 ± 0.02^c
LIN, (%)	0.68 ± 0.019^a	0.51 ± 0.019^b	0.43 ± 0.019^c
Viability (%)	76.40 ± 0.467^a	68.88 ± 0.467^b	60.77 ± 0.467^c
Abnormality (%)	4.75 ± 0.188^a	6.48 ± 0.188^b	8.91 ± 0.188^c
acrosome membrane damage (%)	4.68 ± 0.271^a	6.12 ± 0.271^b	7.97 ± 0.271^c
plasma membrane integrity (%)	70.19 ± 0.475^a	62.06 ± 0.475^a	54.30 ± 0.475^a

Note: Non-like letters in the row indicate a significant difference (P < 0.01).

The results of the effect of adding Nano-selenium on the health of the sperm acrosome membrane showed that the diluents containing 1 and 2 µg/ml of Nano-selenium had (4.98 ± 0.271 and 6.15 ± 0. 271, respectively) reduced damages of acrosome membrane of sperm after freezing and thawing than the control group (P < 0.01). The reason for this reduction can be related to reducing the amount of ROS by adding antioxidants to the diluent. The percentage of spermatozoa with an unhealthy acrosome membrane has also increased significantly during storage time, so that on day zero of experiment, the percentage of unhealthy acrosome membrane of sperm was 4.68 ± 0.271 and on the 30th day of the experiment was 7.97 ± 0.271 (P < 0.01).

The results of Thiobarbituric acid test showed that the amount of Malondialdehyde on day 30 of experiment was significantly lower in Nano-selenium 1 and 2 µg groups than in the control group (P < 0.01).

4. Discussion

The range of variations in the amounts of each of the quantitative and qualitative characteristics of the sperm have already been reported by various reports about the volume of ejaculation (0.6–1.6 ml), sperm concentration (2.6–5.5 × 10⁹), and viability sperm percent (60–90%), the wave motion (4–4.5), the percentage of abnormal sperm (10–14%) (Gundogan 2007). The present examination agreed with above-mentioned reports (Table 1). The initial evaluation of semen samples showed their good quality for freeze-thawing and it was proper for the purpose of this study.

In most studies, the viability rate of sperm in the samples is greater than the percentage of total motility (Sansone et al. 2000; Peris et al. 2007; Mukherjee et al. 2016), that our findings were matches to them.

Cryopreservation reduces the functional and structural integrity of ram spermatozoa, and is associated with reactive oxygen species (ROS) production. Oxidative stress during freezing of mammalian semen can cause functional and structural damage to spermatozoa involving ROS-mediated pathways (Buyukleblebici et al. 2014).

These PUFA particularly of dead spermatozoa bind with oxygen resulting in the production of high level of reactive oxygen species (Banday et al. 2017). The present study showed that using of Nano-Se as a biological antioxidant had a cryoprotective effect on ram sperm and significantly increased the quality of sperm by enhancing motility, viability and membrane integrity. Also significantly reduced the amount of MDA, damage of acrosome membrane and abnormal sperm after the freeze-thawing process. Current findings agreed with most previous studies (Domoslawska et al. 2015; Khoram Abadi et al. 2017; Hozyen et al. 2019; Khalil et al. 2019 and Dorostkar et al. 2013). Selenium, an essential trace element, play important roles in mammalian biology. The best-known role of Selenium is attributed to its presence in glutathione peroxidase (GP_x) and Selenoproteins (Wang et al. 2007). It has also been shown that a reduction in the content of Gluthatione, which plays a role in the antioxidant defence system of sperm, is induced by the freezing and thawing process. Also, in this research, it was demonstrated that the addition of different levels of Nano Selenium to freezing medium was able to reduce free radicals and increase the motility and viability of post-thaw sperm (Keshtgar et al. 2016).

The reduced ram semen MDA when using Nano-Se in the present study is agreed with findings in previous studies (Safa et al., 2016; Morbat et al. 2017; Hozyen et al. 2019; Khalil et al. 2019 and Abd-Allah and Hashem 2015).

Cryopreservation exposes spermatozoa to severe osmotic stress and induces damage of membranes and acrosomes affecting the fertilization capacity of spermatozoa (Hozyen et al. 2019). The obtained results of the present study revealed that modification of extender with Nano-Se improved plasma membrane integrity in ram semen. This result is in agreement to that recently observed in rams by (Hozyen et al. 2019) who reported that supplementation of SeNP_s (0.5 and 1 µg/ml) to extender significantly improved acrosome membrane integrity.

5. Conclusion

In conclusion, the findings in the present study indicated that semen enrichment with Nano selenium would decrease spermatic membrane lipid peroxidation and oxidative damage to sperm. Also decrease sperm acrosome membrane damage and subsequently sperm quality properties increase such as motility, viability, plasma membrane integrity and during freezing 30 days.

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Notes

1. Polyunsaturated fatty acids.

2. Butylated hydroxytoluene.

3. Ethylenediaminetetraacetic acid.