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The aim of the current study was to evaluate the effects of different concentrations of rainbow trout seminal plasma (RTSP) (0.1%, 1% and 10%) in extenders containing either egg yolk or lecithin for use in Awassi ram semen cryopreservation. Pooled sperm were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in egg yolk or lecithin extender containing no RTSP, 0.1%, 1% or 10% RTSP (v/v). Semen samples were assessed for sperm motility, plasma membrane integrity [hypoosmotic swelling test (HOST) and Hoechst 33258] and defective acrosomes [FITC-conjugated Pisum sativum agglutinin (PSA-FITC)] at the following five time points: after dilution with extender A; after equilibration; and post-thaw at 0 h, 3 h and 5 h. Malondialdehyde (MDA) was examined only after thawing. Freezing and thawing procedures (dilution, equilibration and post-thaw incubation at Oh, 3 h and 5 h) negatively affected the motility (P

The aim of the present study was to evaluate the correlation between the routine semen analyses and sperm DNA integrity, as assesed by TUNEL in fresh semen of rams in Kivircik and Awassi breeds in breeding season. Semen was collected by electro-ejaculation five times every other day. For that purpose, 50 fresh ejaculates from 6 Kivircik and 4 Awassi rams were evaluated. The mean percentages of semen volume, concentration, mass activity, motility, acrosomal defects, HOST and DNA fragmentation of Kivircik and Awassi rams were 1.3 ml, 1.9 x109, 3.3 (0-5 scale), 72.6%, 13.2%, 73.6%, 16.2% and 1.0 ml, 1.9 x109, 3.3 (0-5 scale), 72.5%, 11.1%, 77.9%, 15.9%; respectively (P>0.05). Sperm DNA fragmentation was correlated adversely with semen volume (r=-0.329, P0.05), mass activity (r=-0.349, P0.05) and HOS test (r=-0.513, P0.05), mass aktivite (r = -0.349, P0.05) ve HOS testi (r= -0.513, P

The aim of the current study was to evaluate the effect of egg yolk and different soybean lecithin concentrations on the efficiency of ram semen cryopreservation and to test the fertilizing ability of frozen-thawed ram semen. Ejaculates with a thick consistency, rapid wave motion (3-5), and >70% initial motility were pooled. Pooled semen were then divided into four groups and diluted at 1/5 (semen/extender) with 1%, 3%, 6% lecithin (L1, L3 and L6) or 20% egg yolk (EY20) using the two-step dilution method. As expected, the results of the current study showed that both motility and the rates of defective acrosomes in sperm were negatively affected by the cryopreservation procedure (P0.05). The results of in vitro fertilization, as assessed by the rate of blastocyst formation, were more successful in the EY20 group than those noted in different lecithin groups. In conclusion, freezing ram semen with an extender containing egg yolk could yield better post-thaw sperm parameters and embryonic development compared to lecithin containing extenders. Bu çalışmanın amacı, yumurta sarısı ve farklı lesitin konsantrasyonlarının koç spermasının dondurulabilirliği üzerine etkisini değerlendirmek ve dondurup çözdürülen koç spermasının fertilizasyon yeteneğini tespit etmektir. Kitle hareketi (3-5) ve >%70 motiliteye sahip ejakülatlar birleştirildi (pooling). Pooling yapılan sperma dört gruba bölündü ve %1, %3, %6 (L1, L3 ve L6) lesitin veya %20 yumurta sarısı (EY20) içeren sulandırıcılar ile 1/5 (sperma/sulandırıcı) oranında iki aşamalı sulandırma yöntemi kullanılarak sulandırıldı. Bu çalışmanın sonucunda; motilite ve akrozomal bozukluk oranlarının dondurma prosedüründen olumsuz yönde etkilendiği tespit edildi (P0.05). Blastosist oranları bakımından değerlendirilen in vitro fertilizasyon sonuçlarına göre, EY20 grubunun farklı lesitin gruplarından daha başarılı olduğu tespit edildi. Sonuç olarak, yumurta sarısı içeren sulandırıcı ile dondurulan koç spermasının lesitin gruplarına göre eritme sonrası spermatolojik özellikler ve embriyonik gelişim bakımından daha üstün olduğu tespit edildi.

International audience; The aim of the present study was to investigate the suitability of the water test and hypo-osmotic swelling (HOS) test for analyzing honeybee semen. In particular, the relationships between these tests of the integrity of the sperm plasma membrane and tests of sperm motility and sperm viability were measured using SYBR-14/PI. To this end, semen was obtained from mature drones (16 days or older) collected from four colonies. The means of the per-drone sperm concentration, sperm motility, and live spermatozoa were 1.47 × 106, 76.0%, and 87.2%, respectively. The percentage of spermatozoa with swollen tails was 92.2% as measured with the water test. With the HOS tests at 50, 100, and 150 mOsm/kg, the percentages of spermatozoa with swollen tails were 94.2%, 90.5%, and 85.6%, respectively, after a 30-min incubation and 92.2%, 90.5%, and 88.0%, respectively, after a 60-min incubation. It was observed that subjecting honeybee spermatozoa to a hypo-osmotic solution resulted in clearly identifiable swollen-tail spermatozoa. The percentages of swollen-tail spermatozoa obtained using the water and HOS tests were higher than the percentages of viable and motile spermatozoa. Similar results were obtained using the water test and the HOS test at 50 mOsm/kg. It was concluded that HOS and water tests are suitable and simple measures of the functional integrity of the plasma membranes of bee spermatozoa, and they may be useful additions to standard methods of semen evaluation in mammals.

The influence of different extender osmolality levels and the presence of different cryoprotectants on the post-thawed semen's characteristics and post-thawed plasma membrane integrity of ram spermatozoa were studied. Ram semen was frozen with TRIS-egg yolk based extender according to two-step dilution procedures. The final concentrations of the cryoprotectants: 6% glycerol, 6% 1,2-propanediol, 62.5 mM sucrose, and 62.5 mM trehalose were studied in three different extender B osmolality levels (350, 375, and 400 mOsm). The osmolality affected significantly the post-thawed semen's motility, defected acrosomes (DA), total morphological defect (TMD), along with the sperm's plasma membrane integrity (HOST). Type of cryoprotectant exerted significant effect (P

The aim of the study was to evaluate the effect Of the addition of 20% bull seminal plasma to semen extenders on post-thaw sperm motility, morphology, and fertility. Two Merino rains at 3-5 years of age, 43 ewes, and one Holstein bull were used. Fourteen ejaculates Were collected by the use of an artificial vagina, twice a day, at 60 min interval. Six semen ejaculates were extended with Tris-egg yolk extender containing 20% bull seminal plasma (TEY-BSP group), and 8 ejaculates were extended with Tris-egg yolk (TEY group), at a rate to obtain 150 x 10(6) spermatozoa/straw. The owes in oestrous were divided at random into two groups. Twenty-two ewes were inseminated with TEY-BSP and 21 ewes were inseminated with TEY. The mean percentage of motility, defected acrosome, and total morphological defect for the equilibrated and post-thaw semen parameters of the TEY, were 69.4%, 8.6%, and 17.0% and 42.8%, 49.9%, and 51.2%, respectively. The mean percentage of non-return rate (NRR) (n=43) was 30.2% and for the semen frozen with TEY-BSP (n=22) and TEY (n=21) - 31.8%, and 28.6%, respectively. There were no significant differences between TEY-BSP and TEY for NRR. In Conclusion, the addition of the bull seminal plasma to freezing extender has a Supportive effect on extended, equilibrated, and thawed semen. Moreover, the addition of bull seminal plasma to ram semen extender improved fertility.

The objective of the present study was to evaluate some semen characteristics in Saanen bucks during the transition period. A total of 60 ejaculates were obtained from 6 bucks and was subjected to routine semen analysis. In general, all studied semen parameters were similar to the other breeds. Abnormal sperm cells positively correlated with dead spermatozoa and wave activity. Similarly, dead sperm cell correlated with volume and total spermatozoa. Also, wave activity correlated with motility. In contrast, pH negatively correlated with motility and wave activity.

The objective of this study was to investigate the effect of low temperature thawing and post-thaw cold shock application on sperm motility, as well as acrosomal and plasma membrane integrities of cryopreserved bull semen. Frozen semen was thawed in a water bath at 5-7 degrees C for 30, 60 or 90 s (low thawed groups), cold shocked (at 5-7 degrees C for 30, 60, 90 s) after thawing at 37 degrees C for 30 s (cold shocked groups), cold for 30 s (control group). The thawing procedure affected the percentages of motile spermatozoa (P < 0.001)and damaged acrosome (P < 0.001) while the bull factor affected the percentages of motile spermatozoa (P < 0.01) and damaged acrosome (P < 00.01). However, swollen tail spermatozoa rates were not affected by the the two factors. There was a significant interaction between thawing procedures and bulls in terms of the studies sperm parameters (P < 0.001). In conclusion, the percentages of motility, intact acrosome and swollen tail spermatozoa of the control group were higher than low-temperature thawed and post-thaw cold-shocked groups. These results indicate that frozen spermatozoa are significantly sensitive to lower temperature not only before but also after thawing.

The aim of the present study was to assess the relationships between sperm membrane integrity, evaluated by the hypo-osmotic swelling (HOS) and water tests, and sperm quality parameters (percentage of dead, motile and abnormal spermatozoa) in goat bucks. For that purpose, a total of 60 fresh ejaculates from 6 Saanen bucks were evaluated. The mean percentage of dead and motile spermatozoa were 12.7 +/- 5.7% and 62.5 +/- 8.6%, respectively. The mean percentages of abnormal spermatozoa with defected acrosome or total morphological defect were 7.2 +/- 2.9% and 9.7 +/- 3.4%, respectively. The mean percentage of swollen tail spermatozoa obtained with the HOS and water tests were 53.7 +/- 11.3% and 49.8 +/- 9.7%, respectively. The mean percentages of swollen tail spermatozoa obtained with the water test were significantly lower than that evaluated from the HOS test (P < 0.01). The percentage of swollen tail spermatozoa evaluated with the HOS test was correlated with that obtained with the water test (r=0.657, P < 0.001). The percentage of swollen tail spermatozoa evaluated by the water and HOS tests were slightly correlated with the percentage of motile spermatozoa (r=0.554 and r=0.523, P < 0.001 and P < 0.001). In conclusion, a hi-h correlation between the HOS test and water test was observed in this study. The percentage of swollen tail spermatozoa obtained by the water test was significantly lower than that obtained by the HOS test.

Fertility of artificial insemination is relatively low when frozen semen is used in sheep (Ozturkler et al., 1999; Sanchez-Partida et al., 1992). Jones and Mann (1977) have suggested a relation between the lipid peroxidation formation in spermatozoa and quick decrease in motility and phospholipid loss in the membranes. Some researches agreed that dialyzed egg yolk, bull seminal plasma, milk and sodium sulphate could prevent spermatozoa from the post-thaw side effects of lipid peroxidation (Salamon and Maxwell, 1995; Aswort et al., 1994; Barrios et al., 2000). Jones and Mann (loc. cit.) claimed that egg yolk could decrease the peroxidation rate of ram spermatozoa when added to extenders but not as much as the bull seminal plasma. Researchers have found that bull spermatozoa can easily peroxidase due to the high level of unsaturated fatty acids bound to the phospholipids in the seminal plasma. The present study is planned to investigate the effects of bull seminal plasma in semen extenders during freezing of ram semen and its post-thaw motility and morphological characteristics.

The objective of this study was to evaluate effect of different thawing procedure to Hypo-Osmotic Swelling Test (HOST), motility and morphology of bull semen. Straw (0.25 ml) frozen semen from 4 Holstein bulls was used in this study. Post-thaw motility, morphology and hypo-osmotic swelling test (HOST) were carried out at 37degreesC for 30 sec, 50degreesC for 15 sec and 70degreesC for 5 sec. According to these protocols motility, defected acrosome, other morphological defects, total morphological defects, and swollen tail spermatozoa (HOST) values were 56.6 %, 56.7 %, 59.8 %; 3.7 %, 2.6 1.9 %; 10.7 %, 8.8 %, 5.6 %; 14.4 %, 11.5 %, 7.5 %; 37.1 %, 37.4 39.3 % respectively. Thawing protocol has affected all sperm characteristics (P < 0.05) except HOST. There was a high correlation between HOST values and motility (P < 0.01) but no correlation was observed between HOST and acrosome defects, other morphological defects and total morphological defects.

In the present study, ten ejaculates were obtained from each of seven German shepherd dogs. Only the sperm-rich fractions of ejaculates from each dog once a week with a mean interval of 60 min, were used during five weeks. Semen quality was examined in total of 70 semen samples. After this examination, semen samples were centrifuged (5000 g x 10 minutes) and seminal plasma were separated. Plasma total protein, calcium, phosphorus, magnesium, sodium and potassium and alkaline phosphatase were determined. The semen quality and biochemical findings were compared between the first and the second ejaculate following very-short interval ejaculation. There were no significant differences in the percentage of motility, live spermatozoa, acrosomal, tail and total morphological defects of the second ejaculate compared with that of the first ejaculate. However, there were significant lower values for the volume and the spermatozoal concentration in the second ejaculate than that of the first ejaculate (p < 0.05). There were significantly lower values for the total protein, calcium, magnesium and alkaline phoshatase of the second ejaculate compared with that of the first ejaculate. Positive correlations were found between spermatozoal concentration and AP; motility and sodium concentration. The semen quality in the first and the second ejaculate in all dogs was normal for fertility. In conclusion, when the first ejaculate is inadequate, collection of second ejaculate may be useful for cryopreservation or insemination with fresh semen in dogs.

ÖZET Bu çalışmanın amacı dondurmada kullanılan farklı sulandırıcıların spermatolojik özellikler üzerindeki etkilerini karşılaştırarak köpek sperması için en iyi sulandırıcının belirlenmesidir. Araştırmada materyal olarak 10 adet Kurt Köpeği (Alman Çoban Köpeği) kullanıldı. Sperma, köpeklerden penis masajı yöntemi ile İM günde bir ve kızgın bir dişi köpeğin varlığında, alışık oldukları ortamda alındı. Ejakulasyon sırasında renk ve kıvam değişimleri gözlenerek spermatozoadan zengin olan ikinci fraksiyonun alınması sağlandı. Her köpekten 5 'er olmak üzere toplam 50 ejakulat alındı. Alman taze spermaların hacim, renk, pH, motilite, ölü spermatozoa ve morfolojik bozukluk oranlarından oluşan spermatolojik özellikleri incelendi. Köpeklerden alman her bir sperma örneği iki eşit hacme ayrıldı ve bu sperma örnekleri Tris-yumurta şansı (T-YS) ile Sodyum-sitrat yumurta sarısı (SS-YS) sulandırıcıları ile 1:1 oranında sulandırıldı. Sulandırılmış spermalar içinde 37°C su bulunan cam bir behere konularak +5°C'deki buzdolabına yerleştirildi. Bu sırada gliserol içeren T-YS ve SS-YS sulandırıcıları da aynı behere konuldu. Spermanın ısısı 2 saatte +5°C'ye düşürüldü. Isısı +5°C'de olan T-YS ve SS-YS ile sulandırılmış sperma örnekleri kendi hacimleri kadar %8 gliserol içeren T-YS ve SS-YSsulandırıcıları ile finalde %4 gliserol içerecek şekilde sulandmldılar.Gliserolizasyon o işlemi 50 dak.'da tamamlandı. Daha sonra spermalar +5 C'de 2 saat ekılibrasyona bırakıldılar. Ekilibrasyonumı tamamlamış sulandırılmış sperma örnekleri payet o yöntemine göre donduruldu. 0.5 ml.lik payetlerde dondurulan spermalar 37 C'lik su banyosunda 30 sn'de çözüldüler. Çalışmada sulandırma, +5 C'ye soğutma, gliserolizasyon, ekilibrasyon soması yapılan spermatolojik muayenelerde en yüksek motilite Tris sulandıncısmda saptandı (P< 0.05). Sulandırma ve +5°C'ye soğutma aşamalarmda akrozomal ve toplam morfolojik bozukluklar incelendiğinde Tris ve Sodyum sirrat sulandırıcıları arasında istatistiksel açıdan fark saptanmamış, gliserolizasyon, ekilibrasyon ve çözüm sonrası aşamalarda ise fark saptanmıştır (PO.05). Araştırmanın sonucunda çözüm sonrasında Tris sulandıncısmda %53±1.59, Sodyum sitrat sulandırıcısında ise %48±1.46 motilite değeri saptandı ve aralarındaki fark istatistiksel açıdan önemli bulundu (P< 0.05). Anahtar Kelimeler: Köpek, sperma, sulandırma, soğutma, dondurma. SUMMARY Spermatological Characteristics of Dog Semen Diluted with Different Extenders After Freezing. The aim of this study was to determine the best extender for dog sperm by comparing the effects of different extenders used for freezing on spermatolojical characters. In the present study, 10 German Shepherd Dogs were used as materials. The semen was collected by manual massage every other day in the place where the male dogs were accustomed to be in the presence of a bitch in heat. Second sperm- rich fraction of the semen was collected by observing changes of the colour and the density of ejaculate during ejaculation. Five ejaculates per dog were collected and the total ejaculate number used for the study was 50. The semen was examined for volume, colour, pH, motility, dead spermatozoa and morphologically defected ones. Each sperm collected from each dog was divided into two equal parts and one of the parts was diluted with Tris-egg yolk (T-EY) and the other part was diluted with Sodium citrate-egg yolk (SC-EY) extenders by 1:1 ratio. The diluted semen samples were put into a glass cup including 37°C water and then they were fit in refrigerator at 5°C. Meanwhile, T-EY and SC-EY with 8% glycerol were put into the same cup.The semen samples diluted with T-EY and SC-EY at 5°C were mixed with the same amount of T-EY and SC-EY including 8% glycerol. Thus final glycerol concentration became 4%. Glycerolisation was completed within 50 minutes. After this maniuplation, the semen was incubated for equlibration at 5°C for 2 hours. Following equilibration the diluted semen samples were frozen according to the straw method. The freezing semen in 0.5 ml straws was thawed in water bath at 37°C for 30 seconds. In the study, the highest motility was determined with Tris extender (P