Farklı Ekilibrasyon Ortamlarının Çözüm Sonu Boğa Sperması Kalitesi Üzerine Etkisi
Ekilibrasyon, spermanın sıcaklığının düşürüldüğü ve faz değişimlerinin meydana geldiği aşama olarak, sperma dondurulmasında en önemli basamaklardan biridir. Ekilibrasyonun gerçekleştirildiği ortam çoğu zaman göz ardı edilmekte, ekilibrasyon koşullarının başarısı ampirik tecrübelere dayanmaktadır. Bu çalışmanın amacı farklı ekilibrasyon ortamlarının toplam motilite (TMOT), progresif motilite (PMOT), spermatozoa hareket parametleri (VCL, VSL, VAP, LIN, STR, WOB, ALH, BCF), plazma membran ve akrozom bütünlüğü (PMAI) ve DNA fragmentasyon indeksi (DFI) üzerine etkisinin araştırılmasıdır. Çalışmada 4 baş boğadan iki kez sperma alınarak üç eşit hacme bölündü. Andromed® ile sulandırılan spermalar 24 saat 4°C’de payet içinde (Deneme 1), kapta (Deneme 2) ve çalkalayıcıda (Deneme 3) ekilibrasyona bırakıldı. Çözüm sonu sperma kalitesi TMOT, PMOT, VCL, VSL, VAP, LIN, STR, WOB, ALH, BCF, PMAI ve DFI parametreleri değerlendirilerek belirlendi. Farklı ekilibrasyon koşulları arasında, hareket özellikleri olan TMOT, PMOT, VSL, VAP, BCF ve morfolojik özellikler olan PMAI ve DFI açısından istatistiki bir farklılık bulunamadı (p>0,05). En yüksek VCL değeri Deneme 3’te, en yüksek LIN, STR, WOB ve ALH değerleri Deneme 2’de gözlendi (p<0,05). Bu sonuçlar farklı kolullarda yapılan ekilibrasyonun çözüm sonu spermatolojik değerler üzerinde herhangi bir zararlı etkisi olmadığını göstermektedir. Ayrıca boğa spermasının çalkalayıcıda ekilibre edilmesinin kap ve payet içinde ekilibre edilmesine göre spermatozoa hareket özellikleri açısından daha yararlı olduğu söylenebilir.
Effects of Different Equilibration Conditions on Cryopreserved Bovine Sperm Quality
Equilibration is the one of the important steps of semen cryopreservation and it is the stage in which semen is cooled and phase changes occur. The condition of equilibration is mostly regarded; and success of the equilibration conditions depend on empirical experiences. The aim of this study was to investigate the effects of three different equilibration methods on post-thaw total motility (TMOT), progressive motility (PMOT), kinetic parameters of spermatozoa (VCL, VSL, VAP, LIN, STR, WOB, ALH, BCF), plasma membrane and acrosome integrity (PMAI) and DNA fragmentation index (DFI). In each of 4 bulls; two ejaculates were split into three aliquots, diluted by Andromed® and equilibrated for 24h at 4°C in Drawed Straw (Experiment 1), Cups (Experiment 2) and Shaker (Experiment 3). After thawing, sperm quality was determined by examination of TMOT, PMOT, VCL, VSL, VAP, LIN, STR, WOB, ALH, BCF, PMAI and DFI. There were no differences (p>0.05) depending on equilibration conditions for TMOT, PMOT, VSL, VAP, BCF as kinetic parameters and for PMAI and DFI as morphological parameters. The highest VCL value was observed in experiment 3, when the highest LIN, STR, WOB and ALH values were observed in experiment 2 (p<0.05). The results show that changes in equilibrating condition have no detrimental effect on post-thaw bull semen quality. In addition, it can be said that equilibrating bull semen in a shaker presents some kinetic benefits in contrast with cup or straw equilibration methods.
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- Ahmad M, Ahmad N, Riaz A, Anzar M. Sperm
survival kinetics in different types of bull
semen: progressive motility, plasma
membrane integrity, acrosomal status and
reactive oxygen species generation. Reprod
Fert Develop. 2015; 27: 784-793.
- Aires VA, Hinsch KD, Mueller-Schloesser F,
Bogner K, Mueller-Schloesser S, Hinsch
E. In vitro and in vivo comparison of egg
yolk-based and soybean lecithin-based
extenders for cryopreservation of bovine
semen. Theriogenology. 2003; 60(2): 269-279.
- Almadaly E, Farrag F, Shukry M, Murase T.
Plasma membrane integrity and morphology
of frozen-thawed bull spermatozoa
supplemented with desalted and lyophilized
seminal plasma. Global Veterinaria. 2014; 13:
753-766.
- Amirat L, Tainturier D, Jeanneau L, Thorin C,
Gérard O, Courtens JL, Anton M. Bull
semen in vitro fertility after cryopreservation
using egg yolk LDL: A comparison with
Optidyl®, a commercial egg yolk extender.
Theriogenology. 2004; 61: 895-907.
- Anzar M, Kroetsch T, Boswall L.
Cryopreservation of bull semen shipped
overnight and its effect on post-thaw sperm
motility, plasma membrane integrity,
mitochondrial membrane potential and
normal acrosomes. Animal Reproduction
Science. 2011; 126: 23-31.
- Bailey JL, Bilodeau JF, Cormier N. Semen
cryopreservation in domestic animals: A
damaging and capacitating phenomenon.
Journal of Andrology. 2000; 21(1): 1-7.
- Bochenek M, Smorag Z, Pilch J. Sperm
chromatin structure assay of bulls qualified for
artificial insemination. Theriogenology. 2001;
56: 557-567.
- D’Occhio MJ, Hengstberger KJ, Johnston SD.
Biology of sperm chromatin structure and
relationship to male fertility and embryonic
survival. Anim Reprod Sci. 2007; 101: 1-17.
- Evenson D, Jost L. Sperm chromatin structure
assay for fertility assessment, In: Current
protocols in cytometry, Ed; Robinson JP,
John Wiley & Sons, Inc., New York. 2001; pp.
1-7.
- Fetterolf PM, Rogers BJ. Prediction of human
sperm penetrating ability using computerized
motion parameters. Mol Reprod Dev, 1990;
27: 326-331.
- Hammerstedt RH, Graham JK, Nolan JP.
Cryopreservation of mammalian sperm: What
we ask them to survive. Journal of Andrology.
1990; 11(1): 73-88.
- Hering DM, Lecewicz M, Kordan W, Majewska
A, Kaminski S. Missense mutation in
glutathione-S-transferase M1 gene is
associated with sperm motility and ATP
content in frozen-thawed semen of Holstein-
Friesian bulls. Animal Reproduction Science.
2015; 159: 94-97.
- Karabinus DS, Evenson DP, Jost LK, Baer RK.
Comparison of semen quality in young and
mature Holstein bulls measured by light
microscopy and flow cytometry. J Dairy Sci.
1990; 73: 2364-2371.
- Karoui S, Díaz C, González-Marín C, Amenabar
ME, Serrano M, Ugarte E, Gosálvez J, Roy
R, López-Fernández C, M. J. Carabaño
MJ. Is sperm DNA fragmentation a good
marker for field AI bull fertility? J Anim Sci.
2012; 90: 2437-2449.
- Kumar S, Millar JD, Watson PF. The effect of
cooling rate on the survival of cryopreserved
bull, ram, and boar spermatozoa: a
comparison of two controlled-rate cooling
machines. Cryobiology. 2003; 46(3): 246-253.
- Layek SS, Mohanty TK, Kumaresan A, Parks JE.
Cryopreservation of bull semen: Evolution
from egg yolk based to soybean based
extenders. Animal Reproduction Science.
2016; 172: 1-9.
- Leite TG, Filho VRV, Arruda RP, Andrade AFC,
Emerick LL, Zaffalon FG, Martins JAM.
Effects of extender and equilibration time on
post-thaw motility and membrane integrity of
cryopreserved Gyr bull semen evaluated by
CASA and flow cytometry. Animal
Reproduction Science. 2010; 120(1-4): 31-38.
- Liu DY, Clarke GN, Baker HG. Relationship
between sperm motility assessed with the
Hamilton‐Thorn motility analyzer and
fertilization rates in vitro. J Androl. 1991; 12:
231-239.
- Moallem U, Neta N, Zeron Y, Zachut M, Roth
Z. Dietary a-linolenic acid from flaxseed oil or
eicosapentaenoic and docosahexaenoic acids
from fish oil differentially alter fatty acid
composition and characteristics of fresh and
frozen-thawed bull semen. Theriogenology.
2015; 30: 1-11.
- Murphy EM, Murphy C, O'Meara C, Dunne G,
Eivers B, Lonergan P, Fair S. A
comparison of semen diluents on the in vitro
and in vivo fertility of liquid bull semen.
Journal of Dairy Science. 2017; 100(2): 1541-
1551.
- Nagy S, Jansen J, Topper EK, Gadella BM. A
triple-stain flow cytometric method to assess
plasma-and acrosome-membrane integrity of
cryopreserved bovine sperm immediately after
thawing in presence of egg-yolk particles. Biol
Reprod. 2003; 68: 1828-1835.
- Stadnik L, Rajmon R, Beran J, Simonik O,
Dolezalova M, Sichtar J, Stupka R,
Folkova P. Influence of selected factors on
bovine spermatozoa cold shock resistance.
Acta Vet Brno. 2015; 84: 125-131.
- Sukcharoen N, Keith J, Irvine DS, Aitken RJ.
Prediction of the in-vitro fertilization (IVF)
potential of human spermatozoa using sperm
function tests: the effect of the delay between
testing and IVF. Hum Reprod. 1996; 11:
1030-1034.
- Sukcharoen N, Sithipravej T, Promviengchai S,
Chinpilas V, Boonkasemsanti W. Sperm
morphology evaluated by computer (IVOS)
cannot predict the fertilization rate in vitro
after intracytoplasmic sperm injection. Fertil
Steril. 1998; 69: 564-568.
- Tartaglionea CM, Ritta MN. Prognostic value of
spermatological parameters as predictors of in
vitro fertility of frozen-thawed bull semen.
2004; 62(7): 1245-1252.
- Thun R, Hurtado M, Janett F. Comparison of
Biociphos-Plus® and TRIS-egg yolk extender
for cryopreservation of bull semen.
Theriogenology. 2002; 57(3): 1087-1094.
- Watson PF. The causes of reduced fertility with
cryopreserved semen. Animal Reproduction
Science. 2000; 60-61: 481-492.