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In vivo elde edilen Saanen keçisi embriyolarının yavaş dondurulması üzerine farklı kriyoprotektanların etkisinin karşılaştırılması

Year 2018, Volume: 65 Issue: 2, 171 - 178, 01.06.2018

Sakine Ülküm Çizmeci Mehmet Güler Mustafa Kaymaz

https://doi.org/10.1501/Vetfak_0000002843

Abstract

Bu çalışmada farklı kriyoprotektanların, yavaş dondurma yöntemi ile dondurulup çözdürülmüş Saanen keçisi embriyolarının canlılığı üzerine etkilerinin belirlenmesi amaçlanmıştır. Çalışmanın hayvan materyalini 15 baş Saanen ırkı keçi ve 3 baş teke oluşturdu. Keçilere siklusun dönemi gözetilmeksizin 12 gün süreyle fluorogeston asetat (20 mg) emdirilmiş intravaginal sünger yerleştirildi. Sünger uygulamasının 9. gününden itibaren 3 gün süreyle 12 saat arayla azalan dozlarda folikül uyarıcı hormon (FSH) (5050; 30-30; 20-20 mg) kas içi yolla enjekte edildi. Sünger çıkarıldıktan 24 saat sonra doğal aşım yaptırıldı. İlk aşımdan sonraki 7. gün laparatomik uterus yıkaması yapılarak embriyolar elde edildi. Toplanan embriyolar 3 farklı kriyoprotektan [etilen glikol (EG), gliserol ve dimetil sulfoksit (DMSO)] kullanılarak yavaş dondurma yöntemiyle donduruldu. Çözdürülen embriyolar 38.5 ºC’de %5 CO2’de inkübasyona bırakıldı ve 24, 48 ve 72. saatlerde canlılık muayeneleri yapıldı. Çalışmada süperovulasyon cevabı (≥4 Cl) %93.3, embriyo toplama oranı %72.3, transfer edilebilir embriyo oranı ise %58 olarak belirlendi. Dondurulup çözdürülen embriyoların 24, 48 ve 72. saatlerdeki canlılık oranları EG’de %64.86; %56.76; %54.05, gliserolde %54.55; %45.45; %36.36, DMSO da ise %46.88; %40.63; %28.13 olarak belirlendi. Embriyoların 72. saate ulaşmalarında EG ile dondurulanların gliserol ve DMSO ile dondurulanlardan istatistiki olarak daha iyi olduğu tespit edildi (P<0.05). Saatlere göre blastosistlerin yaşama oranları EG’de %76; %64; %60, gliserolde %54.6; %45.5; %36.4 ve DMSO’da %42.1; %36.8; %21.1 olarak belirlendi ve 72. saatte EG ve DMSO arasında farkın önemli olduğu görüldü (P<0.05). Yapılan çalışma sonucunda çözdürme sonrası 24, 48 ve 72. saatlerdeki canlılık oranları EG ile dondurulan embriyolarda diğer kriyoprotektanlarla dondurulanlardan daha yüksek olduğu belirlendi

Keywords

DMSO etilen glikol gliserol keçi yavaş dondurma

References

  • Ağaoğlu AR, Karakaş K, Kaymaz M (2014): In vivo embryo production in some native goats breeds in Turkey. Turk J Vet Anim Sci, 38, 1-7.
  • Al Yacoub AN, Gauly M, Holtz W (2010): Open pulled straw vitrification of goat embryos at various stages of development. Theriogenology, 73, 1018-1023.
  • Amiridis GS, Cseh S (2012): Assisted reproductive technologies in the management of small ruminants. Anim Reprod Sci, 130, 152-161.
  • Cocero MJ, Procureur E, De La Fuente J ve ark. (1988): Glycerol or ethylene glycol for cryoprotection of deep frozen ewe embryos. Theriogenology, 29, 238.
  • De Paz P, Sanchez AJ, Fernandez JG ve ark. (1994): Sheep embryo cryopreservation by vitrification and conventional freezing. Theriogenology, 42, 327-338.
  • Frank GC, Coley SL, Betterbed B ve ark. (1985): The effects of cryoprotective agents, dilution rates, freezing rates, and freezing units on the survival of bovine embryos. Theriogenology, 23, 194.
  • Gal FL, Baril G, Vallet JC ve ark. (1993): In vivo and in vitro survival of goat embryos after freezing with ethylene glycol or glycerol. Theriogenology, 40, 771-777.
  • Garcia-Garcia RM, Gonzales-Bulnes A, Dominquez V ve ark. (2006): Survival of frozen thawed sheep embryos cryopreserved at cleavage stages. Cryobiology, 52, 108-113.
  • Gordon I (1997): Reproduction in sheep and goats. CABI publishing, USA.
  • Greyling JPC, ven der Nest M, Schwalbach LMJ ve ark. (2002): Superovulation and embryo transfer in South African Boer and Indigenous feral goats. Small Ruminant Res, 43, 54-51.
  • Grizelj J, Vince S, Tomaskoviç A ve ark. (2006): Superovulation response and embryo recovery of donor does Boer race in embryo transfer procedures. 14 Congreso Internacional de la Federacion Mediterranea de Sanidad y Produccion de Rumiantes.
  • Han YM, Kim SJ, Park JS ve ark. (2000): Blastocyts viability and generation of
  • transgenic cattle following freezing of in vitro produced, DNA- injected embryos. Anim Reprod Sci, 63, 53-63.
  • International Embryo Transfer Society (IETS). Available http://www.iets.org/index.asp?autotry=true&ULnotkn=true (Accessed 03.04.2011). at
  • Kanagawa H, Shimohira I, Saitoh N (1995): Manual of Bovine Embryo Transfer. National Livestock Breeding Center MAFF JICA- JAPAN.
  • Kaymaz M (2012): Yardımcı Üreme Teknikleri, 695-811. In. A Semacan, M Kaymaz. M Fındık, A Rişvanlı, A Köker (Ed). Çiftlik Hayvanlarında Doğum ve Jinekoloji, Medipress, Malatya.
  • Kılboz Eİ, Karaca F (2010): Üreme mevsimi dışında genç keçilerde flugeston asetat vaginal sünger ve norgestomet kulak implantı uygulamalarıyla östrüslerin uyarılması. Van Vet J, 21, 1-6.
  • Lehloenya KC, Greyling JPC, Grobler S (2008): Effect of season on the superovulatory response in Boer goat does. Small Ruminant Res, 78, 74-79.
  • Lehloenya KC, Greyling JPC (2010a): The ovarian response and embryo recovery rate in Boer goat does following different superovulation protocols, during the breeding season. Small Ruminant Res, 88, 38-43.
  • Lehloenya KC, Greyling JPC (2010b): Embryo transfer using cryopreserved boer goats blastocyts. S Afr J Anim Sci, 5, 446-450.
  • Liu L (2009): Development of novel cryopreservation method for mammalian oocytes. Szent Istvan University. (Accessed 10.05.2011).
  • Mapletoft RJ, Kristina BS, Gregg PA (2002): Recent advances in the superovulation in cattle. Reprod Nutr Dev, 42, 601-611.
  • Maracek I, Krajnicakova M, Kostecky M ve ark. (2002): Tertiary follicular growth wave dynamics after oestrus synchronization and superovulation in ewes and goats. Acta Vet Brno, 71, 481-486.
  • Martemucci G, D’Alessandro AG (2013): Efficiancy of FSH/LH treatments for in vivo production of embryos and their cryopreservation by different methods in goats. Small Ruminant Res, 114, 264-271.
  • Martinez AG, Matkoviç M (1998): Cryopreservation of ovine Theriogenology, 49, 1039-1049. freezing and vtirification.
  • Mayorga I, Maraa L, Sannaa D ve ark. (2011): Good quality sheep embryos produced by superovulation treatment without the use of progesterone devices. Theriogenology, 75, 1661-1668.
  • Menchaca A, Vilarino M, Crispo M ve ark. (2007): Day 0 protocol: Superstimulatory treatment initiated in the absence of a large follicle improves ovarian response and embryo yield in goats. Theriogenology, 68, 1111-1117.
  • Palasz AT, Mapletoft RJ (1996): Cryopreservation of mammalian embryos and
  • oocytes: Recent advances. Biotech Adv, 14, 127-149.
  • Paramio MT (2010): In vivo and in vitro embryo production in goats. Small Ruminant Res, 89, 144-148.
  • Prentice JR, Anzar M (2011): Cryopreservation of mammalian oocyte for conservation of animal genetics. Veterinary Medicine International, 1-11. Available at http://dx.doi.org/10.4061/2011/146405 13.03.2014). (Accessed
  • Quana F, Zhang Z, Anc Z ve ark. (2010): Effect of transporting donor or recipient does and their embryos on the outcome of fresh embryo transfer in Boer goats. Small Ruminant Res, 88,1-5.
  • Sağırkaya H, Bağış H (2003): Memeli embriyolarının kriyoprezervasyonu. Uludağ Üniv Vet Fak Derg, 22, 127-135.
  • Sarıbay MK, Doğruer G, Ergün Y ve ark. (2008): Üreme sezonu dışında laktasyondaki kıl keçilerinde flourogestone acetat içeren vaginal süngerlerle östruslerin uyarılması; GnRH ve hCG uygulamalarının döl verimi üzerine etkisi. Vet Bil Derg, 24, 21-27.
  • Seidel GE (1991): Training manual for embryo transfer in cattle. Food and agriculture organization of the united nations (FAO), Rome, [Cited 11.03.2014]. Available at http://www.fao.org/docrep/004/t0117e/t0117e00.htm.
  • Smith GD, Silva CA, Silva E (2004): Developmental consequences of cryopreservation of mammalian oocytes and embryos. Reprod BioMed Online, 9, 171-178.
  • Stringfellow DA (2010): Recommendations for the sanitary handling of in vivo derived embryos, 65-68. In: DA Stringfellow, MD Givens (Ed), Manual of the international embryo transfer society (International Embryo Transfer Society), IETS publishing, United States of America.
  • Taşdemir U, Ağaoğlu AR, Kaymaz M ve ark. (2011): Ovarian response and embryo yield of Angora and Kilis goats given the day 0 protocol for superovulation in the non- breeding season. Trop Anim Health Prod, 43, 1035-1038.
  • Tekeli T (2010): Embriyo Nakli, 81-97. In: E Alaçam (Ed), Evcil Hayvanlarda Doğum ve İnfertilite, Medisan, Ankara.
  • Van Wagtendonk-De Leeuw AM, Den Daas JHG, Kruip TH ve ark. (1995): Comparison of the efficacy of conventional slow freezing rapid cryopreservation methods for bovine embryos. Cryobiology, 32,157-167.
  • Wright JM, Appendix D (2010): Photographic illustrations of embryo developmental stage and quality codes, 141-144. In: DA Stringfellow, MD Givens (Ed), Manual of the international embryo transfer society (International Embryo Transfer Society), IETS publishing, United States of America.
  • Wurth YA, Reinders JMC, Rall WF ve ark. (1994): Developmental potential of in vitro produced bovine embryos following cryopreservation and single-embryo transfer. Theriogenology, 42,1275-1284.

Comparision of different cryoprotectants on slow freezing of in vivo derived Saanen goats embryos

Year 2018, Volume: 65 Issue: 2, 171 - 178, 01.06.2018

Sakine Ülküm Çizmeci Mehmet Güler Mustafa Kaymaz

https://doi.org/10.1501/Vetfak_0000002843

Abstract

This study aimed to determine the effects of different cryoprotectants on the viability of Saanen goats embryos which were frozen and thawed with slow freezing method. The study was conducted on 15 Saanen goats and 3 bucks. Fluorogeston acetate (20 mg) impregnated intravaginal sponges were inserted in goats for 12 days regardless of the sexual cycle. Starting on the 9thday of intravaginal sponge administration, follicle stimulating hormone (FSH) was injected intramuscularly, every 12 hours at decreasing doses (50-50; 30-30; 20-20 mg) for 3 days. Goats were mated naturally 24 hours after removal of the sponges. Embryos were recovered by laparotomic uterine flushing on the 7th day after the first mating. Collected embryos were frozen by using 3 different cryoprotectants [ethylene glycol (EG), glycerol, and dimetil sulfoksit DMSO)] with slow freezing technique. Thawed embryos were incubated at 38.5 ºC and 5% CO2. The viability of embryos was evaluated at the 24th, the 48th, the 72nd hours after thawing. Superovulation response (≥4 Cl), embryo recovery rate and transferable embryo rate were found to be 93.3%, 72.3% and 58%, respectively. Viability rates of frozen and thawed embryos at the 24th, the 48th, the 72nd hours were found respectively to be 64.86%; 56.76%; 54.05% in EG group, 54.55%, 45.45%; 36.36% in glycerol group and 46.88%; 40.63%; 28.13% in DMSO group. Viability rates of the frozen embryos with EG were statistically better than embryos frozen with glycerol and DMSO (P<0.05) at 72nd hour. Survival rates of blastocysts frozen were 76%; 64%; 60%; 54.6% in EG group 45.5%; 36.4% in glycerol group, and 42.1%; 36.8%; 21.1% in DMSO group and at 72nd hour the difference between EG and DMSO group was significant (P<0.05). In conclusion, viability of embryos at the 24th, the 48th, the 72nd hours after thawing in EG group was significantly higher than the embryos frozen with other cryoprotectants

Keywords

DMSO ethylene glycol glycerol goat slow freezing

References

  • Ağaoğlu AR, Karakaş K, Kaymaz M (2014): In vivo embryo production in some native goats breeds in Turkey. Turk J Vet Anim Sci, 38, 1-7.
  • Al Yacoub AN, Gauly M, Holtz W (2010): Open pulled straw vitrification of goat embryos at various stages of development. Theriogenology, 73, 1018-1023.
  • Amiridis GS, Cseh S (2012): Assisted reproductive technologies in the management of small ruminants. Anim Reprod Sci, 130, 152-161.
  • Cocero MJ, Procureur E, De La Fuente J ve ark. (1988): Glycerol or ethylene glycol for cryoprotection of deep frozen ewe embryos. Theriogenology, 29, 238.
  • De Paz P, Sanchez AJ, Fernandez JG ve ark. (1994): Sheep embryo cryopreservation by vitrification and conventional freezing. Theriogenology, 42, 327-338.
  • Frank GC, Coley SL, Betterbed B ve ark. (1985): The effects of cryoprotective agents, dilution rates, freezing rates, and freezing units on the survival of bovine embryos. Theriogenology, 23, 194.
  • Gal FL, Baril G, Vallet JC ve ark. (1993): In vivo and in vitro survival of goat embryos after freezing with ethylene glycol or glycerol. Theriogenology, 40, 771-777.
  • Garcia-Garcia RM, Gonzales-Bulnes A, Dominquez V ve ark. (2006): Survival of frozen thawed sheep embryos cryopreserved at cleavage stages. Cryobiology, 52, 108-113.
  • Gordon I (1997): Reproduction in sheep and goats. CABI publishing, USA.
  • Greyling JPC, ven der Nest M, Schwalbach LMJ ve ark. (2002): Superovulation and embryo transfer in South African Boer and Indigenous feral goats. Small Ruminant Res, 43, 54-51.
  • Grizelj J, Vince S, Tomaskoviç A ve ark. (2006): Superovulation response and embryo recovery of donor does Boer race in embryo transfer procedures. 14 Congreso Internacional de la Federacion Mediterranea de Sanidad y Produccion de Rumiantes.
  • Han YM, Kim SJ, Park JS ve ark. (2000): Blastocyts viability and generation of
  • transgenic cattle following freezing of in vitro produced, DNA- injected embryos. Anim Reprod Sci, 63, 53-63.
  • International Embryo Transfer Society (IETS). Available http://www.iets.org/index.asp?autotry=true&ULnotkn=true (Accessed 03.04.2011). at
  • Kanagawa H, Shimohira I, Saitoh N (1995): Manual of Bovine Embryo Transfer. National Livestock Breeding Center MAFF JICA- JAPAN.
  • Kaymaz M (2012): Yardımcı Üreme Teknikleri, 695-811. In. A Semacan, M Kaymaz. M Fındık, A Rişvanlı, A Köker (Ed). Çiftlik Hayvanlarında Doğum ve Jinekoloji, Medipress, Malatya.
  • Kılboz Eİ, Karaca F (2010): Üreme mevsimi dışında genç keçilerde flugeston asetat vaginal sünger ve norgestomet kulak implantı uygulamalarıyla östrüslerin uyarılması. Van Vet J, 21, 1-6.
  • Lehloenya KC, Greyling JPC, Grobler S (2008): Effect of season on the superovulatory response in Boer goat does. Small Ruminant Res, 78, 74-79.
  • Lehloenya KC, Greyling JPC (2010a): The ovarian response and embryo recovery rate in Boer goat does following different superovulation protocols, during the breeding season. Small Ruminant Res, 88, 38-43.
  • Lehloenya KC, Greyling JPC (2010b): Embryo transfer using cryopreserved boer goats blastocyts. S Afr J Anim Sci, 5, 446-450.
  • Liu L (2009): Development of novel cryopreservation method for mammalian oocytes. Szent Istvan University. (Accessed 10.05.2011).
  • Mapletoft RJ, Kristina BS, Gregg PA (2002): Recent advances in the superovulation in cattle. Reprod Nutr Dev, 42, 601-611.
  • Maracek I, Krajnicakova M, Kostecky M ve ark. (2002): Tertiary follicular growth wave dynamics after oestrus synchronization and superovulation in ewes and goats. Acta Vet Brno, 71, 481-486.
  • Martemucci G, D’Alessandro AG (2013): Efficiancy of FSH/LH treatments for in vivo production of embryos and their cryopreservation by different methods in goats. Small Ruminant Res, 114, 264-271.
  • Martinez AG, Matkoviç M (1998): Cryopreservation of ovine Theriogenology, 49, 1039-1049. freezing and vtirification.
  • Mayorga I, Maraa L, Sannaa D ve ark. (2011): Good quality sheep embryos produced by superovulation treatment without the use of progesterone devices. Theriogenology, 75, 1661-1668.
  • Menchaca A, Vilarino M, Crispo M ve ark. (2007): Day 0 protocol: Superstimulatory treatment initiated in the absence of a large follicle improves ovarian response and embryo yield in goats. Theriogenology, 68, 1111-1117.
  • Palasz AT, Mapletoft RJ (1996): Cryopreservation of mammalian embryos and
  • oocytes: Recent advances. Biotech Adv, 14, 127-149.
  • Paramio MT (2010): In vivo and in vitro embryo production in goats. Small Ruminant Res, 89, 144-148.
  • Prentice JR, Anzar M (2011): Cryopreservation of mammalian oocyte for conservation of animal genetics. Veterinary Medicine International, 1-11. Available at http://dx.doi.org/10.4061/2011/146405 13.03.2014). (Accessed
  • Quana F, Zhang Z, Anc Z ve ark. (2010): Effect of transporting donor or recipient does and their embryos on the outcome of fresh embryo transfer in Boer goats. Small Ruminant Res, 88,1-5.
  • Sağırkaya H, Bağış H (2003): Memeli embriyolarının kriyoprezervasyonu. Uludağ Üniv Vet Fak Derg, 22, 127-135.
  • Sarıbay MK, Doğruer G, Ergün Y ve ark. (2008): Üreme sezonu dışında laktasyondaki kıl keçilerinde flourogestone acetat içeren vaginal süngerlerle östruslerin uyarılması; GnRH ve hCG uygulamalarının döl verimi üzerine etkisi. Vet Bil Derg, 24, 21-27.
  • Seidel GE (1991): Training manual for embryo transfer in cattle. Food and agriculture organization of the united nations (FAO), Rome, [Cited 11.03.2014]. Available at http://www.fao.org/docrep/004/t0117e/t0117e00.htm.
  • Smith GD, Silva CA, Silva E (2004): Developmental consequences of cryopreservation of mammalian oocytes and embryos. Reprod BioMed Online, 9, 171-178.
  • Stringfellow DA (2010): Recommendations for the sanitary handling of in vivo derived embryos, 65-68. In: DA Stringfellow, MD Givens (Ed), Manual of the international embryo transfer society (International Embryo Transfer Society), IETS publishing, United States of America.
  • Taşdemir U, Ağaoğlu AR, Kaymaz M ve ark. (2011): Ovarian response and embryo yield of Angora and Kilis goats given the day 0 protocol for superovulation in the non- breeding season. Trop Anim Health Prod, 43, 1035-1038.
  • Tekeli T (2010): Embriyo Nakli, 81-97. In: E Alaçam (Ed), Evcil Hayvanlarda Doğum ve İnfertilite, Medisan, Ankara.
  • Van Wagtendonk-De Leeuw AM, Den Daas JHG, Kruip TH ve ark. (1995): Comparison of the efficacy of conventional slow freezing rapid cryopreservation methods for bovine embryos. Cryobiology, 32,157-167.
  • Wright JM, Appendix D (2010): Photographic illustrations of embryo developmental stage and quality codes, 141-144. In: DA Stringfellow, MD Givens (Ed), Manual of the international embryo transfer society (International Embryo Transfer Society), IETS publishing, United States of America.
  • Wurth YA, Reinders JMC, Rall WF ve ark. (1994): Developmental potential of in vitro produced bovine embryos following cryopreservation and single-embryo transfer. Theriogenology, 42,1275-1284.
There are 42 citations in total.

Details

Primary Language English
Subjects Veterinary Surgery
Other ID JA68AZ44NG
Journal Section Research Article
Authors

Sakine Ülküm Çizmeci

Mehmet Güler

Mustafa Kaymaz

Publication Date June 1, 2018
Published in Issue Year 2018Volume: 65 Issue: 2

Cite

APA Çizmeci, S. Ü., Güler, M., & Kaymaz, M. (2018). Comparision of different cryoprotectants on slow freezing of in vivo derived Saanen goats embryos. Ankara Üniversitesi Veteriner Fakültesi Dergisi, 65(2), 171-178. https://doi.org/10.1501/Vetfak_0000002843
AMA Çizmeci SÜ, Güler M, Kaymaz M. Comparision of different cryoprotectants on slow freezing of in vivo derived Saanen goats embryos. Ankara Univ Vet Fak Derg. June 2018;65(2):171-178. doi:10.1501/Vetfak_0000002843
Chicago Çizmeci, Sakine Ülküm, Mehmet Güler, and Mustafa Kaymaz. “Comparision of Different Cryoprotectants on Slow Freezing of in Vivo Derived Saanen Goats Embryos”. Ankara Üniversitesi Veteriner Fakültesi Dergisi 65, no. 2 (June 2018): 171-78. https://doi.org/10.1501/Vetfak_0000002843.
EndNote Çizmeci SÜ, Güler M, Kaymaz M (June 1, 2018) Comparision of different cryoprotectants on slow freezing of in vivo derived Saanen goats embryos. Ankara Üniversitesi Veteriner Fakültesi Dergisi 65 2 171–178.
IEEE S. Ü. Çizmeci, M. Güler, and M. Kaymaz, “Comparision of different cryoprotectants on slow freezing of in vivo derived Saanen goats embryos”, Ankara Univ Vet Fak Derg, vol. 65, no. 2, pp. 171–178, 2018, doi: 10.1501/Vetfak_0000002843.
ISNAD Çizmeci, Sakine Ülküm et al. “Comparision of Different Cryoprotectants on Slow Freezing of in Vivo Derived Saanen Goats Embryos”. Ankara Üniversitesi Veteriner Fakültesi Dergisi 65/2 (June 2018), 171-178. https://doi.org/10.1501/Vetfak_0000002843.
JAMA Çizmeci SÜ, Güler M, Kaymaz M. Comparision of different cryoprotectants on slow freezing of in vivo derived Saanen goats embryos. Ankara Univ Vet Fak Derg. 2018;65:171–178.
MLA Çizmeci, Sakine Ülküm et al. “Comparision of Different Cryoprotectants on Slow Freezing of in Vivo Derived Saanen Goats Embryos”. Ankara Üniversitesi Veteriner Fakültesi Dergisi, vol. 65, no. 2, 2018, pp. 171-8, doi:10.1501/Vetfak_0000002843.
Vancouver Çizmeci SÜ, Güler M, Kaymaz M. Comparision of different cryoprotectants on slow freezing of in vivo derived Saanen goats embryos. Ankara Univ Vet Fak Derg. 2018;65(2):171-8.