افزایش انجمادپذیری اسپرم گاو با افزودن شیر پس چرخ و پلاسمای منی گاو ‎به ترکیب یک رقیق کننده محافظ انجمادی بر پایه تریس

نوع مقاله : مقاله پژوهشی

نویسندگان

1 استادیار، گروه علوم دامی، دانشکده کشاورزی، دانشگاه یاسوج

2 دانشیار، گروه علوم دامی، دانشکده کشاورزی، دانشگاه ارومیه

چکیده

اثر افزودن شیر پس‎چرخ و پلاسمای منی گاو به ترکیب یک رقیق‎کننده‎ تریس-سیترات-LDL بر ویژگی‏های حرکت‎شناسی و زنده‎مانی اسپرم منجمد-یخ­گشایی شده‎ گاوهای هلشتاین به­منظور تخفیف اثر رقت بررسی شد. نمونه منی از چهار گاو نر، هفته‏ای یک‎بار و به­مدت چهار هفته متوالی جمع‎آوری شد. مخلوط منی به چهار قسمت مساوی تقسیم شد و هر قسمت به­طور جداگانه با رقیق‎کننده‎های آزمایشی تا رسیدن به غلظت 16 میلیون اسپرم در هر میلی ‎لیتر رقیق‎سازی شد. آزمایش در قالب طرح کاملاً تصادفی با آرایش فاکتوریل 3×3 شامل سه سطح شیر (0، 5 و 10 درصد) و سه سطح پلاسمای منی (0، 2 و 5 درصد) انجام شد. بنا به نتایج، جنبایی پیش‎رونده، راستی حرکت (STR) و فراسنجه‎های سرعت اسپرم‎ها [سرعت در مسیر میانگین (VAP)، سرعت در مسیر منحنیِ طی­شونده به­وسیله اسپرم (VCL) و سرعت در مسیر مستقیم (VSL)] در رقیق‏کننده‏های حاوی شیر (در هر دو سطح 5 و 10 درصد) در مقایسه با شاهد به­طور معنی‏داری بیشتر بود (05/0˂P). افزودن پلاسمای منی به ترکیب رقیق‎کننده پایه در سطح 5 درصد، منجر به افزایش شاخص‎های جنبایی (کل و پیش‎رونده)، سرعت، جهت‎داری [راستی مسیر طی شده (STR)، خطی بودن (LIN)، لرزش (WOB) و آمپلیتود جابجایی جانبی سر اسپرم (ALH) شد (05/0˂P). در هنگام نیاز به کاهش تعداد اسپرم‎ها در هر دوز در کارهای برون‏تنی گنجاندن شیر در سطوح 5 یا 10 درصد و یا پلاسمای منی در سطح 5 درصد در ترکیب رقیق‎کننده منی توصیه می‎شود. انتخاب رقیق‎کننده مناسب برای انجماد اسپرم با تعداد اندک در هر دوز تلقیحی، منوط به انجام آزمون‎های بیشتر است.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Increasing the freezeability of bovine sperm by adding skimmed cow's milk and seminal plasma to the composition of a Tris-based cryoprotective medium

نویسندگان [English]

  • F. Samadian 1
  • M. Memar 1
  • F. Farrokhi Ardebili 2
1 Assistant Professor, Department of Animal Science, Faculty of Agriculture, Yasouj, Iran
2 Associate Professor, Department of Animal Science, Faculty of Agriculture, Urmia University, Urmia, Iran
چکیده [English]

Introduction: The decrease in the sperm number in each dose of artificial insemination (AI) has led to the deterioration of sperm quality in different species. Decreasing sperm quality could be a major obstacle to the commercial application of flow-sorted sperm for gender selection. The reduction in the viability of cryopreserved spermatozoa at high dilution rates (low number of sperm/dose) has been attributed to the lack of useful components of the seminal plasma in the final freezing medium. Although the effect of adding or removing seminal plasma or its proteins on sperm activity has been widely investigated, the results of these studies are contradictory. Factors such as the final dilution rate and the extenders used in each work may influence the results. On the other hand, a milk-based extender was reported to be better than a Tris-citrate-fructose extender for a percentage of uncapacitated and linear motile spermatozoa after freezing-thawing of ovine semen. However, milk-based diluents interfere with the evaluation of sperm kinematics by the computer-assisted sperm analysis (CASA) systems. It is speculated that by adding milk to the composition of Tris diluents while taking advantage of the positive effects of milk, problems related to sperm evaluation disorders by CASA will not occur. Therefore, this study aimed to investigate the impacts of adding bovine skim milk and seminal plasma into the formulation of a Tris-citrate-egg yolk LDL extender on the kinematics and viability of frozen-thawed bull sperm when sub-optimal sperm numbers were packed in straws.
Materials and methods: Semen samples were collected from four bulls once a week for four consecutive weeks. The ejaculates collected on the same day were pooled provided that the semen concentration and progressive motility in each ejaculate were ≥2×109 cells/mL and ˃70%, respectively. The semen pool was divided into four equal aliquots and diluted with experimental extenders to obtain a final sperm concentration of 15×106 spermatozoa per mL. The base extender [made by Tris buffer (80% v/v), liquid ammonium sulfate-insoluble yolk fraction (20%), and glycerol (7% v/v)] was supplemented with 0, 5, and 10% (v/v) milk, and 0, 2 and 5% bovine seminal plasma. Therefore, the experiment was conducted in a completely randomized design with a factorial arrangement of 3×3. Diluted semen samples were frozen and stored in liquid nitrogen after being cooled and packed in 0.5 mL straws. The percentage of live cells in thawed samples was determined after Eosin-nigrosin staining and motility and kinematic parameters were analyzed by CASA. Data were subjected to analysis of variance using the general linear model of SAS software (version 9.2). Significant differences among treatment means were evaluated using the Tukey test. The statistical significance level was set at 0.05.
Results and discussion: The results indicated that the addition of milk at both 5 and 10% led to an increase in progressive motility, straightness, and sperm velocity parameters (Average path velocity, curvilinear velocity, and straight-line velocity). Adding 5% seminal plasma to the basal extender formulation increased the motility indexes (total and progressive motility), velocity indexes, and directional indices of cryopreserved spermatozoa. Among the parameters reflecting sperm wobble characteristics, wobble and amplitude of literal head increased; however, beat cross frequency and mean angular displacement decreased in the freeze-thawed spermatozoa diluted in the extenders containing 5% seminal plasma compared to those in the control straws. The average concentrations of both factors (5% milk and 2% seminal plasma), when present together in the base composition of the diluent, have shown a better result in terms of percentage sperm viability than when present alone.
Conclusions: According to the results, adding 5% seminal plasma to the extender formulation increased the kinematic parameters of frozen-thawed sperms. However, it should be noted that these increases do not seem to be desirable in sperm doses for artificial insemination but could be acceptable for in vitro fertilization (IVF). However, the 2% level of seminal plasma reduced the mean angular displacement parameter without affecting the velocity indices, which may indicate an improvement in sperm fertility. The addition of 2% semen plus 5% milk in bull semen diluent has resulted in better sperm viability. In addition, the 5% proportion of milk improved many parameters of sperm motility and kinematics, as did the 10% proportion. Therefore, the addition of 2% bovine seminal plasma plus 5% skimmed cow's milk to a Tris-LDL-citrate extender may positively affect the viability of frozen-thawed bull sperm and kinematic values ​​associated with fertility when semen is diluted in small amounts/doses.

کلیدواژه‌ها [English]

  • Dilution effect
  • Freezing-thawing
  • Seminal plasma
  • Sperm quality
  • Cow

مراجع

Al-Somai, N., Molan, P. C., Vishwanath, R., & Shannon, P. (1994). Anionic and cationic components from protein aggregates in bovine seminal plasma and their effects on sperm motility. Molecular Reproduction Development, 39, 328-336. doi: 10.1071/RD9940165
Arjun, V., Kumar, P., Dutt, R., Kumar, A., Bala, R., Verma, N., Jerome, A., Virmani, M., Patil, C. S., Singh, S., & Kumar, D. (2021). Is addition or removal of seminal plasma able to compensate for the dilution effect of buffalo semen? Andrologia, 53(8), e14123. doi: 10.1111/and.14123
Barrios, B., Pérez-Pé, R., Gallego, M., Tato, A., Osada, J., Muiño-Blanco, T., & Cebrián-Pérez, J. A.  (2000). Seminal plasma proteins revert the cold-shock damage on ram sperm membrane. Biology of Reproduction, 63, 1531-1537. doi: 10.1095/biolreprod63.5.1531
Batellier, F., Magistrini, M., Fauquant, J., & Palmer, E. (1997). Effect of milk fractions on survival of equine spermatozoa. Theriogenology, 48(3), 391-410. doi: 10.1016/S0093-691X(97)00250-1
Bergeron, A., & Manjunath, P. (2006). New insights towards understanding the mechanisms of sperm protection by egg yolk and milk. Molecular Reproduction and Development, 73(10), 1338-1344. doi: 10.1002/mrd.20565
Bernardini, A., Hozbor, F., Sanchez, E., Fornes, M. W., Alberio, R. H., & Cesari, A. (2011). Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage. Theriogenology, 76(3), 436-447. doi: 10.1016/j.theriogenology.2011.02.020
Bromfield, J. J. (2016). A role for seminal plasma in modulating pregnancy outcomes in domestic species. Reproduction, 152(6), R223-R232. doi: 10.1530/REP-16-0313
Brunner J. R. 1969. Milk lipoproteins. In: Tria, E., Scanu, A.M. (Eds.), Structural and functional aspects of lipoproteins in living system.  London and New York: Academic Press. Pp. 545-615.
Colás, C., Junquera, C., Pérez‐Pé, R., Cebrián‐Pérez, J. A., & Muiño‐Blanco, T. (2009). Ultrastructural study of the ability of seminal plasma proteins to protect ram spermatozoa against cold‐shock. Microscopy Research and Technique, 72(8), 566-572. doi: 10.1002/jemt.20710
Choong, C. H., & Wales, R. G. (1963). The use of various diluents for deep-freezing bull spermatozoa. Australian Journal of Biological Sciences, 16(4), 896-904. doi: 10.1071/BI9630896
Christensen, P., Labouriau, R., Birck, A., Boe-Hansen, G. B., Pedersen, J., & Borchersen, S. (2011). Relationship among seminal quality measures and field fertility of young dairy bulls using low-dose inseminations. Journal of Dairy Science, 94(4), 1744-1754. doi: 10.3168/jds.2010-3087
D'Amours, O., Frenette, G., Fortier, M., Leclerc, P., & Sullivan, R. (2010). Proteomic comparison of detergent-extracted sperm proteins from bulls with different fertility indexes. Reproduction, 139(3), 545-556. doi: 10.1530/REP-09-0375
D’Alessandro, A. G., & Martemucci, G. (2003). Evaluation of seasonal variations of semen freezability in Leccese ram. Animal Reproduction Science, 79, 93-102. doi: 10.1016/S0378-4320(03)00113-1
Dominguez Rebolledo, A., Navarrete Sierra, L., Cruz Tamayo, A., Aguiar Loria, A., Erosa Denis, S., Bolio Oses, R., González Parra, E., Paredes Monsreal, L., & Ramón Ugalde, J. (2007). Fertility in hair sheep inseminated with freeze spermatozoa rediluited with seminal plasma. Revista Científica, 17(1), 73-76.
Foote, R. H., Brockett, C. C., & Kaproth, M. T. (2002). Motility and fertility of bull sperm in whole milk extender containing antioxidants. Animal Reproduction Science, 71(1-2), 13-23. doi: 10.1016/S0378-4320(02)00018-0
Fréour, T., Jean, M., Mirallié, S., Dubourdieu, S., & Barrière, P. (2010). Computer-Assisted Sperm Analysis (CASA) parameters and their evolution during preparation as predictors of pregnancy in intrauterine insemination with frozen-thawed donor semen cycles. European Journal of Obstetrics & Gynecology and Reproductive Biology, 149(2), 186-189. doi: 10.1016/j.ejogrb.2009.12.029
Garcia, M. A., & Graham, E. F. (1987). Effects of low-molecular-weight fractions (LMWF) from milk, egg yolk, and seminal plasma on freezability of bovine spermatozoa. Cryobiology, 24(5), 429-436. doi: 10.1016/0011-2240(87)90046-0
Garner, D. L., Thomas, C. A., Gravance, C. G., Marshall, C. E., DeJarnette, J. M., & Allen, C. H. (2001). Seminal plasma addition attenuates the dilution effect in bovine sperm. Theriogenology, 56(1), 31-40. doi: 10.1016/S0093-691X(01)00540-4
Gil, J., Söderquist, L., & Rodriguez-Martinez, H. (2000). Influence of centrifugation and different extenders on post-thaw sperm quality of ram semen. Theriogenology, 54(1), 93-108. doi: 10.1016/S0093-691X(00)00328-9
Gillan, L., & Maxwell, W. M. C. (1999). The functional integrity and fate of cryopreserved ram spermatozoa in the female tract. Journal of Reproduction and Fertility Supplement, 54, 271-283. doi: 10.1530/biosciprocs.4.021
Heitland, A. V., Jasko, D. J., Squires, E. L., Graham, J. K., Pickett, B. W., & Hamilton, C. (1996). Factors affecting motion characteristics of frozen‐thawed stallion spermatozoa. Equine Veterinary Journal, 28(1), 47-53. doi: 10.1111/j.2042-3306.1996.tb01589.x
Holden, S. A., Fernandez-Fuertes, B., Murphy, E. M., Lonergan, P., & Fair, S. (2017). Effect of seminal plasma from high-and low-fertility bulls on cauda epididymal sperm function. Reproduction, Fertility and Development, 29(12), 2457-2465. doi: 10.1071/RD17136
Joint, F. A. O. (2005). Improving artificial breeding of cattle and buffalo in Asia. Guidelines and recommendations. A manual prepared under the framework of an IAEA Technical Cooperation Regional RCA Project on 'Improving Animal Productivity and Reproductive Efficiency', with technical support of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture (No. IAEA-TECDOC--1480). Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. P. 54.
Juyena, N. S., & Stelletta, C. (2012). Seminal plasma: an essential attribute to spermatozoa. Journal of Andrology, 33(4), 536-551. doi: 10.2164/jandrol.110.012583
Kakar, S. S., & Ganguli, N. C. (1978). Milk as an extender for semen: a review. The Indian Journal of Animal Sciences, 48(11), 777-790.
King, L. M., Holsberger, D. R., & Donoghue, A. M. (2000). Correlation of CASA velocity and linearity parameters with sperm mobility phenotype in turkeys. Journal of Andrology, 21(1), 65-71. doi: 10.1002/j.1939-4640.2000.tb03277.x
Larsen, L., Scheike, T., Jensen, T. K., Bonde, J. P., Ernst, E., Hjollund, N. H., Zhou, Y., Skakkebaek, N. E., & Giwercman, A. (2000). Computer-assisted semen analysis parameters as predictors for fertility of men from the general population. Human Reproduction, 15(7), 1562-1567. doi: 10.1093/humrep/15.7.1562
Leboeuf, B., Guillouet, P., Batellier, F., Bernelas, D., Bonne, J. L., Forgerit, Y., & Magistrini, M. (2003). Effect of native phosphocaseinate on the in vitro preservation of fresh semen. Theriogenology, 60(5), 867-877. doi: 10.1016/S0093-691X(03)00095-5
Manjunath, P., & Thérien, I. (2002). Role of seminal plasma phospholipid-binding proteins in sperm membrane lipid modification that occurs during capacitation. Journal of Reproductive Immunology, 53(1-2), 109-119. doi: 10.1016/S0165-0378(01)00098-5
Maxwell, W. M., Welch, G. R., & Johnson, L. A. (1996). Viability and membrane integrity of spermatozoa after dilution and flow cytometric sorting in the presence or absence of seminal plasma. Reproduction, Fertility and Development, 8(8), 1165-1178. doi: 10.1071/RD9961165
Maxwell, W. M. C., Evans, G., Mortimer, S. T., Gillan, L., Gellatly, E. S., & McPhie, C. A. (1999). Normal fertility in ewes after cervical insemination with frozen-thawed spermatozoa supplemented with seminal plasma. Reproduction Fertility Development, 11, 123-126. doi: 10.1071/RD99046
Maxwell, W.M.C., De Graaf, S. P., Ghaoui, RE-H., & Evans, G. (2007). Seminal plasma effects on sperm handling and female fertility. Society of Reproduction and Fertility Supplement, 64, 13-38. doi: 10.5661/RDR-VI-13
Mdhluli, M. C., & Van der Horst, G. (2002). The effect of oleanolic acid on sperm motion characteristics and fertility of male Wistar rats. Laboratory Animals, 36(4), 432-437. doi: 10.1258/002367702320389107
Morrell, J. M., Rodriguez‐Martinez, H., & Johannisson, A. (2010). Single-layer centrifugation of stallion spermatozoa consistently selects the most robust spermatozoa from the rest of the ejaculate in a large sample size. Equine Veterinary Journal, 42(7), 579-585. doi: 10.1111/j.2042-3306.2010.00101.x
Morrell, J. M., Madej, M., Madej, A., & Stalhammar, H. (2015). Fast Protein Liquid Chromatography of Swedish Red and Holstein bull seminal plasma proteins in relation to bull fertility. Reproduction in Domestic Animals, 50, 67-68.
Muiño-Blanco, T., Pérez-Pé, R., & Cebrián-Pérez, J. A. (2008). Seminal plasma proteins and sperm resistance to stress. Reproduction in Domestic Animals, 4, 18-31. doi: 10.1111/j.1439-0531.2008.01228.x
Nongbua, T., Goodla, L., Johannisson, A., & Morrell, J. M. (2014). Relationship between 56-day non-return rate and the quality of frozen-thawed bull semen. Reproduction in Domestic Animals, 49, 84-84.
Nongbua, T., Al-Essawe, E. M., Edman, A., Johannisson, A., & Morrell, J. M. (2018). Adding bovine seminal plasma prior to freezing improves post-thaw bull sperm kinematics but decreases mitochondrial activity. Systems Biology in Reproductive Medicine, 64(3), 183-190. doi: 10.1080/19396368.2018.1455245
Okazaki, T., Abe, S., Yoshida, S., & Shimada, M. (2009). Seminal plasma damages sperm during cryopreservation, but its presence during thawing improves semen quality and conception rates in boars with poor post-thaw semen quality. Theriogenology, 71(3), 491-498. doi: 10.1016/j.theriogenology.2008.08.014
Oliveira, L. Z., de Arruda, R. P., de Andrade, A. F. C., Celeghini, E. C. C., dos Santos, R. M., Beletti, M. E., & de Lima, V. F. M. H. (2012). Assessment of field fertility and several in vitro sperm characteristics following the use of different Angus sires in a timed-AI program with suckled Nelore cows. Livestock Science, 146(1), 38-46. doi: 10.1016/j.livsci.2012.02.018
Oyewumi, S. O., Akintunde, A. O., Tayo, G. O., Ndubuisi-Ogbonna, L. C., & Abdullah, A. R. (2024). Review of livestock semen extension and cryopreservation of spermatozoa. Bima Journal of Science and Technology, 8(1B), 13-22. doi: 10.56892/bima.v8i1.609
Patil, S., Kumar, P., Singh, G., Bala, R., Jerome, A., Patil, C. S., Kumar, D., Singh, S., & Sharma, R. K. (2020). ‘Semen dilution effect’ on sperm variables and conception rate in buffalo. Animal Reproduction Science, 214, 106304. doi: 10.1016/j.anireprosci.2020.106304
Pickett, B. W., & Amann, R. P. (1987). Extension and storage of stallion spermatozoa: a review. Journal of Equine Veterinary Science, 7(5), 289-302. doi: 10.1016/S0737-0806(87)80049-7
Poiani, A. (2006). Complexity of seminal fluid: a review. Behavioral Ecology and Sociobiology, 60, 289-310. doi: 10.1007/s00265-006-0178-0
Pérez-Pé, R., Cebrián-Pérez, J. A., & Muino- Blanco, T. (2001). Semen plasma proteins prevent cold-shock membrane damage to ram spermatozoa. Theriogenology, 56, 425-434. doi: 10.1016/S0093-691X(01)00574-X
Rodríguez‐Martínez, H., Kvist, U., Ernerudh, J., Sanz, L., & Calvete, J. J. (2011). Seminal plasma proteins: what role do they play? American Journal of Reproductive Immunology, 66, 11-22. doi: 10.1111/j.1600-0897.2011.01033.x
Stachecki, J. J., Ginsburg, K. A., Leach, R. E., & Armant, D. R. (1993). Computer‐assisted semen analysis (CASA) of epididymal sperm from the domestic cat. Journal of Andrology, 14(1), 60-65. doi: 10.1002/j.1939-4640.1993.tb00370.x
Suarez, S. S., & Ho, H. C. (2003). Hyperactivated motility in sperm. Reproduction in Domestic Animals, 38(2), 119-124. doi: 10.1046/j.1439-0531.2003.00397.x
Ustuner, B., Gunay, U., & Nur, Z. (2009). Effect of seminal plasma, egg yolk, and season on the fereezability of Saanen buck semen.  Bulletin of the Veterinary Institute in Puławy, 53, 369-374.
Yi, Y. J., Im, G. S., & Park, C. S. (2002). Lactose-egg yolk diluent supplemented with N-acetyl-D-glucosamine affect acrosome morphology and motility of frozen-thawed boar sperm. Animal Reproduction Science, 74(3-4), 187-194. doi: 10.1016/S0378-4320(02)00187-2
تحقیقات تولیدات دامی
دوره 13، شماره 3
آذر 1403
صفحه 47-60

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