Farelerde Sperma Sulandırıcısına Düşük Yoğunluklu Lipoprotein İlavesinin Dondurma Çözdürme Sonrası Sperm Kalitesi ve Nükleer DNA Bütünlüğü Üzerine Etkileri

Öz

Fareler genetik ve moleküler biyoloji için önemli bir araştırma aracıdır ve araştırmacıların çeşitli insan hastalığı modellerini keşfetmesine olanak tanır. Yumurta sarısı, evcil hayvanlara yönelik sperma sulandırıcılarının yaygın bir bileşenidir ve yumurta sarısından elde edilen düşük yoğunluklu lipoproteinlerin (LDL) bazı kriyoprotektif özellikleri vardır. Bu çalışma, farede farklı konsantrasyonlarda LDL (%2.5, %5.0, %7.5, %10) ilaveli sperma sulandırıcısında (%18 rafinoz + %3 yağsız süt) dondurma sonrası sperm kalite özelliklerini ve nükleer DNA bütünlüğünü araştırmayı amaçladı. LDL içermeyen kontrol grubu olarak %18 Raffinoz+%3 yağsız süt sulandırıcısı kullanıldı. Çalışmada CD-1 fareler kullanıldı ve kauda epididimisten sperma toplandı ve sperma sulandırıcısı ile sulandırıldı, payetlere çekilerek motilite, canlılık, plazma zarı (HOST), akrozom ve nükleer DNA bütünlüğü parametreleri dondurma sonrası değerlendirildi. Nativ sperma, HTF çözeltisinde 4 saat boyunca en yüksek motilite, canlılık, plazma zarı bütünlüğüne ve dayanıklılığa sahip bulundu. Nükleer DNA bütünlüğü taze spermada en yüksek belirlendi (p<0.05). Çözdürme sonrası en yüksek motilite (%56.7±1.3), canlılık (%11.6±2.9), membran bütünlüğü (%63.0±2.7) ve yaşam süresi (%31.3±2.1) %2.5 LDL takviyeli grupta, en düşük progresif motilite, canlılık ve plazma membran bütünlüğü ise %10 LDL grubunda belirlendi (p<0.05). Kriyoprezervasyon işlemi, taze sperm ile karşılaştırıldığında tüm LDL ve kontrol gruplarında parçalanmış DNA oranlarını arttırdı (p<0.05). Kontrol ve deney grupları arasında donma ve çözdürme sonrası nükleer DNA hasarı açısından istatistiksel fark yoktu (p<0.05). Sulandırıcıya %2.5 LDL ilavesinin dondurma sonrası spermatolojik kalite parametrelerini iyileştirdiği ve fare sperminin dondurulması ve korunmasında daha yüksek oranda soğuk şokuna karşı koruyucu etki gösterdiği için kullanılabileceği sonucuna varıldı.

Anahtar Kelimeler

• Fare spermi
• Düşük Yoğunluklu Lipoprotein
• Sperm analizi

The Effects on Post-Thaw Sperm Quality and Nuclear DNA Integrity of Supplementation of Low-Density Lipoprotein to Freezing Extender in the Mouse

Öz

Mice are an important research tool for genetic and molecular biology, allowing researchers to explore a variety of human illness models. Egg yolk is a common component of semen extenders for domestic animals and low-density lipoproteins (LDL) from egg yolk have some cryoprotective properties. This study aimed to investigate sperm quality characteristics and nuclear DNA integrity after post-thawing in an extender (18% raffinose + 3% skim milk) supplemented with different concentrations of LDL (2.5%, 5.0%, 7.5%, or 10%) in mice. 18% Raffinose+3% skim milk extender was used as a control group without LDL. CD-1 mice were used in the study, and semen was collected from the cauda epididymis and diluted with the extender. The straws were then frozen and thawed to evaluate progressive motility, viability, plasma membrane (HOST), acrosome, and nuclear DNA integrity parameters. Fresh sperm had the highest progressive motility, viability, plasma membrane integrity, and longevity (endurance) of progressive motility for 4 h in HTF solution. The greatest spermatologic results, including nuclear DNA integrity, were determined in fresh sperm (p<0.05). The greatest post-thaw progressive motility (56.7±1.3%), viability (11.6±2.9%), membrane integrity (63.0±2.7%), and longevity at the end of the 4 h (31.3±2.1%) were found in the group with supplementation of 2.5% LDL compared with the control group. The lowest progressive motility, viability, and plasma membrane integrity were determined in the 10% LDL group (p<0.05). The cryopreservation process increased the rates of fragmented DNA in all tested LDL and control groups compared with fresh sperm (p<0.05). There was no statistical difference between the control and experimental groups in terms of nuclear DNA damage after freezing and thawing (p<0.05). It was concluded that the addition of 2.5% LDL to the extender improved the spermatological quality parameters after freezing and could be used in the freezing and preservation of mouse sperm as it showed a higher protective effect against cold shock.

Anahtar Kelimeler

• Mouse sperm
• Low Density Lypoprotein
• Sperm analysis

Kaynakça

1. Akhter S, Ansari MS, Rakha BA et al. (2011). Effect of low-density lipoproteins in extender on freezability and fertility of buffalo (Bubalus bubalis) bull semen. Theriogenology, 76 (4), 759-764.
2. Amirat L, Anton M, Tainturier D et al. (2005). Modifications of bull spermatozoa induced by three extenders: Biociphos, low density lipoprotein and Triladyl, before, during and after freezing and thawing. Reproduction, 129 (4), 535-543.
3. Amirat L, Tainturier D, Jeanneau L et al. (2004). Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl, a commercial egg yolk extender. Theriogenology, 61 (5), 895-907.
4. Ansari MS, Rakha BA, Anrabi SMH, Akhter S (2010). Usefulness of powdered and fresh egg yolk for cryopreservation of Zebu bull spermatozoa. Reprod Biol, 10 (3), 235-240.
5. Anton M, Martinet V, Dalgalarrondo V et al. (2003). Chemical and structural characterisation of low-density lipoproteins purified from hen egg yolk. Food Chem, 83 (2), 175-183.
6. Bencharif D, Amirat L, Anton M et al. (2008). The advantages of LDL (low-density lipoproteins) in the cryopreservation of canine semen. Theriogenology, 70 (9), 1478-1488.
7. Bencharif D, Amirat L, Garand A et al. (2010). Freezing canine sperm: Comparison of semen extenders containing Equex® and LDL (Low-Density Lipoproteins). Anim Reprod Sci, 119 (3-4), 305-313.
8. Bergeron A, Crête MR, Brindle Y, Manjunath P (2004). Low-density lipoprotein fraction from hen’s egg yolk decreases the binding of the major proteins of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane. Biol Reprod, 70 (3), 708-717.

9. Chohan KR, Griffin JT, Carrell DT (2004). Evaluation of chromatin integrity in human sperm using acridine orange staining with different fixatives and after cryopreservation. Andrologia, 36 (5), 321-326.
10. Dalal J, Chandolia RK, Pawaria S et al. (2020). Low‐density lipoproteins protect sperm during cryopreservation in buffalo: Unraveling mechanism of action. Mol Reprod Dev., 87 (12), 1231-1244.
11. Dong QX, Rodenburg SE, Hill D, VandeVoort CA (2011). The role of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in comparison with whole egg yolk for sperm cryopreservation in rhesus monkeys. Asian. J Androl, 13 (3), 459.
12. Donnelly ET, McClure N, Lewis SE (2001). Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity. Fertil steril, 76 (75), 892-900.
13. Evenson DP (2016). The Sperm Chromatin Structure Assay (SCSA®) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility. Anim Reprod Sci, 169, 56-75.
14. Flaherty SP, Dianna P, Swann NJ, Mattews CD (1995). Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection. Hum Reprod, 10 (10), 2623-2629.
15. Fraser L, Strzezek J (2004). The use of comet assay to assess DNA integrity of boar spermatozoa following liquid preservation at 5 degrees C and 16 degrees C. Folia Histochem Cytobiol, 42 (1), 49-55.
16. Hammadeh ME, Greiner S, Rosenbaum P, Schmidt W (2001). Comparison between human sperm preservation medium and TEST‐yolk buffer on protecting chromatin and morphology integrity of human spermatozoa in fertile and subfertile men after freeze‐thawing procedure. J Androl, 22 (6), 1012-1018.
17. Hu JH, Jiang ZL, Lv RK et al. (2011). The advantages of low-density lipoproteins in the cryopreservation of bull semen. Cryobiology, 62 (1), 83-87.
18. Hu JH, Li QW, Li G et al. (2006). The cryoprotective effect on frozen-thawed boar semen of egg yolk low-density lipoproteins. Asian-Aust J Anim Sci, 19 (4), 486-490.
19. Iaffaldano N, Iorio MD, Rosato MP, Manchisi A (2014). Cryopreservation of rabbit semen using non-permeable cryoprotectants: effectiveness of different concentrations of low-density lipoproteins (LDL) from egg yolk versus egg yolk or sucrose. Anim Reprod Sci, 151 (3-4), 220-228.
20. Jamieson BG, Leung LP (1991). Fish evolution and systematics: evidence from spermatozoa: with a survey of lophophorate, echinoderm and protochordate sperm and an account of gamete cryopreservation. Cambridge University Press, England.
21. Jeyendran RS (1984). Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. Reproduction, 70 (1),219-228.
22. Jiang ZL, Li QW, Li WY et al. (2006). Effect of low-density lipoprotein on DNA integrity of freezing-thawing boar sperm by neutral comet assay. Anim Reprod Sci, 99 (3-4), 401-407.
23. Junior ASV, Corcini CD, Ulguim RR et al. (2009). Effect of low-density lipoprotein on the quality of cryopreserved dog semen. Anim Reprod Sci, 115 (1-4), 323-327.
24. Li HG, Liao AH, Ding XF, Zhou H, Xiong CL (2006). The expression and significance of CATSPER1 in human testis and ejaculated spermatozoa. Asian J Androl, 8 (3), 301-306.
25. Mahiddine FY, Kim MJ (2021). Overview on the Antioxidants, Egg Yolk Alternatives, and Mesenchymal Stem Cells and Derivatives Used in Canine Sperm Cryopreservation. Animals, 11 (7), 19-30.
26. Moreno D, Bencharif D, Amirat L et al. (2013). Preliminary results: the advantages of low-density lipoproteins for the cryopreservation of equine semen. J Equine Vet Sci, 33 (12), 1068-1075.
27. Moussa M, Martinet V, Trimeche A, Tainturier D, Anton M (2002). Low density Lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull semen. Theriogenology, 57 (6), 1695-1706.
28. Moustacas VS, Zaffalon FG, Lagares MA et al. (2011). Natural, but not lyophilized, low density lypoproteins were an acceptable alternative to egg yolk for cryopreservation of ram semen. Theriogenology, 75 (2), 300-307.
29. Nakagata N (2000). Cryopreservation of mouse spermatozoa. Mamm Genome, 11 (7), 572-576.
30. Patil S, Kumar P, Singh G et al. (2020). Semen dilution effect on sperm variables and conception rate in buffalo. Anim Reprod Sci, 214, 106304.
31. Peris S, Morier A, Dufour M, Bailey JL (2004). Cryopreservation of ram semen facilitates sperm DNA damage: relationship between sperm andrological parameters and the sperm chromatin structure assay. J Androl, 25(2), 224-233.
32. Perumal P, Srivastav SK, Ghosh SH et al. (2016). Effects of low‐density lipoproteins as an additive on quality parameters and oxidative stress following cryopreservation of Mithun (Bos frontalis) spermatozoa. Reprod Domest Anim, 51 (5), 708-716.
33. Quinn P, Kerin JF, Warnes GM (1985). Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril, 44 (4), 493-498.
34. Royere D, Hamamah S, Nicolle JC, Barthelemy C, Lansac J (1988). Freezing and thawing alter chromatin stability of ejaculated human spermatozoa: fluorescence acridine orange staining and Feulgen- DNA cytophotometric studies. Gamete Res, 21, 51-57.
35. Scott AP, Baynes SM (1980). A review of the biology, handling and storage of salmonid spermatozoa. J Fish Biol, 17, 707-739.
36. Shahverdi A, Sharafi M, Gourabi H et al. (2015). Fertility and flow cytometric evaluations of frozen-thawed rooster semen in cryopreservation medium containing low-density lipoprotein. Theriogenology, 83, 78-85.
37. Sharma R, Masaki J, Agarwal A (2013). Sperm DNA fragmentation analysis using the TUNEL assay In Spermatogenesis. Humana Press, Totowa NJ, 121-136.
38. Somfai T, Bodo SZ, Nagy SZ et al. (2002). Simultaneous evaluation of viability and acrosome integrity of mouse spermatozoa using light microscopy. Biotec Histochem, 77 (3), 117-120.
39. Souza ALP, Lima GL, Peixoto GCX et al. (2015). Sperm characteristics following freezing in extenders supplemented with whole egg yolk and different concentrations of low-density lipoproteins in the collared peccary (Pecari tajacu). Reprod Biol, 15 (4), 223-228.
40. Takeo T, Nakagata N (2018). Mouse sperm cryopreservation using cryoprotectant containing l-glutamine. Cold Spring Harb Protoc, (6).
41. Takeo T, Nakagata N (2010). Mouse sperm cryopreservation and effective embryo production using cryopreserved C57BL/6 mouse sperm. J Mamm Ova Res, 27, 70-78.
42. Thérien I, Moreau R, Manjunat P (1999). Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm. Biol Reprod, 61 (3), 590-598.
43. Tonieto RA, Goularto KL, Gastal GDA et al. (2010). Cryoprotectant effect of trehalose and low-density lipoprotein in extenders for frozen ram semen. Small Rumin Res, 93 (2-3), 206-209.
44. Varela E, Rojas M, Restrepo G (2020). Membrane stability and mitochondrial activity of bovine sperm frozen with low‐density lipoproteins and trehalose. Reprod Domest Anim, 55 (2), 146-153.
45. Wang P, Wang YF, Wang CW et al. (2014). Effects of low-density lipoproteins extracted from different avian yolks on boar spermatozoa quality following freezing-thawing. Zygote, 22 (2), 175-181.
46. Ward MA, Kaneko T, Husakabe H et al. (2003). Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection. Biol Reprod, 69 (6), 2100-2108.
47. Witte TS, Schäfer‐Somi S, Kuchar A et al. (2009). Effect of hen's egg yolk on capacitation and acrosome reaction of diluted canine spermatozoa. Anim. Reprod. Sci, 110 (3-4), 293-305.
48. World Health Organization (1999). WHO laboratory manual for the examination of human semen and sperm‐cervical mucus interactions. 4th edt. Cambridge University Press, Cambridge, UK.
49. Yamauchi S, Nakagura S, Lay K et al. (2009). Characteristics of Okinawan native Agu pig spermatozoa after addition of low-density lipoprotein to the freezing extender. J Reprod Dev, 0906260187-0906260187.
50. Yildiz C, Bozkurt Y, Yavas I (2013). An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm. Cryobiology, 67 (1), 91-94.
51. Yildiz C, Law N, Ottaviani P et al. (2010). Comparison of sperm quality and DNA integrity in mouse sperm exposed to various cooling velocities and osmotic stress. Theriogenology, 74 (8), 1420-1430.
52. Yildiz C, Ottaviani P, Law N et al. (2007). Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development in the mouse. Reproduction, 33 (3), 585-595.