Optimizing the conventional method of sperm freezing in liquid nitrogen vapour for Wallachian sheep conservation program


Savvulidi F.G., Ptacek M., Malkova A., Beranek J., Stadnik L. (2021): Optimizing the conventional method of sperm freezing in liquid nitrogen vapour for Wallachian sheep conservation program. Czech J. Anim. Sci., 66: 55–64.

download PDF

The aim of the present study was to optimize the conventional method of sperm freezing in liquid nitrogen (LN2) vapour for successful cryopreservation of Wallachian ram sperm, the genetic resources of the Czech Republic. Sperm in straws were frozen using the conventional freezing method via a static exposure of sperm doses to LN2 vapour, or by four different modified freezing methods. Under modified freezing, straws were frozen by a discontinuous, time-dependent decremental change in the distance between the straws and the surface of LN2. The viability of sperm was evaluated by flow cytometry after sperm equilibration, and immediately after thawing. Besides the observed inter-sire and daily variation, the obtained results suggest the methodological weakness of the conventional freezing method via the static exposure of sperm doses to LN2 vapour. With the use of the optimized freezing procedure, all parameters of thawed sperm were significantly (P < 0.05) improved in comparison with the conventional method: percentage of thawed sperm viability increased up to 48.3%, percentage of sperm with plasma membrane damage after thawing decreased to 6.58%, percentage of sperm with acrosome damage decreased to 24.4%, and percentage of sperm with deteriorated mitochondrial activity decreased to 6.28%. In conclusion, our results suggest that an optimized freezing procedure should be routinely used instead of the conventional method to cryopreserve Wallachian ram sperm.

Akcay E, Kulaksiz R, Daskin A, Cebi C, Tekin K. The effect of different dilution rates on post-thaw quality of ram semen frozen in two different eggyolk free extenders. Slov Vet Res. 2012 Jan 1;49(2):97-102.
Alvarez M, Tamayo-Canul J, Anel E, Boixo JC, Mata-Campuzano M, Martinez-Pastor F, Anel L, Paz P. Sperm concentration at freezing affects post-thaw quality and fertility of ram semen. Theriogenology. 2012 Apr 1;77(6):1111-8.
Anel L, Paz P, Alvarez M, Chamorro CA, Boixo JC, Manso A, Gonzalez M, Kaabi M, Anel E. Field and in vitro assay of three methods for freezing ram semen. Theriogenology. 2003 Oct 15;60(7):1293-308. https://doi.org/10.1016/S0093-691X(03)00140-7
Barbas JP, Mascarenhas RD. Cryopreservation of domestic animal sperm cells. Cell Tissue Bank. 2009 Feb 1;10(1):49-62.
Beran J, Stadnik L, Duchacek J, Tousova R, Louda F, Stolc L. Effect of bulls’ breed, age and body condition score on quantitative and qualitative traits of their semen. Acta Univ Agric et Silvic Mendel Brun. 2011;59(6):37-44. https://doi.org/10.11118/actaun201159060037
Busa D, Machac M, Balazi A, Svoradova A, Vasicek J, Jurcik R, Chrenek P. Sperm motility of rams from two Slovak sheep breeds: Short communication. Slovak J Anim Sci. 2019 Dec 18;52(4):178-80.
Byrne GP, Lonergan P, Wade M, Duffy P, Donovan A, Hanrahan JP, Boland MP. Effect of freezing rate of ram spermatozoa on subsequent fertility in vivo and in vitro. Anim Reprod Sci. 2000 Sep 1;62(4):265-75.
Choe CY, Kim JG, Cho SR, Son DS, Kim YK, Balasubramanian S, Choe SE, Rho GY. Influence of seasons, extenders, slow and rapid freezing on seminal characters in Korean native bucks. Reprod Domest Anim. 2006 Feb;41(1):55-60. https://doi.org/10.1111/j.1439-0531.2006.00652.x
D’Alessandro A, Martemucci G. Evaluation of seasonal variations of semen freezability in Leccese ram. Anim Reprod Sci. 2003 Nov 20;79(1-2):93-102. https://doi.org/10.1016/S0378-4320(03)00113-1
David I, Druart X, Lagriffoul G, Manfredi E, Robert-Granie C, Bodin L. Genetic and environmental effects on semen traits in Lacaune and Manech tete rousse AI rams. Genet Sel Evol. 2007 Jul-Aug;39(4):405-19. https://doi.org/10.1186/1297-9686-39-4-405
David I, Kohnke P, Lagriffoul G, Praud O, Plouarboue F, Degond P, Druart X. Mass sperm motility is associated with fertility in sheep. Anim Reprod Sci. 2015 Oct;161:75-81. https://doi.org/10.1016/j.anireprosci.2015.08.006
Dolezalova M, Stadnik L, Biniova Z, Duchacek J, Stupka R. Equilibration and freezing interactions affecting bull sperm characteristics after thawing. Czech J Anim Sci. 2016 Nov 17;61(11):515-25.
Domingo P, Olaciregui M, Gonzalez N, Blas I, Gil L. Comparison of different semen extenders and cryoprotectant agents to enhance cryopreservation of rabbit spermatozoa. Czech J Anim Sci. 2019 Feb 14;64(2):59-66.
Galarza DA, Lopez-Sebastian A, Woelders H, Blesbois E, Santiago-Moreno J. Two-step accelerating freezing protocol yields a better motility, membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols. Cryobiology. 2019 Dec 1;91:84-9.
Jandurova OM, Kott T, Kottova B, Czernekova V, Milerski M. Genetic relationships among Sumava, Valachian and Improved Valachian sheep. Small Rumin Res. 2005 Mar 1;57(2-3):157-65.
Jha PK, Alam MGS, Mansur AA, Naher N, Islam T, Bhuiyan MU, Bari FY. Cryopreservation of Bangladeshi ram semen using different diluents and manual freezing techniques. Cryobiology. 2019 Aug 1;89:35-41.  https://doi.org/10.1016/j.cryobiol.2019.06.001
Jovicic M, Chmelikova E, Sedmikova M. Cryopreservation of boar semen. Czech J Anim Sci. 2020 Apr 30;65(4):115-23.
Lv C, Wu G, Hong Q, Quan G. Spermatozoa cryopreservation: State of art and future in small ruminants. Biopreserv Biobank. 2019 Apr 1;17(2):171-82. https://doi.org/10.1089/bio.2018.0113
Martinez-Pastor F, Mata-Campuzano M, Alvarez-Rodriguez M, Alvarez M, Anel L, Paz P. Probes and techniques for sperm evaluation by flow cytometry. Reprod Domest Anim. 2010 Jun;45 Suppl 2:67-78.
Olah J, Kusza S, Harangi S, Posta J, Kovacs A, Pecsi A, Budai C, Javor A. Seasonal changes in scrotal circumference, the quantity and quality of ram semen in Hungary. Arch Anim Breed. 2013 Oct 10;56(1):102-8.  https://doi.org/10.7482/0003-9438-56-010
Ptacek M, Milerski M, Duchacek J, Schmidova J, Tancin V, Uhrincat M, Stadnik L, Michlova T. Analysis of fatty acid profile in milk fat of Wallachian sheep during lactation. J Dairy Res. 2019a May 1;86(2):233-7.
Ptacek M, Milerski M, Stadnik L, Duchacek J, Tancin V, Schmidova J, Uhrincat M, Michlova T, Nohejlova L. Effect of milk intake, its composition, and fatty acid profile distribution on live weight of suckling Wallachian lambs until their weaning. Animals. 2019b Oct;9(10): [7 p.]. https://doi.org/10.3390/ani9100718
Ptacek M, Stadnikova M, Savvulidi F, Stadnik L. Ram semen cryopreservation using egg yolk or egg yolk-free extenders: Preliminary results. Sci Agric Bohem. 2019c Jun 1;50(2):96-103.  https://doi.org/10.2478/sab-2019-0014
Purdy P. The post-thaw quality of ram sperm held for 0 to 48 h at 5 °C prior to cryopreservation. Anim Reprod Sci. 2006 Jun;93(1-2):114-23. https://doi.org/10.1016/j.anireprosci.2005.07.002
Ramon M, Perez-Guzman MD, Jimenez-Rabadan P, Esteso MC, Garcia-Alvarez O, Maroto-Morales A, Anel-Lopez L, Soler AJ, Fernandez-Santos MR, Garde JJ. Sperm cell population dynamics in ram semen during the cryopreservation process. PloS One. 2013 Mar 27;8(3): [8 p.].
Russel AJF, Doney JM, Gunn RG. Subjective assessment of body fat in live sheep. J Agric Sci. 1969 Jun;72(3):451-4. https://doi.org/10.1017/S0021859600024874
Salamon S, Maxwell WMC. Storage of ram semen. Anim Reprod Sci. 2000 Aug 18;62(1-3):77-111. https://doi.org/10.1016/S0378-4320(00)00155-X
Stolc L, Stadnik L, Jezkova A, Louda F. Relationships among herd, ram breeds, age of rams, sperm density before diluting and sperm motility during thermal survival test. Acta Univ Agric et Silvic Mendel Brun. 2009 Mar;57(4):109-16. https://doi.org/10.11118/actaun200957040109
download PDF

© 2021 Czech Academy of Agricultural Sciences | Prohlášení o přístupnosti