The effect of supplementation of vitamin D to the egg-yolk extender on cryopreservation of ram semen

doi:10.21203/rs.3.rs-3013445/v1

Download PDF

Research Article

The effect of supplementation of vitamin D to the egg-yolk extender on cryopreservation of ram semen

https://doi.org/10.21203/rs.3.rs-3013445/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 01 Jul, 2024

Read the published version in Veterinary Medicine and Science →

Version 1

posted

You are reading this latest preprint version

This study aimed to examine the effects of supplementation of vitamin D to the egg-yolk extender on characteristics of frozen-thawed ram semen. Semen samples obtained from adult rams were pooled and divided into five equal volumes. It was reconstituted with extenders containing different concentrations of vitamin D (0 (control), 12.5 (VITD 12.5), 25 (VITD 25), 50 (VITD 50), and 100 ng/ml (VITD 100)) and then they were frozen. Sperm motility, plasmamembrane functionalintegrity, acrosomal integrity, DNA fragmentation and mitochondrial membrane potential of the groups were determined. Among motility values after thawing, the VITD50 group was found to be superior to the other groups (P < 0.05). When the plasma membrane integrity was evaluated, VITD25 and VITD50 groups were found better than the other groups (P < 0.05). Acrosomal integrity, DNA fragmentation, and mitochondrial membrane potential rates were found better in VITD25, VITD50 and VITD100 groups when compared to all other groups, but no significant difference was found among all groups (P > 0.05). In the study, it was observed that adding vitamin D to the extender had a beneficial effect on ram spermatological parameters. In addition, it was concluded that the use of the 50 ng/ml vitamin D in the extender provided more effective protection than the other doses.

Ram

Semen

Cryopreservation

Vitamin D

Flow Cytometer

Nowadays, the success achieved in artificial insemination applications with frozen ram semen is not satisfactory. One of themost important reasons for this is due to intracellular ice crystallization, coldshock, oxidativestress and reactive oxygen species (ROS) that occur as a result offreezing-thawing, as in other species. Depending on these, sperm motility, mitochondrial injury, DNA fragmentation, viability and fertilization capacity of spermatozoa decrease (Desai et al., 2009; Agarwal et al., 2014; Moghadam et al., 2020). To eliminate all these problems and to further the success achieved, it is possible to freeze the semen for a long time by using different chemicals and antioxidants (Trounson, 2020).

Vitamin D, which is a prohormone in steroid structure, exists in two forms, D3 and D2 (Gil et al., 2018). A large part of the D3 form is synthesized in the skin by ultraviolet-B (UVB) rays, while a very small part is taken from the diet (Gil et al., 2018; Uitterlinden et al., 2004). The D2 form is provided from plant-based foods. There are results from experimental animal studies that vitamin D regulates testicularfunction. Also, vitaminD receptor (VDR)and its metabolizingenzymes are expressed in germcells and mature spermatozoa (Jensen et al., 2010).

In addition to its effects on the immune system, it is known to have a protectiveeffect against oxidative stress (Dong et al., 2012; Berridge, 2015). This effect is indicated by binding to the vitamin D receptor (VDR), which is a nuclear receptor (Berridge, 2015). Oxidative stress occurs as a result of the increase in free radicals, especiallyreactive oxygen species(ROS), and the deterioration of the balance between antioxidants that detoxify prooxidants (Grygiel-Gorniak and Puszczewicz, 2014). The reactions that form ROS under physiological conditions are reversible, and after an oxide protein completes its function, it is reduced to its inactive form. Vitamin D regulates theexpression of antioxidant genes thatprevent oxidative stress through transcription factors such as Klotho and nuclear factorerythroid 2-related factor 2 (Nrf2), by removing ROS or reversing the oxidativechanges that occur in the normalROS signaling process (Berridge, 2015). Vitamin D increases the expression of gammaglutamyl transpeptidase (GGT), which increases glutathione (GSH) synthesis, and increased GSH synthesis decreases NOX (NADPH oxidase) expression, which is one of the main sources of ROS production (Dong et al., 2012). VitaminD also increasesthe production of GSH, which is the major redox buffer, by increasing theexpression of antioxidantenzymes such as glucose 6 phosphate hydrogenase (G6PD), glutamate cysteine ligase, and glutathione reductase; by increasing the expression of glutathione peroxidase, it provides the conversion of H2O2 to water (Jain and Micinski, 2013; Briones and Darwish, 2014).

Although some studies were showing that vitamin D has significant effects on the cryopreservation of human, bull and pig semen, there is no study conducted on ram semen. Our study, it was aimed to examine the effects of different ratios of vitamin D supplementation to the extender on the spermatological parameters of the frozen/thawed ram semen.

The study was carried out with the scientific ethics committee document approved by Bursa Uludag University (No: 2021-13/03). Work was completed with chemicals from Merck (Darmstadt Germany) and Sigma (St. Louis, MO, USA).

2.1. Experimental design

This planned study was conducted to investigate the effects of different doses of vitamin D added to the extender used for the frozen storage of ram semen. Taking into account the previous works, vitamin D doses were determined in groups as no vitamin D (control), 12.5 ng/ml (VITD 12.5), 25 ng/ml (VITD 25), 50 ng/ml (VITD 50) and 100 ng/ml (VITD 100). Each trial was repeated 5 times under the same conditions.

2.2. Semen collection, extenders preparation and dilution

Six different Merinos rams under the same care and feeding conditions were used to collect fresh semen. Semen samples were collected twice a week for 3 weeks using through an electroejaculation method (Ruakura GoatProbe PlasticProducts, Hamilton, NewZealand) and then ejaculates were kept in a waterbath at 37°C at the laboratory. After an initial evaluation, ram semen samples having motility > 80%, rapid wave > + 3 and ≥ 1.5x 10⁹ spermatozoa/mL wereused to categorize semen.

Semen extenders 223.7 mmol/L Tris, 66.6 mmol/L citric acid, 55.5 mmol/L fructose, 4.03 mmol/L EDTA, 4 g/L penicillin G, 100.4 mmol/L trehalose, Contains 3 g/L dihydrostreptomycin, 20% egg yolk. According to the experimental design, each group was supplemented with vitamin D. Vitamin D3 (Vitamin D) was dissolved in ethanol to achieve concentrations of 12.5, 25, 50, and 100 ng/mL for the experimental groups. Then, distilled water was used to dilute to obtain the desired doses.

All in all, ejaculates from rams were pooled and split into fiveequal aliquots. They were diluted to a final concentration of 200x10⁶ (spermatozoon/mL) withextenders having no vitamin D (control), 12.5 ng/ml (VITD 12.5), 25 ng/ml (VITD 25), 50 ng/ml (VITD 50) and 100 ng/ml (VITD 100). They were then gradually cooled to 4°C andequilibrated at 4°C for 2 hours.

2.3. Semen Freezing and Thawing

As in a previous study that was conducted by Alcay et al., equilibrated semen was placed into 0.25 mL straws and they were cooled in liquid nitrogen vapor at 3°C/min from 5°C to -8°C and at 25°C/min cooling rate using Nicool Plus PC (Air Liquide, Marne-la-Vallée Cedex 3, France) (Alcay et al., 2022). After that, they were cooled from − 8°C to -120°C. Thestraws were thenplunged into liquid nitrogen at − 196^οC wherethey were stored for at least one month. At least three straws fromeach group were thawed at 37°Cfor 30s in a water bath to evaluate post-thaw semen characteristics.

2.4. Examination of Frozen Sperm

After thawing, sperm motility and kinematicparameters (A computer-assistedsperm analyzer [CASA]), plasma membrane integrity (Hyposmotic swellingtest [HOST]), DNA damage (TUNEL), acrosomal integrity (FITC-PSA), mitochondrial membrane activation potential (CytoFLEX Flow Cytometer (5,5′,6,6′-tetrachlor1,1′3,3′-tetramethyl benzimidazolylcarbocyanine iodide[(JC-1)-PI]) were evaluated. All of the applications were made by the same person from start to finish.

2.4.1. Sperm Motility and Kinematic Parameters

Ram sperm motility and kinematic parameters consideration was executed using a computer-assisted sperm analyzer (CASA - Ceros II, IMV Technologies) using leja slides that were pre-heated to 37°C. Total motility (%), rapid motility (%), progressive motility (%), velocity average path (VAP, µm/s), velocity straight line (VSL, µm/s), velocity curve linear (VCL, µm/s), straightness (STR, VSL/VAP × 100), linearity (LIN, VSL/VCL × 100), and wobble (WOB, %) were assessed. Foreach sample, 250 to 400 sperm wereevaluated in eight fields (Inanc et al., 2019).

2.4.2. Plasma membrane integrity

Plasma membraneintegrity was tested using the hypoosmotic swellingtest (HOST). This method was conducted as Alcay et al. indicated before and sperm with a curled tail were counted and evaluated (Alcay et al., 2015)

2.4.3. Fragmentation of DNA

For evaluation, 10 µL of sperm samples were placed in 100 µL of PBS and then centrifuged at 2500 rpm for 5min. The supernatant was discarded and 75 µL of PBS was added and centrifuged again. Then, a drop was smeared on a slideand fixed with10% formaldehyde for 20min. After fixation, the slides were washed with PBS and then dried. Prepared slides were incubatedfor 1hour at 37°C with a TUNEL reaction mixturecontaining Terminal deoxynucleotidyltransferase (TdT) plus dUTP tag. TUNEL positive sperm cells were first detected under a fluorescent microscope, and then the total number of cells in the area was determined under a phase contrast microscope.

2.4.4. Acrosomal Integrity

Acrosomal integrities of sperm cells were evaluated by FITC conjugated Pisum sativum agglutinin (FITC-PSA) method (Alcay et al., 2022) 10 µl of sperm sample was added to 100 µl of PBS and centrifuged. The supernatant was discarded and then100 µl of PBS was added and centrifuged again. After centrifuge, a drop from the pellet was smeared on the slide. Then the prepared slides were fixed with acetone. After staining, 200 spermatozoa cells wereevaluated under a fluorescentattachment microscope (Alcay et al., 2015).

2.4.5. Mitochondrial membrane activation potential

Mitochondrial membrane potential assessment was performed with a flow cytometer analyzer, Cytoflex Flow Cytometer (BeckmanCoulter, Fullerton, USA). Sperm samples were evaluated with a 488 nm blue laser beam (50mW laser output), emission filters of 525 ± 40, 585 ± 42 and 610 ± 20, and data were taken from 10000 events.

Frozen-thawed spermatozoa were determinedwith 5,5′,6,6′-tetrachloro1,1′3,3′ tetramethyl benzimidazolylcarbocyanine iodide (JC-1)-PImolecular probes. Straws were thawed at 37°C, 10 µl of JC-1 and 3 µl of PI were added to 10 µl of sperm sample in 487 µl Tris solution. It was then incubated at 37°C for 30 min. (in a dark environment). Damaged and intact spermatozoa were determined by PI. Following this, cells withhigh mitochondrial membranepotential with JC-1 fluorescent orange and cellswith low mitochondrialmembrane potential fluorescent green (Inanc et al., 2019). (Fig. 1).

2.5. Statistical analysis

SPSS 28 was used for statistical analysis in the study. Firstly, the normality test Shapiro-Wilk was used and the distributions were determined. The mean values of the parameters were obtained and the One Way ANOVA test and Kruskal Wallis test were applied. The groups with differences were determined by Tukey and Mann-Whitney U tests, respectively. All obtained data were given as mean ± standarddeviation, and P-values less than 0.05 were considered to be significantly different.

Six parameters were considered in the post frozen-thawed evaluation in this study. These are motility, kinetic parameters, plasmamembrane integrity, acrosomal integrity, DNA fragmentation and mitochondrial membranepotential. There was a decrease after cryopreservation in all spermatological parameters pooling concerning fresh semen (P < 0.05). According to the motility results, all groups had higher values than the control group (Table 1.) However, only VITD 50 group was significantly higher than all groups (P < 0.05). Although VAP, VSL and VCL ratios in kinematic parameters werebetter than the control group, no significant difference was found among the groups. (P > 0.05). VITD 12.5, VITD 25 and VITD 50 were significantlydifferent in STR and LIN compared to the control and VITD 100 (P < 0.05). WOB values of VITD 12.5 and VITD 50 were higher thanthe othergroups. (P < 0.05). The plasma membrane integrity of VITD 25 and VITD 50 was significantly different from the other groups (P < 0.05). VITD 25 in acrosomal integrity and VITD 50 in DNA fragmentation were higher than the other groups, but there was no significant difference among the groups (P > 0.05). Mitochondrial membrane potential was found to be higher in VITD 50 than in other groups. (P < 0.05) (Table 2).

Table 1

The mean of studied ram sperm post-thawing motility and kinematic parameters on different extender groups.
Kinematics / Grups	Control	12,5 ng/ml	25 ng/ml	50 ng/ml	100 ng/ml
VAP (µm/s)	84.87 + 23.33^a	89.70 + 16.16^a	101.36 + 21.14^a	109.39 + 21.82^a	91.68 + 18.15^a
VSL (µm/s)	65.96 + 18.07^a	68.27 + 10.32^a	76.38 + 14.19^a	79.01 + 14.95^a	61.46 + 13.70^a
VCL (µm/s)	159.21 + 46.27^a	173.12 + 41.72^a	192.76 + 50.70^a	227.34 + 45.85^a	198.40 + 31.24^a
STR (%)	70.38 + 2.58^a	76.87 + 6.60^a,d	76.65 + 4.85^a,d	74.72 + 1.65^b,d	68.71 + 1.77^c
LIN (%)	41.17 + 5.12^a	45.80 + 11.44^a,d	45.81 + 9.04^a,d	47.83 + 2.13^b,d	35.27 + 1.03^c
WOB (%)	54.41 + 4.34^a	56.84 + 9.29^a,d	57.46 + 8.10^a	58.42 + 1.36^b,d	48.94 + 0.51^b
Total Motility (%)	37 + 6.70^a	43 + 5.70^a	44 + 4.18^a	55 + 2^b	45 + 3.53^a
Data is presented in Mean ± S.D.
Different letters within the same column show significant differences among the groups (P < 0.05).

Table 2

The mean of studied ram sperm post-thawing parameters on different extender groups.
Grups	DNA Fragmentation(%)	Acrosomal Integrity(%)	Host (%)	HMMP (%)
Control	13.6 ± 5.72^a	70 ± 4.89^a	83.6 ± 3.78^a	72.88 ± 15.73^a
12.5 ng/ml	12.80 ± 5.58^a	70 ± 2.34^a	82.4 ± 1.81^a	69.41 ± 24.11^a
25 ng/ml	14.40 ± 9.12^a	75 ± 4.24^a	88 ± 0.70^b	68.39 ± 20.39^a
50 ng/ml	16 ± 4.72^a	74 ± 2.64^a	87.4 ± 2.19^b	79.49 ± 6.48^b
100 ng/ml	15,8 ± 5.01^a	74.40 ± 3.36^a	84.8 ± 2.38^a	64.12 ± 25.15^a
Data is presented in Mean ± S.D.
Different letters within the same line show significant differences among the groups (P < 0.05).

The aim of this studywas to investigate whether vitamin D protects sperm cells against harmful effects exposed during the cryopreservation process. Vitamin D indicates these effects by binding to nuclear receptors against oxidative stress (Wang et al., 2017; Chen and Zhi, 2020). They express antioxidant genes that prevent oxidative stress by eliminating ROS or reversing the normal ROS signaling process (Mokhtari et al., 2017). Due to this feature, vitamin D was added to diluents in different doses as the main materials and their effects were investigated. In many studies, the effects of vitamin D on semen have been investigated by adding it to the diets of animals or adding it to the extenders. Studies have been made in some animal species such as bulls and pigs as well as in humans. However, there is no study on rams as far as our investigations (Moghadam et al., 2020; Lin et al., 2017; Moghadam et al., 2019; Asadpour et al., 2021).

Cryopreservation of sperm ensures the long-term preservation of genetic material. However, it is known that freezing has negative effects on the fertilization ability of semen (Agarwal et al., 2005). These undesirable effects show by reducing sperm viability, motility, plasma membrane integrity, acrosomal integrity, DNA fragmentation and mitochondrial membrane potential (Donnelly et al., 2000).

Motility is one of the basic quality parameters in the evaluation of semen. In the present study, it was observed that the addition of vitamin D increased the motility rate of ram semen after frozen thawing. The total motility rate of VITD 50, which is one of the vitamin D groups added to the extenders at different rates, is significantly important compared to the other groups (P < 0.05). When the kinematic parameters are examined, there is no significant difference among the groups in VAP, VSL, and VCL values. In STR, LIN, WOB values, VITD 12.5, VITD 25 and VITD 50 added groups were significantly different from control and VITD 100 groups. Considering these results, the results of this study show parallelism with other studies (Moghadam et al., 2020; Moghadam et al., 2019; Asadpour et al., 2021).

Spermatozoon plasma membrane integrity is positively related to the capacitation of sperm cells which has a very important effect on acrosome reaction and sperm fusion with the egg during fertilization (Ustuner et al., 2016). However, the integrity of the spermatozoon plasma membrane is adversely affected by the cryopreservation process (Forouzanfar et al., 2010; Emamverdi et al., 2013; Najafi et al., 2013). The known negative effects are the effects of cold shock, ice crystallization, osmotic stress, and lipid peroxidation formed during cryopreservation on sperm membrane permeability and morphology (Taylor et al., 2009; El-

Kon, 2011). Therefore, it is very important to maintain membrane integrity during the freezing process to prevent cellular damage. HOST is an optimized test to detect subtle changes in spermatozoon membrane functionality (Jeyendran et al., 1984; Maxwell and Salamon, 1993). In our study, it was observed that vitamin D preserved the integrity of the plasma membrane after thawing. In the present study, VITD 25and VITD 50 are statistically higher than the other groups. (P < 0.05) These data are in agreement with other mentioned studies.

Acrosomal defect and DNA fragmentation are one of undesirable effects of the cryopreservation process (Nur et al., 2010). These effects are caused by ROS emerging after cryopreservation. Therefore, the extenders used for cryopreservation should protect the semen against freezing (Kumar et al., 2015). The addition of vitamin D has a positive effect on the ROS andresults of some studies support this claim (Jain and Micinski, 2013; Moghadam et al., 2019). In our study, it was seen that vitamin D had also the same positive effects as mentioned in other studies against acrosomal damage and DNA fragmentation. However, there was not found any statistical difference among the groups (P > 0.05).

There is a positive correlation between mitochondrial membrane potential and plasma membrane integrity and motility. Mitochondria have axonemes that produce intracellular ATP (Inanc et al., 2019; Gürler et al., 2016). When these are damaged as a result of cryopreservation, ATP production and motility are reduced. In our study, mitochondrial membrane potential was high in the VITD 25 and VITD 50 groups, and accordingly, plasma membrane integrity and motility values were also high.

As a result of all parameters checked after cryopreservation, it was observed that the addition of vitamin D to the extender protected the sperm against the harmful effects of cryopreservation. The semen protection effect was found generally higher in VITD 50 when compared with other groups. In future studies, post-incubation parameters and the effect of vitamin D on different animal species should be examined. In addition, semen frozen with vitamin D should be used for reproduction and its success should be evaluated.

Acknowledgements

All procedures used were approved by Uludag University committee of ethics (No: 2021-13/03).

Financial support

This work was supported by the Bursa Uludağ University Scientific Research Projects Unit (Project Code: TOA-2022-670).

Author Contributions

AA, MBT designed the experiment. AA, SA, DK, UCU performed the experiment. AA, MBT analyzed the data. AA, MBT made tables, and wrote the paper. All authors reviewed and approved the final manuscript.

Conflict of Interest

The authors declared that there is no conflict of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Agarwal, A., Prabakaran, S.A., Said, T.M. (2005). Prevention of oxidative stress injury to sperm. J Androl. 26, 654-660. https://doi.org/10.2164/jandrol.05016.
Agarwal, A., Sharma, R.K., Sharma, R., Assidi, M., Abuzenadah, A.M., Alshahrani, S., Durairajanayagam, D., Sabanegh, E., 2014. Characterizing semen parameters and their association with reactive oxygen species in infertile men. Reprod Biol Endocrinol. 12, 33. https://doi.org/10.1186/1477-7827-12-33.
Alcay, S., Toker, M.B., Gokce, E., Ustuner, B., Onder, N.T., Sagirkaya, H., Nur, Z., Soylu, M.K., 2015. Successful ram semen cryopreservation with lyophilized egg yolk-based extender. Cryobiology. 71, 329-333. https://doi.org/10.1016/j.cryobiol.2015.08.008.
Alçay, S., Aktar, A., Koca, D., Kılıc, M.A., Akkasoglu, M., Yılmaz, M.M., Sagırkaya, H., 2022. Autologous platelet rich plasma have positively effect on ram spermatozoa during cryopreservation in non-breeding season. Kafkas Univ. Vet. Fak. Derg. 28, 229-234. https://doi.org/10.9775/kvfd.2021.26763.
Asadpour, R., Tavarat, M., Rahbar, M., Khoshniyat, M., Hamidian, G., 2021. Effects of vitamin D supplementation in extender on sperm kinematics and apoptosis following the freeze-thaw process in normozoospermic and asthenozoospermic Holstein bulls. Basic Clin Androl. 31, 20. https://doi.org/10.1186/s12610-021-00137-5.
Berridge, M.J., 2015. Vitamin D cell signalling in health and disease. Biochem. Biophys. Res. Commun. 460, 53–71. https://doi.org/10.1016/j.bbrc.2015.01.008.
Briones, T.L., Darwish, H., 2014. Decrease in age-related tau hyperphosphorylation and cognitive improvement following vitamin D supplementation are associated with modulation of brain energy metabolism and redox state. Neuroscience. 262, 143–155. https://doi.org/10.1016/j.neuroscience.2013.12.064.
Chen, Y., Zhi, X., 2020. Roles of vitamin D in Reproductive Systems and Assisted Reproductive Technology. Endocrinology. 161, 1-12. https://doi.org/10.1210/endocr/bqaa023.
Desai, N., Sharma, R., Makker, K., Sabanagh, E., Agarwal, A., 2009. Physiologic and pathologic levels of reactive oxygen species in neat semen of infertile men. Fertil Steril. 92, 1626-1631. https://doi.org/10.1016/j.fertnstert.2008.08.109.
Dong, J., Wong, S.L., Lau, C.W., Lee, H.K., Ng, C.F., Zhang, L., Yao, X., Chen, Z.Y., Vanhoutte, P.M., Huang, Y., 2012. Calcitriol protects renovascular function in hypertension by downregulating angiotensin II type 1 receptors and reducing oxidative stress. Eur. Heart. J. 33, 2980–2990. https://doi.org/10.1093/eurheartj/ehr459.
Donnelly, E.T., Mcclure, N., Lewis, S.E.M., 2000. Glutathione and hypotaurine in vitro: motility, DNA integrity and production of reactive oxygen species. Mutagenesis. 15, 61-68. https://doi.org/10.1093/mutage/15.1.61.
El-Kon I., 2011, Testing usability of bovine serum albumin (BSA) for preservation of Egyptian buffalo semen. American-Eurasian J Agric Environ Sci. 11, 495-502.
Emamverdi, M., Zhandi, M., Shahneh, A.Z., Sharafi, M., Akbari-Sharif, A., 2013. Optimization of ram semen cryopreservation using a chemically defined soybean lecithin-based extender. Reprod. Dom. Anim. 48, 899-904. https://doi.org/10.1111/rda.12183.
Forouzanfar, M., Sharafi, M., Hosseini, S.M., Ostadhosseini, S., Hajian, M., Hosseini, L., Abedi, P., Nili, N., Rahmani, H.R., Nasr-Esfahani, M.H., 2010. In vitro comparison of egg yolk-based and soybean lecithinbased extenders for cryopreservation of ram semen. Theriogenology. 73, 480–487. https://doi.org/10.1016/j.theriogenology.2009.10.005.
Gil, A., Plaza-Diaz, J., Mesa, M.D., 2018. Vitamin D: Classic and Novel Actions. Ann. Nutr. Metab. 72, 87–95. https://doi.org/10.1159/000486536.
Gürler, H., Malama, E., Heppelmann, M., Calisici, O., Leiding, C., Kastelic, J.P., Bollwein, H., 2016. Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species and DNA damage of bovine sperm. Theriogenology. 86, 562-571. https://doi.org/10.1016/j.theriogenology.2016.02.007.
Grygiel-Gorniak, B., Puszczewicz, M., 2014. Oxidative damage and antioxidative therapy in systemic sclerosis. Mediators Inflamm. 389582. https://doi.org/10.1155/2014/389582.
Inanc, M.E., Gungor, S., Ozturk, C., Korkmaz, F., Bastan, I., Cil, B., 2019. Cholesterol-loaded cyclodextrin plus trehalose improves quality of frozen-thawed ram sperm. Veterinarni Medicina. 64, 118-124. https://doi.org/10.17221/146/2018-vetmed.
Jain, S.K., Micinski, D., 2013. Vitamin D upregulates glutamate cysteine ligase and glutathione reductase, and GSH formation, and decreases ROS and MCP-1 and IL-8 secretion in high-glucose exposed U937 monocytes. Biochem. Biophys. Res. Commun. 437, 7–11. https://doi.org/10.1016/j.bbrc.2013.06.004.
Jensen, B.M., Nielsen, J.E., Jorgensen, A., Meyts, E.R., Kristensen, D.M., Jorgensen, N., Skakkebaek, N., Juul, A., Leffers, H., 2010. Vitamin D receptor and vitamin D metabolizing enzymes are expressed in the human male reproductive tract. Hum. Reprod. 25, 1303-1311. https://doi.org/10.1093/humrep/deq024.
Jeyendran, R.S., Van-Der-Ven, H.H., Perez-Pelaez, M., Crabo, B.G., Zaneveld, L.J., 1984. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil. 70, 219-228. https://doi.org/10.1530/jrf.0.0700219.
Kumar, P., Saini, M., Kumar, D., Balhara, A.K., Yadav, S.P., Singh, P., Yadav, P.S., 2015. Liposome-based semen extenders is suitable alternative to egg yolk-based exrender for cryopreservation of buffalo (bubalos bubalis) semen. Animal Repro Sci. 159, 38-45. https://doi.org/10.1016/j.anireprosci.2015.05.010.
Lin, Y., Lv, G., Dong, H.J., Wu, D., Tao, Z., Xu, S., Che, L., Fang, Z., Bai, S., Feng, B., Li, J., Xu, X.Y., 2017. Effects of the different levels of dietary vitamin D on boar performance and semen quality. Livestock Science. 203, 63-68. https://doi.org/10.1016/J.LIVSCI.2017.07.003.
Maxwell, W.M., Salamon, S., 1993. Liquid storage of ram semen: A review. Reprod Fertil Dev. 5, 613-638. https://doi.org/10.1071/rd9930613.
Moghadam, M.T., Yousef, A.F., Saki, G., Nikbaht, R., 2019. Effect of vitamin D on apoptotic marker, reactive oxygen species and human sperm parameters during the process of cryopreservation. Iran. J. Basic. Med. Sci. 22, 1036-1043. https://doi.org/10.22038/ijbms.2019.36258.8634.
Moghadam, M.T., Hosseini, G., Absalan, F., Tabar, M.H., Nikbakht, R., 2020. Effects of vitamin D on apoptosis and quality of sperm in Asthenozoospermia. JBRA Assisted Reptoduction. 24, 316-323. https://doi.org/10.5935/1518-0557.20200009.
Mokhtari, Z., Hekmatdoost, A., Nourian, M., 2017. Antioxidant efficacy of vitamin D. J Parathry Dis. 5, 11-16.
Najafi, A., Zhandi, M., Towhidi, A., Sharafi, M., Sharif, A.A., Motlagh, M.K., Martinez-Pastor, F., 2013. Trehalose and glycerol have a dose-dependent synergistic effect on the post-thawing quality of ram semen cryopreserved in a soybean lecithin-based extender. Cryobiology. 66, 275-282. https://doi.org/10.1016/j.cryobiol.2013.03.002.
Nur, Z., Zık, B., Ustuner, B., Sagirkaya, H., Ozguden, C.G., 2010. Effects of different cryoprotective agents on ram sperm morphology and DNA integrity. Theriogenology. 73, 1267-1275. https://doi.org/10.1016/j.theriogenology.2009.12.077.
Taylor, M.A., Guzmán-Novoa, E., Morfin, N., Buhr, M.M., 2009. Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios. Theriogenology. 7, 149-159. https://doi.org/10.1016/j.theriogenology.2009.02.012.
Trounson, A.O., 1990. Cryopresrevation. Br. Med. Bull. 46, 695-708. https://doi.org/10.1093/oxfordjournals.bmb.a072425.
Uitterlinden, A.G., Fang, Y., Van-Meurs, J.B.J., Pols, H.A.P., Leeuwen, J.P.T.M., 2004. Genetics and biology of vitamin D receptor polymorphisms. Gene. 338, 143–156. https://doi.org/10.1016/j.gene.2004.05.014.
Ustuner, B., Alcay, S., Toker, M.B., Nur, Z., Gokce, E., Sonat, F.A., Gul, Z., Duman, M., Ceniz, C., Uslu, A., Sagirkaya, H., Soylu, M.K., 2016. Effect of rainbow trout (Oncorhynchus mykiss) seminal plasma on the post-thaw quality of ram semen cryopreserved in a soybean lecithin-based or egg yolk-based extender. Animal Reproduction Science. 164, 97-104. https://doi.org/10.1016/j.anireprosci.2015.11.017.
Wang, E.W., Siu, P.M., Pang, M.Y., Woo, J., Collins, A.R., Benzie, F.F., 2017. Vitamin D deficiency, oxidative stress and antioxidant status: only weak association seen in the absence of advanced age, obesity or pre-existing disease. British Journal of Nutrition. 118, 11-16. https://doi.org/10.1017/S000711451700188X.

Download PDF

Journal Publication

published 01 Jul, 2024

Read the published version in Veterinary Medicine and Science →

Version 1

posted

You are reading this latest preprint version

The effect of supplementation of vitamin D to the egg-yolk extender on cryopreservation of ram semen

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Material and Method

2.1. Experimental design

2.2. Semen collection, extenders preparation and dilution

2.3. Semen Freezing and Thawing

2.4. Examination of Frozen Sperm

2.4.1. Sperm Motility and Kinematic Parameters

2.4.2. Plasma membrane integrity

2.4.3. Fragmentation of DNA

2.4.4. Acrosomal Integrity

2.4.5. Mitochondrial membrane activation potential

2.5. Statistical analysis

3. Results

4. Discussion

Declarations

References

Status:

Journal Publication

Version 1