Effects of Different Antifreeze Protective Agents on Hu Sheep Semen Storage at 4 ˚C

Iranian Journal of Applied Animal Science

دوره 13، شماره 1، خرداد 2023، صفحه 77-87   

نوع مقاله: Research Articles

نویسندگان

L. Zhang؛ X. Sun؛ Y. Kang؛ T. Sohail؛ X. Wang؛ Y. Li^*

College Animal Science and Technology, Yangzhou University, Yangzhou, China

چکیده

The present study aimed to investigate the effects of different antifreeze protectants on the preservation of Hu sheep semen at 4 ˚C. The semen was diluted with Tris extender at room temperature, supplemented with dimethyl sulfoxide (DMSO) (0, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%), glycerol (1.5%, 3.0%, 4.5% and 6.0%), glycol (2.50%, 5.00%, 7.50% and 10.00%), skimmed milk powder (SMP) (0.50%, 1.50%, 2.50% and 3.00%), soy lecithin (SL, 0.15%, 0.30%, 0.45% and 0.60%) and five optimal concentrations of antifreeze protectants (0.4% DMSO, 4.5% glycerol, 7.5% glycol, 1.5% SMP and 0.15% SL), and stored at 4 ˚C refrigerator. Spermatozoa motility parameters (spermatozoa viability, spermatozoa progressive motility, straight line velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP)) were evaluated during the preservation of semen. The addition of antifreeze protectants, especially the 0.4% DMSO, 4.5% glycerol, 7.50% glycol, 1.50% SMP and 0.15% SL exerted the best effects on spermatozoa viability and progressive motility compared to the spermatozoa without antifreeze protectants (control group). In the experiment of antifreeze protective agents with the optimal concentration, the spermatozoa viability and progressive motility of 1.5% SMP were significantly higher than those of other antifreeze groups during storage. In conclusion, the addition of 1.5% SMP to semen spermatozoa preserved at 4 ˚C refrigerator can most effectively enhance the semen preservation quality compared to the other optimal concentration of antifreeze protectant groups.

کلیدواژه‌ها

computer‐assisted spermatozoa analysis؛ DMSO؛ glycerol؛ glycol؛ SMP؛ soy leci-thin

اصل مقاله

INTRODUCTION

Assisted reproductive technologies (ART) including artificial insemination (AI), in vitro fertilization (IVF) and embryo transfer (ET), which are used to collect and process oocytes, spermatozoa and embryos in vitro to improve the chances of conception (Esteves et al. 2019). In the field of livestock, ART can quickly spread and preserve the genetic material of cherished species and avoid the extinction of endangered species (Daly et al. 2020; Figueiredo et al. 2020). At the same time, ART can also improve breeding efficiency, reduce breeding costs and reduce the risk of disease transmission (Rodriguez, 2012). The core of ART is to prolong the preservation time of semen and to improve the quality of semen preservation by adding various substances, such as antioxidants (taurine (Zhang et al. 2021)), antibacterial agents (nisin (Shin et al. 2016)), antifreeze protection agents (yolk (Garde et al. 2008)), etc. In the process of semen preservation, with the influence of spermatozoa metabolic activity and external environment, it will inevitably be affected by reactive oxygen species (ROS) and bacterial microorganisms (Rezaie et al. 2021; Tvrdá et al. 2021). Therefore, it is very important to add antioxidants that can remove ROS and antimicrobial agents that inhibit the proliferation of microorganisms. Semen is easily affected by low temperature and cold during cryopreservation which seriously affects the quality of semen preservation (Gloria et al. 2020). Therefore, the addition of antifreeze protectant is very important to improve the preservation quality of semen. Antifreeze protectant is divided into permeable antifreeze protectant and non-permeable antifreeze protectant (Diaz et al. 2019). Dimethyl sulfoxide (DMSO), glycerol, glycol and soy lecithin (SL) are permeable antifreeze protectants. Skimmed milk powder (SMP) and bovine serum albumin are non-permeable antifreeze protectants. The permeable antifreeze protectant can pass through the spermatozoa cell membrane and enter into the spermatozoa, rearrange the fat and protein on the spermatozoa plasma membrane, increase the fluidity, reduce the formation of intracellular ice crystals, and then improve the survival rate after freezing and thawing (Blanco et al. 2011). Non-permeable antifreeze protectant cannot pass through the spermatozoa cell membrane, but can only play a role in the outside. It can change the semen osmotic pressure, make the cells dehydrated, reduce the possibility of the formation of intracellular ice crystals, and then improve the spermatozoa survival rate after thawing (Rosato and Iaffaldano, 2013). The most commonly used antifreeze protectant is egg yolk, but egg yolk is an animal-derived additive that possess a risk of biological infection as it may contain toxins, viruses, etc (Yildiz et al. 2013; Mehdipour et al. 2016). On the other hand, the properties of egg yolk may be variable depending on the breeds and feeding conditions. Therefore, it is necessary to find a safe, consistent and effective antifreeze protectant that can replace yolk. It is reported that different concentrations of DMSO, glycerol, glycol and SL play different roles in different species, such as Australian flat oyster (Hassan et al. 2017), bison (Hussain et al. 2013), stallion (Gonzalez et al. 2019), dog (Rota et al. 2006), Buffalo (Swelum et al. 2011), goat (Küçük et al. 2014), human (Reed et al. 2009) and Brown-bear (Alvarez et al. 2013). However, there are few reports on DMSO, Glycerol, Glycol and SL to be used as antifreeze protectants for preservation of Hu sheep semen at 4 ˚C. Some researches reported that the addition of 6% glycerol could improve the cryopreservation of Hu sheep semen. Also reported that the addition of 6% glycerol and some egg yolk could improve the cryopreservation of Hu sheep semen. Therefore, the present study aimed to determine the effect and the optimum added concentration of DMSO, Glycerol, Glycol, SL and SMP for preservation of Hu sheep semen of 4 ˚C.

 

MATERIALS AND METHODS

All experimental procedures were approved bythe Yangzhou University for protection of experimental animals. (SYXK [Su] 2017-0044).

 

Experimental design

Effect of dimethyl sulfoxide (DMSO) (0, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%), glycerol (1.5%, 3.0%, 4.5% and 6.0%), glycol (2.50%, 5.00%, 7.50% and 10.00%), SMP (0.50%, 1.50%, 2.50% and 3.00%), SL (0.15%, 0.30%, 0.45% and 0.60%) and five optimal concentrations of antifreeze protectants (0.4% DMSO, 4.5% glycerol, 7.5% Glycol, 1.5% SMP and 0.15% SL) on spermatozoa motility parameters [Spermatozoa viability, spermatozoa progressive motility, straight line velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP)] of stored Hu sheep semen at 4 ˚C were evaluated every day during preservation and compared with spermatozoa motility parameters without antifreeze protectants (control group).

 

Animals and semen collection

Five sexually mature Hu rams, aged 2-3 years and body weighted about 75 kg, were used in this study from October to December 2021. Hu sheep were fed by stallfeeding. The rams were fed 0.2 kg concentrate/every time, twice a day, and ad libitum hay and water. Alfalfa was supplemented every day and feeding of corn kernels was increased during semen collection. The sheep shed is equipped with suitable types of licking bricks to supplement various trace elements. These rams were in good condition and free from any disease. A total number of 90 ejaculates were collected from the rams twice weekly with the artificial vagina. The semen volume of each ram was from 0.5 mL to 1.0 mL, which had normal smell and color. The quality assessment was carried for the motility (>80%), the spermatozoa deformity (<15%) and the spermatozoa concentration (2.5×10⁹/mL). The qualified semen was diluted for conducting the experiments.

 

Semen extender

The base extender consisted of 3.07 g Tris, 2.0 g fructose, 1.64 g citric acid, 31.18 mg penicillin and 69.44 mg streptomycin in 100 mL distilled water. DMSO was added to the base extender at concentrations of 0.10%, 0.20%, 0.30%, 0.40% and 0.50%, while the control was the base extender without DMSO. Glycerol was added to the base extender at concentrations of 1.50%, 3.00%, 4.50% and 6.00%, while the control was the base extender without glycerol. Glycol was added to the base extender at concentrations of 2.50%, 5.00%, 7.50% and 10.00%, while the control was the base extender without glycol. SMP was added to the base extender at concentrations of 0.50%, 1.50%, 2.50% and 3.00%, while the control was the base extender without SMP. Soy lecithin was added to the base extender at concentrations of 0.15%, 0.30%, 0.45% and 0.60%, while the control was the base extender without SL. When preparing SL extender, it is necessary to dissolve 30 min in a water bath at 60 ˚C. Finally, all extenders were fully oscillated so that they are completely dissolved. The pH of each extender was between 7.4 and 7.5.

 

Dilution and evaluation of semen

The semen was diluted at 1:9 ratio with the extender preheated at 37 ˚C and preserved in 2 mL centrifuge tube. The diluted semen was kept at room temperature for 1 h. Then wrap it with 8 layers of cotton and put it in the refrigerator at 4 ˚C after wrapping. During semen preservation, the centrifuge tube for semen preservation is slowly reversed every day to prevent spermatozoa deposition. The semen preserved by 20 μL was diluted at 1:4 ratio and placed in a 37 ˚C water bath for 3min. Then 1.8 μL was dropped on a special computer spermatozoa counting board and placed on a 37 ˚C constant temperature stage. Computer-assisted spermatozoa analyse (ML-608JZ II Mailang, Nanning, China) (CASA) was used to analyse spermatozoa motility parameters such as spermatozoa viability (%), spermatozoa progressive motility (%), VSL (μm/s), VCL (μm/s) and VAP (μm/s).

 

Statistical analysis

The experiment was repeated for six times. Data were analyzed using SPSS 25.0 statistical software (SPSS, 2011). The Shapiro–Wilk test was performed to detect whether the data conform to the normal distribution. One-way ANOVA tests were performed to assess the difference in these parameters. Significance was set at P ≤ 0.05 unless otherwise specified. The results are expressed as the mean ± SEM.

 

RESULTS AND DISCUSSION

The effects of different concentrations of DMSO on Hu sheep spermatozoa motility parameters during storage at 4 ˚C are shown in Table1. The spermatozoa viability in the 0.4% DMSO supplemented semen was the highest and significantly (P≤0.05) higher than that of the 0.5% group within 1 to 3 days. The spermatozoa viability of the 0.4% group was significantly (P≤0.05) higher than that of the other groups within 4 to 6 days. The spermatozoa progressive motility of the 0.4% group was the highest and significantly (P≤0.05) higher than that of the 0.5% group on the 1^st and 3^rd day. The spermatozoa progressive motility of the 0.4% group was significantly (P≤0.05) higher than that of the other groups within 4 to 6 days. The spermatozoa VSL of the 0.4% group was not significantly (P>0.05) different from the other groups on the 4th and 6th day. The spermatozoa VCL and VAP of the 0.4% group were higher (P>0.05) than that of the other groups within 4 to 6 days. The effects of different concentrations of glycerol on Hu sheep spermatozoa motility parameters during storage at 4 ˚C are shown in Table 2. The spermatozoa viability of the 4.5% group was the highest and significantly (P≤0.05) higher than that of the control group within 1 to 4 days. The spermatozoa viability of the 4.5% group was significantly (P≤0.05) higher than that of the other groups within 5 to 6 days. The spermatozoa progressive motility of the 4.5% group was the highest and significantly (P≤0.05) higher than that of the control and 1.5% groups within 3 to 6 days. The spermatozoa VSL of the 4.5% group was higher (P>0.05) than that of the control group within 1 to 5 days and significantly (P≤0.05) higher than that of the control group on the 2^nd, 4^th and 5^th day. The spermatozoa VCL and VAP of the 4.5% group were significantly (P≤0.05) higher than that of the control group within 2 to 5 days. The effects of different concentrations of glycol on Hu sheep spermatozoa motility parameters during storage at 4 ˚C are shown in Table 3. The spermatozoa viability and progressive motility of the 7.5% group were significantly (P≤0.05) higher than that of the control group within 1 to 6 days and significantly (P≤0.05) higher than that of the 2.5% group within 1 to 4 days. The spermatozoa VCL and VAP of the 7.5% group were significantly (P≤0.05) higher than that of the control group on the 1st day. The spermatozoa VSL, VCL and VAP of the 7.5% group were significantly (P≤0.05) higher than that of the other groups on the 3^rd day. The effects of different concentrations of SMP on Hu sheep spermatozoa motility parameters during storage at 4 ˚C are shown in Table 4. The spermatozoa viability of the 1.5% group was the highest and significantly (P≤0.05) higher than that of the control group on the 1st, 2nd, 4th and 6th day. The spermatozoa viability of the 1.5% group was significantly (P≤0.05) higher than that of the other groups on the 3^rd and 5^th day. The spermatozoa progressive motility of the 1.5% group was significantly (P≤0.05) higher than that of the control group within 1 to 5 days. The spermatozoa progressive motility of the 1.5% group was significantly (P≤0.05) higher than that of the other groups on the 6^th day. The spermatozoa VSL of the 1.5% group was significantly (P≤0.05) higher than that of the control group on the 3^rd day. The spermatozoa VSL of the 1.5% group was higher (P>0.05) than that of the control group within 4 to 6 days.

 

Table 1 Effects of different concentrations of DMSO on Hu ram spermatozoa motility parameters stored at 4 ˚C

VSL: straight line velocity; VCL: curvilinear velocity and VAP: average path velocity.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

 

The spermatozoa VCL and VAP of the 1.5% group were higher (P>0.05) than that of the control group within 1 to 2 days. The spermatozoa VCL and VAP of the 1.5% group were significantly (P≤0.05) higher than that of the control group within 3 to 5 days. The effects of different concentrations of SL on Hu sheep spermatozoa motility parameters during storage at 4 ˚C are shown in Table 5. The spermatozoa viability and progressive motility of the 0.15% group were significantly (P≤0.05) higher than that of the other groups within 1 to 6 days. The spermatozoa VSL of the 0.15% group were significantly (P≤0.05) higher than that of the control group on the 1^st day. The spermatozoa VSL of the 0.15% group were higher (P>0.05) than that of the control group on the 2^nd and 6^th day. The spermatozoa VCL and VAP of the 0.15% group were the highest and significantly (P≤0.05) higher than that of the control and 0.6% groups within 1 to 6 days. The effects of different antifreeze protective agents on Hu sheep spermatozoa motility parameters during storage at 4 ˚C are shown in Table 6. The spermatozoa viability and progressive motility of the 1.5% SMP group were the highest within 1 to 6 days and significantly (P≤0.05) higher than that of the other groups within 1 to 2 days and 4 to 5 days. The spermatozoa viability of the 0.15% SL group was significantly (P≤0.05) higher than that of the 0.4% DMSO, 4.5% glycerol and 7.5% glycol groups within 1 to 3 days. The spermatozoa viability of the 0.4% DMSO group was significantly (P≤0.05) lower than that of the other groups within 4 to 5 days.

 

Table 2 Effects of different concentrations of glycerol on Hu ram spermatozoa motility parameters stored at 4 ˚C

VSL: straight line velocity; VCL: curvilinear velocity and VAP: average path velocity.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

 

However, there was no significant difference among other groups (except 1.5% SMP group). The spermatozoa viability and progressive motility of the 4.5% glycerol group were significantly (P≤0.05) higher than that of the 0.4% DMSO, 7.5% Glycol and 0.15% SL groups on the 6th day. The spermatozoa progressive motility of the 0.15% SL group was significantly (P≤0.05) higher than that of the 0.4% DMSO, 4.5% glycerol and 7.5% glycol groups within 2 to 3 days. The spermatozoa progressive motility of the 0.4% DMSO group was significantly (P≤0.05) lower than that of the other groups on the 5^th day. The spermatozoa VSL, VCL and VAP of the 1.5% SMP group were significantly (P≤0.05) higher than that of the 0.4% DMSO and 7.5% glycol groups on the 4^th day. The spermatozoa VSL, VCL and VAP of the 1.5% SMP group were significantly (P≤0.05) higher than that of the 0.4% DMSO group but was no significantly different from the other groups on the 5^th day. In this study, the effects of DMSO, glycerol, glycol, SMP and SL on the preservation of Hu sheep semen at 4 ˚C were analyzed. It was showed that adding appropriate concentration of the above five cryoprotectants can improve spermatozoa viability, progressive motility, and movement rate. It is reported that DMSO can penetrate the spermatozoa cell membrane and reduce the concentration of salt at a certain temperature, thus reducing the damage of spermatozoa at this temperature (Lovelock, 1953). In rabbits, Vicente and Viudes (1996) reported that the addition of DMSO could improve the cryopreservation of rabbit semen.

 

Table 3 Effects of different concentrations of glycol on Hu ram spermatozoa motility parameters stored at 4 ˚C

 

VSL: straight line velocity; VCL: curvilinear velocity and VAP: average path velocity.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

 

In goats, Kundu et al. (2000) reported that the addition of DMSO could improve the cryopreservation of goat semen. In this study, the addition of DMSO improved the preservation effect of semen, of which 0.4% DMSO had the best effect on spermatozoa motility, progressive motility and movement rate, and 0.5% DMSO also played a certain protective effect, but the effect was not as good as 0.4% DMSO. The molecular weight of DMSO is relatively low and its permeation rate in spermatozoa is faster, so the high concentration of DMSO does not further improve the preservation effect of semen, which may be due to the toxicity of DMSO rather than its osmotic effect. Studies have reported that glycerol can rearrange membrane lipids and proteins, increase membrane fluidity, and bind to metal ions to dehydrate cells and prevent the fracture in the frozen solutions by reducing the total ice volume expansion during water solidification (Lohmann et al. 1964; Maxwell and Salamon, 1979; Gao et al. 1995). In boars, Pursel and Johnson (1975) reported that the addition of glycerol could improve the cryopreservation of porcine semen. In bulls, Polge (1953) reported that the addition of glycerol could improve the cryopreservation of bovine semen. In this study, the addition of glycerol improved the preservation effect of semen, of which 4.5% glycerol had the best effect on the kinematic performance of spermatozoa, and 6% glycerol also had a certain protective effect, but the effect was not as good as 4.5% glycerol. This may be because the relative molecular weight of glycerol is large, which will have a certain penetration and toxic pressure on spermatozoa, resulting in spermatozoa damage. On the other hand, it may be that higher concentration of glycerol can promote programmed death of spermatozoa (Wündrich et al. 2006).

 

Table 4 Effects of different concentrations of skimmed milk powder (SMP) on Hu ram spermatozoa motility parameters stored at 4 ˚C

VSL: straight line velocity; VCL: curvilinear velocity and VAP: average path velocity.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

 

The study reported that glycolis less likely to cause osmotic shock to the spermatozoa (Moore et al. 2006). In this study, 7.5% glycol had the best protective effect on cryopreserved Hu ram spermatozoa. Rota et al. (2006) reported that the addition of 5% glycol could improve the cryopreservation of dog semen. The addition of 5% of the report is different from that of 7.5% in this study, which may be due to different species. In this study, all concentrations of SMP improved the preservation effect of semen, but the higher concentration of SMP did not have the best preservation effect. This may be due to the addition of too much SMP, which increases the viscosity of the solution, makes spermatozoa more prone to agglutination and reduces its preservation effect. Skimmed milk powder may contain antioxidants, membrane stabilizers and carbohydrate nutrients, so it can improve the preservation effect of semen in many ways (Vernet et al. 2001; Chen et al. 2002). It is reported that SL contains low-density lipoprotein components similar to yolk, which can protect the integrity of phospholipid membrane at low temperature and may also be an antioxidant (Dalmazzo et al. 2018). Zhao et al. (2021) reported that the addition of SL had a good effect on the preservation of Duolangrams semen at 0 ˚C. In this study, the optimum concentration of SL was 0.15%. Although the addition of higher concentration could also have a beneficial effect on semen preservation, it did not further improve its preservation effect.

 

Table 5 Effects of different concentrations of soy lecithin on Hu ram spermatozoa motility parameters stored at 4 ˚C

VSL: straight line velocity; VCL: curvilinear velocity and VAP: average path velocity.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

 

It could be that the decreased spermatozoa motility in samples with increased extender concentrations could be related both to the high viscosity of SL preventing spermatozoa movement and to the formation of extender debris (Forouzanfar et al. 2010). Another hypothesis is that excessive addition of SL impairs the function of spermatozoa mitochondria (Del et al. 2012). In this study, the protective effect of 7.5% glycol was better than 4.5% glycerol, and 4.5% glycerol was better than 0.4% DMSO. Gilmore et al. (2000) reported that the protective effect of adding glycol was better than that of glycerol in the freezing of human semen. Guthrie et al. (2002) reported that the protective effect of glycol was also better than that of glycerol in the cryopreservation of bovine semen. Najafi et al. (2017) reported the least protective effect of DMSO in the cryopreservation of Ghezel ram semen. The results of this study are consistent with those reported above. However, Silva et al. (2012) also reported that the protective effect of glycerol was stronger than that of glycol in Morada Nova ram semen cryopreservation, which may be related to sheep breed, preservation method and added concentration. In this study, 1.5% SMP had the best effect on cryopreservation of Hu ram spermatozoa, which was better than 0.15% SL group. Küçük et al. (2014) reported that the protective effect of SMP was better than that of egg yolk in cryopreservation of goat semen. The main substance of egg yolk to protect spermatozoa is lecithin, SL is considered as a substitute for egg yolk (Kmenta et al. 2011).

 

Table 6 Effects of different antifreeze protection agents on Hu ram spermatozoa motility parameters stored at 4 ˚C

DMSO: dimethyl sulfoxide; SMO: skimmed milk powder; VSL: straight line velocity; VCL: curvilinear velocity and VAP: average path velocity.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

 

SMP may regulate osmotic pressure and reduce the concentration of electrolyte in semen. On the other hand, it can also attach to the surface of spermatozoa cell membrane, protect spermatozoa membrane and maintain the normal morphological structure and physiological function of spermatozoa. The protective ability of various antifreeze protectants depends on their permeability, solubility, the number of unpaired electrons in the compound and their effect on the membrane structure (Wu et al. 2015). Some authors stated that the addition of cryoprotectants modified water permeability, lowering the hydraulic conductivity and thus limiting volumetric excursion and osmotic stress (Swelum et al. 2011). Therefore, according to the different species, preservation conditions and experimental methods, the type and concentration of antifreeze protectants are very important.

 

CONCLUSION

In conclusion, when DMSO, glycerol, glycol, SMP, SL were respectively added to Hu ram semen at 4 ˚C, the most suitable concentration was 0.4%, 4.5%, 7.5%, 1.5% and 0.15%. When each optimal concentration of antifreeze protectant was added to Hu ram semen at 4 ˚C, 1.5% SMP had the best effect on the preservation of Hu ram semen at 4 ˚C.

 

ACKNOWLEDGEMENT

The authors thank all of the individuals who participated in this work. Liuming Zhang: Conceptualization, Methodology, Investigation, Formal analysis, Writing- original draft. Tariq Sohail: Conceptualization, Writing-original draft, Writing-review and editing. Yan Kang and Xuyang Wang: Methodology, Investigation. Xiaomei Sun: Formal analysis, Writing-review and editing. Yongjun Li: Writing-review and editing, Visualization, Supervision, Project administration. All authors read, revised, and approved the final manuscript.

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