Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa

Pontbriand, D.; Howard, Jeremy; Schiewe, M.C.; Stuart, L.D.; Wildt, David E.

doi:10.1016/0011-2240(89)90058-8

Cited by 60 publications

(31 citation statements)

References 44 publications

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“…This result agrees with our preliminary findings in Tris-citrate-based diluents (Landers et al, 1992). Pellet freezing is generally superior to straw freezing, largely because of its simplicity and reliability (Evans, 1988), although some workers report the opposite (Pontbriand et al, 1989). However, it is likely that the poor results obtained with minitube and straw freezing in this study were partly due to variability in freezing rates in nitrogen vapour (Evans, 1988).…”

Section: Discussionmentioning

confidence: 81%

In vitro evaluation of zwitterion buffers in diluents for freezing ram spermatozoa

Molinia

Evans²,

Maxwell³

1994

Reprod. Nutr. Dev.

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Summary ― Diluents based on the zwitterion buffers Tes, Hepes and Pipes titrated to pH 7.0 with NaOH or Tris were compared with Tris-citrate diluents by assessment of post-thawing motility and acrosome integrity of frozen ram spermatozoa. Varying buffer osmolalities between 300 and 360 mosmol had no effect but there was a decrease (P < 0.001 ) in motility between 360 and 420 mosmol. There was a quadratic effect (P < 0.001 ) on motility of increasing glucose concentration in the diluent (30, 85, 140, 195 and 250 mM) with a maximum at 85 mM. At this sugar concentration, motility was higher (P < 0.001 ) for diluents containing glucose and fructose than lactose, sucrose or trehalose. Inclusion of egg yolk (optimum at 13.5% v/v; P < 0.05), centrifugation of the diluents (P < 0.01 use of low dilution rates (3 or 6-fold; P < 0.05) and freezing in pellets rather than minitubes or straws (P < 0.001 ) improved both the motility and acrosome integrity of spermatozoa in the zwitterion diluents. It is concluded that zwitterion buffers are superior to Tris-citrate in ram semen freezing diluents. zwitterion buffer / motility / acrosome integrity / frozen ram spermatozoa Résumé ― Effet de l'addition de tampons amphotères à des dilueurs sur la congélabilité des spermatozoïdes de bélier. Nous avons comparé l'efficacité de dilueurs à base de tampons amphotères TES, HEPES et PIPES ajustés à pH 7,0 avec NaoH ou tris, à celle de milieux à base de tris-citrate, en évaluant la motilité des spermatozoïdes de bélier et l'intégrité de leur acrosome après congélation. Entre 300 et 360 milliosmoles, l'osmolarité des tampons n'a aucun effet. Au-delà, et jusqu'à 420 milliosmoles, on observe une baisse significative (P < 0, 001) de la motilité. On obtient un effet biphasique sur la motilité en augmentant la concentration de glucose dans le dilueur (30, 85, 140, 195, 250 mM) avec un maximum pour 85 mM. À cette concentration, la motilité est plus élevée avec le milieu contenant du glucose et du fructose qu'avec ceux contenant du lactose, du sucrose ou du tréhalose (P < 0,001). L'addition de jaune d'œuf (optimum à 13,5% vlv; P < 0,05), la centrifugation des dilueurs (P < 0,01), l'utilisation d'un taux de dilution peu élevé (3 ou 6 fois ; P < 0,05) et la congélation en pastilles plutôt qu'en minitubes ou en minipaillettes (P < 0,001) améliorent la motilité et l'intégrité de l'acrosome des spermatozoïdes congelés dans des milieux contenant un tampon amphotère. Cette étude montre donc que les tampons amphotères sont plus efficaces que le tampon tris-citrate pour les dilueurs utilisés pour la congélation du sperme de bélier.bélier / mobilité l acrosome / sperme congelé / dilueur / tampon * Current address:

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Section: Discussionmentioning

confidence: 81%

In vitro evaluation of zwitterion buffers in diluents for freezing ram spermatozoa

Molinia

Evans²,

Maxwell³

1994

Reprod. Nutr. Dev.

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“…Üstelik sulandırıcılara katılan glyserol, DMSO ve yumurta sarısı gibi kryoprotektanlar bile, koç spermasının dondurulmasında alışım ve çözdürme sonrası spermatozoa motilitesi ve plazma membranlarındaki hasarı dolayısıyla fertilitenin düşmesini önleyememekte-dir (18,16).…”

Section: Tartışma Ve Sonuçunclassified

Farklı taurin dozlarının ve dondurma hızının koç spermasının dondurulması üzerine etkileri

Tekin

2006

Ankara Üniversitesi Veteriner Fakültesi Dergisi

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Özet: Çalışmada koç ejakülatlarının dondurulması ve çözdürülmesi sürecinde sperma sulandırıcısına katılan farklı dozlarda antioksidan (taurin) ile değişik hızlarda soğutmanın başlıca spermatolojik değerler üzerine etkilerinin araştırılması amaçlanmıştır. Araştırmada toplam 3 koçtan (Akkaraman, Sakız, Ramlıç) sun'i vagina ile alınan ejekülatlar kullanılmıştır. Çift ejekülat olarak alınan ve başlıca spermatolojik özellikleri belirlenen normospermi kalitesindeki ejekülatlar birleştirilerek kullanılmıştır. Araştırma ve kontrol gruplarında spermalar split ejekülat biçiminde değerlendirilmiştir. Effects of different taurine doses and freezing rate on freezing of ram semenSummary: In this study, it was aimed to investigate of effects of different doses antioxidant (taurine) added to extender and various cooling rates on some spermatological parameters during freezing and thawing of ram ejaculates.Ejaculates collected by artificial vagina from 3 rams (Akkaraman, Sakız, Ramlıç) were used in the study. Ejaculates collected double ejeculation, determined principle spermatological properties and having/with normospermie quality were pooled. Samples were evaluated as split ejeculate in the experiment and control groups. Samples were extended with Tris extender containing 4 % glycerol and different taurine doses (20, 50 ve 80 mM). It was also formed control group that is not contain taurine. After dilution and dose processing, samples withdrawed to 0.25ml straws were frozen in programmable and automatic freezer at different cooling rates (I-15 o C/min; II-10 o C/min; III-5 o C/min).Frozen semen were thawed in a water bath at 40 ºC for 10 seconds. According to post-thawing assessing, in the semen which was diluted with tris diluent containing 20, 50, 80 mM taurine and without taurine (control) groups, the sperm motility values were recorded as 38.7, 31.1, 26.5 and 34.4 % respectively. The percentages of abnormal spermatozoa were found 18.7, 18.4, 17.0, and 19.0 %, the percentage of dead spermatozoa were determined 52.2, 60.3, 61.5 and 54.7 % respectively. In I, II and III groups of cooling rates, post-thawing sperm motility values were determined 30.0, 33.1, 32.0 %, the percentages of abnormal spermatozoa were found 17.7, 17.7, 23.0 % and the percentages of dead spermatozoa were recorded as 62.0, 54.0, 56.8 % respectively. According to the obtained results, it was determined that different taurine doses and cooling rates did not showed significant differences as statistically in the cryopreservation of ram semen.

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“…Cooling and freezing rates (7,15,18,22,25), type (5,16,25) and concentration (7,25,27) of cryoprotective agents (5,16,25), dilution rates (15,18,20), temperature at which glycerol is added to the semen, equilibration period, and thawing rate interact with the success of post-thaw recovery of spermatozoa (1,2,7,19) and fertilizing capability (19,26). Some authors explain these phenomena with reducing transport ability and survival of ram spermatozoa in the female reproductive tract and loss of viability, morphological and DNA integrity during freezing and thawing procedures (7,16,26).…”

Section: Introductionmentioning

confidence: 99%

“…Several researchers have developed different extenders and protocols for freezing ram semen, but the fertility results are not comparable to those obtained with fresh semen and natural mating (7,18,22,25). Thermal shock, formation of ice crystals, dehydration, increased salt concentration and osmotic shock that occur during freeze-thawing have detrimental effects on spermatozoa (22).…”

Section: Introductionmentioning

confidence: 99%

Effect of freezing rate on acrosome and chromatin integrity in ram semen

;ZIK¹

2011

Ankara Üniversitesi Veteriner Fakültesi Dergisi

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Summary:The objective of the present study was to investigate the effect of different freezing rates on post-thaw sperm motility, acrosome defect, and sperm chromatin structure and apoptotic activity in ram semen. Collected semen was diluted at 1:5 (semen/extender) with Bioxel® (IMV technologies France) at 30ºC and then cooled to 5ºC within 1h. Cooled semen was subjected to the equilibration for 2 hours. Equilibrated semen was frozen in 0.25 ml straw at two different cooling rates (slow: 0.5°C/min from 5 to -20°C and fast: 5°C/min from 5 to -20°C). Both groups were frozen from -20 to -120°C at 25°C/min and stored in liquid nitrogen until use. Post-thaw (37°C/30 min) sperm motility, defected acrosome (Pisum sativum agglutinin fluorescein conjugate, FITC PSA), sperm chromatin structure determined by Acridin Orange (AO) and apoptotic activity using terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL) were evaluated. Post-thaw sperm motility, acrosome defect, AO and TUNEL for slow frozen semen were 42.8±8.8%, 31.6±12.9%, 2.9±2.4% and 2.8±1.6%, and for fast frozen semen were 36.5±9.9%, 24.7±11.1%, 3.3±2.2% and 6.3±3.4%, respectively. Post-thaw semen analyses showed that there was no significant difference between two freezing curves in terms of acrosome defect, sperm chromatin damage (AO). However, a significant difference was found for postthaw semen motility between two groups (P<0.05). In conclusion, while the slow freezing procedure improved post-thaw sperm motility, acrosome and chromatin integrities and apoptotic index in ram spermatozoa did not show any significant difference between freezing rates.Key words: Apoptosis, freezing rate, ram semen. Koç spermasında dondurma hızının akrozom ve kromatin bütünlüğü üzerine etkisiÖzet: Bu çalışma, farklı dondurma hızlarının, eritme sonrası motilite, akrozom bütünlüğü, kromatin yapısı ve apoptozis üzerine olan etkilerini araştırmak amacıyla gerçekleştirildi. Toplanan sperma örnekleri 30ºC'de 1/5 oranında (sperma/sulandırıcı) Bioxel® (IMV, Fransa) ile sulandırıldı ve 1 saatte +5ºC'ye düşürülerek 2 saat süre ile ekilibrasyona bırakıldı. Ekilibre edilen sperma 0.25 ml'lik payetlere çekilerek iki farklı soğutma hızında (yavaş: +5°C'tan -20°C'a 0.5°C/dak ve hızlı: +5°C'tan -20°C'a 5°C/dak) donduruldu. Her iki grup -20°C'tan -120°C'a 25°C/dak hızla donduruldu. Eritme sonrası (37°C/30 dak) aşamada; akrozom bozukluğu Pisum sativum agglutinin fluorescein conjugate ( FITC PSA) ile kromatin yapısı Acridin Orange (AO) ile ve apoptozis ise terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling tekniği (TUNEL) kullanarak belirlendi. Motilite muayeneleri ısıtma tablalı faz kontrast mikroskop ile gerçekleştirildi. Eritme sonrası motilite, akrozom bozukluğu, AO ve TUNEL değerleri sırasıyla yavaş dondurma için %42.8±8.8, %31.6±12.9, %2.9±2.4 ve 2.8±1.6 hızlı dondurma için ise %36.5±9.9, %24.7±11.1, %3.3±2.2 ve %6.3±3.4 olarak saptandı. Spermanın eritme sonrası bulgularının değerlendirilmesinde akrozom bozukluğu ve sperma kromatin yapısı (AO) yönünde...

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Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa

Cited by 60 publications

References 44 publications

In vitro evaluation of zwitterion buffers in diluents for freezing ram spermatozoa

In vitro evaluation of zwitterion buffers in diluents for freezing ram spermatozoa

Farklı taurin dozlarının ve dondurma hızının koç spermasının dondurulması üzerine etkileri

Effect of freezing rate on acrosome and chromatin integrity in ram semen

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