Prediction of male fertility with in vitro semen quality parameters remains a challenge for the bull industry. Fluorescein staining and computer-assisted semen analysis (CASA) ensure kinetically proper and functionally objective results to improve spermatological parameter control. Therefore, we aimed to examine the CASA kinetic parameters and fluorescein staining of cryopreserved bull semen and its relationship with fertility. A total of 90 frozen straws from 5 Simmental bulls were evaluated. After semen was thawed, the motility, kinetic parameters, and insemination doses were evaluated by CASA; fluorescein stainings were evaluated using a fluorescein microscope. The 60th-day nonreturn rates (NRRs) were recorded for determination of pregnancy rates. There were significant differences in motility and kinetic parameters among bulls. Bull 3 had the lowest percentage of NRR and curvilinear velocity, straight line velocity, beat-cross frequency, and lateral head displacement parameters (P < 0.05). A positive correlation was detected between the pregnancy rate and some kinetic parameters. The correlation between the CASA parameters was determined as well. We concluded that various kinetic parameters obtained with CASA software system algorithms and fluorescein stainings can be linked to fertility. However, further research is needed to acquire a more precise link with fertility.

Use of computer-assisted sperm analyzers (CASAs) to evaluate sperm fertilizing ability has great importance for the artificial insemination industry. For motility evaluation, different CASA systems and diluents are being used. These assays are poor in predicting sperm fertility, because only the samples with markedly poor quality can be detected. However, there is not a unitary extender worldwide. Therefore, we aimed to evaluate the postthaw kinetic parameters of curvilinear velocity (VCL), straight-line velocity (VSL), and averagepath velocity (VAP) with two different extenders (skim milk, SM; Tris, TS). Semen was collected twice a week with an artificial vagina from four adult Norduz bucks. The ejaculates having normospermic quality were split into two equal aliquots and frozen. Sperm motility (%), VCL, VSL, VAP, viability (%), and abnormal spermatozoa (%) were evaluated. Regarding the abnormal spermatozoa rates and viability, percentages of motility were significantly higher in SM extender (P < 0.05), and VCL, VSL, and VAP rates were found higher with TS. In conclusion, according to the in vitro findings of this study, motility rates are more favorable in SM extenders than TS, and spermatozoa swim significantly faster in TS extender compared to SM.

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Citation of This AbstractIn vitro toxic effects of selected pesticides (cypermethrin, flumethrin, propoxur, carbaryl, chlorpyrifos, metamidophos) were evaluated on dog and buck fresh spermatozoa using MTT (mitochondrial activity/viability), NeutralRed-NR (lysosomal activity/viability) and CASA (motility parameters/VCL, VSL, VAP, linearity, straightness, wobble) tests. For buck, the most toxic compounds were carbamates followed by organophosphate and pyrethroids. Carbamates, again induced the highest toxicity in dog sperm, followed by pyrethroids. In general, all pesticide treatments were found to increase the hyperactivity of buck spermatozoa except flumethrin; whereas a decrease was present for dog spermatozoa (except carbaryl). A decrease of VCL, VSL and VAP parameters was evident in buck (P<0.05), whereas no difference was found in dog sperm (P>0.05). Overall, flumethrin was found to induce less effect on motility parameters compared to cypermethrin. As a conclusion, the combination of MTT and CASA along with NR would provide more accurate data for the in vitro evaluation of chemicals on spermatozoa; where alternative testing strategies for the ReproTox tests (especially for cosmetic product safety assessment where animal testing is banned) are recommended nowadays with battery strategies and species specific differences would play a key role for understanding the defense mechanisms. Keywords: Pesticides, Buck spermatozoa, Dog spermatozoa, In vitro toxicity, MTT, CASA, Neutral red Bazı Pestisitlerin Teke ve Köpek Sperması Üzerine İn Vitro Toksisiteleri ÖzetBu çalışmada, bazı pestisitlerin (sipermetrin, flumetrin, propoksur, karbaril, klorprifos, metamidofos) köpek ve teke taze spermaları üzerindeki etkileri, MTT (mitokondriyal etkinlik/canlılık), Nötral Kırmızı-NK (lizozomal etkinlik/canlılık) ve Bilgisayar Destekli Semen Analizi-CASA (motilite parametreleri, VCL, VSL, VAP, lineerlik, doğrusallık, yalpalama) kullanılarak in vitro olarak araştırıldı. Teke sperması üzerine canlılık ve motilite bakımından en toksik bileşiğin karbamatlar, ardından organofosfatlar ve piretroitler olduğu; köpek spermasında ise aynı şekilde karbamatların, ardından da piretroitlerin toksik etki gösterdiği tespit edildi. Genel olarak tüm pestisit uygulamalarının teke spermasında hiperaktiviteyi (flumetrin dışında) arttırdığı, köpek spermasında ise azalttığı tespit edildi (karbaril dışında). Tekede VCL, VSL ve VAP parametrelerinde azalma gözlenirken (P<0.05), köpek spermasında anlamlı bir fark gözlenmedi (P>0.05). Piretroitler arasında flumetrinin, motilite parametreleri üzerine etkisi sipermetrine göre daha az bulundu. Sonuç olarak üreme toksisite testlerinde (özellikle kozmetik ürünlerin toksisite testlerinde hayvan deneylerinin yasaklandığı göz önünde bulundurularak) alternatif testler arasında bulunan ve batarya testleri içerisinde yer alan in vitro sperm toksisite araştırmalarında; MTT ve CASA ile birlikte NK'nın birlikte kullanılmasının kimyasalların sperma üzerine etkilerinin değerlendirilmesinde daha etkili olacağı ve türe özgü...

SummaryThe study was conducted to evaluate the eff ects of diff erent extenders and inositol additions on post-thaw semen quality, lipid peroxidation (LPO) and antioxidant activities. Semen was collected from four Karayaka rams from by artifi cial vagina three times a week. Semen samples showing normospermy quality were pooled. The pooled semen samples were extended in three extenders (Tris, T-, skimmed milk, M-and sodium citrate, NaC) with myo-inositol at two diff erent doses (5 mM, 10 mM) and no antioxidant (control). Nine experimental groups were assigned as follows: T-5I, T-10I, T (control); M-5I, M-10I, M (control); Na-5I, Na-10I, NaC (control). Straws containing extended semen were equilibrated at 4°C for 2 h, frozen in vapor of (15 min at -120°C) liquid nitrogen and stored in liquid nitrogen. Frozen semen was thawed in a water bath at 37°C for 30 seconds. The use of all the extenders supplemented with diff erent doses of myo-inositol did not lead to any significant improvement in microscopic sperm and oxidative stress parameters (P>0.05). Extenders of T and M resulted in higher sperm motility (50.00±2.24% and 55.00±0.42%) and HOST (49.00 3.32% and 48.17±2.97%) rates, compared to NaC (37.00±3.74% and 31.80±2.96%, P<0.01), following the freeze/thawing process. Extenders supplementated with myo-inositol not significantly aff ect malondialdehyde (MDA) levels and activities of catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GSH-PX) in comparison to the control groups (P>0.05), except for MDA level of T extender containing 10 mM inositol. MDA level was found lower (1.22±0.07 nmol/ml) in T than those of the M and NaC (P<0.05). For GSH and GSH-PX activities, T and NaC gave the higher values, compared to M, following the freeze/thawing process (P<0.01). Keywords: Ram semen, Inositol, Cryopreservation, Semen parameters, Antioxidant activities Koç Spermasının Çözüm Sonu Kalitesi Üzerine Farklı Sulandırıcıların ve Myo-İnositolün Etkileri ÖzetBu çalışma koç spermasının çözüm sonu kalitesi, lipit peroksidasyonu ve antioksidan aktiviteleri üzerine farklı sulandırıcıların ve inositolün etkilerini değerlendirmek amacıyla yapıldı. Sperma 4 baş Karayaka koçundan suni vajen yardımıyla haftada üç kez alındı. Alınan sperma örneklerinden normospermi özellik gösterenler birleştirildi. Birleştirilen sperma örnekleri iki farklı dozda myo-inositol (5, 10 mM) içeren ve içermeyen (kontrol) üç farklı sulandırıcı (tris, yağsız süt tozu, sodyum sitrat) ile sulandırıldı. Örnekler dokuz ayrı çalışma grubuna ayrıldı: T-5I, T-10I, T (kontrol); M-5I, M-10I, M (kontrol); Na-5I, Na-10I, NaC (kontrol) sulandırılmış sperma içeren payetler 4°C'de 2 saat ekilibre edildi, sıvı azot buharında (-120°C'da 15 dakika) donduruldu ve sıvı azot (-196°C) içinde saklandı. Dondurulmuş spermalar su banyosunda 37°C'de 30 saniyede çözdürüldü. Sulandırıcılara eklenenen myo-inositol mikroskopik sperm ve oksidatif stres parametelerine önemli bir etkiye neden olmadı (P>0.05). T ve M sulandırıcıları, NaC sulandırıcısına göre ...