In-vitro evaluation of ram sperm, frozen in tris-EGG yolk extender supplemented with methionine, cysteine and bht

Bu çalışmada TRIS-sitrik asit yumurta sarısı (%20) temel sulandırıcısına 2,5mM, 5mM metiyonin, 1mM, 2mM sistein, 1mM, 2mM bütil hidroksitolüen (BHT) eklenerek sulandırılan ve dondurulan koç spermasında çözüm sonrası bazı spermatolojik parametreler üzerine antioksidanların etkileri ve karşılaştırılması amaçlandı. Sperma, beş baş Kıvırcık ırkı koçtan üreme sezonunda, elektro-ejakülatör ile haftada iki kez alındı ve birleştirildi. Pooling sonrası ortalama hacim (ml), motilite (%), yoğunluk (x109/ml), plazma membran bütünlüğü (HOST, %), akrozomal (%), diğer morfolojik (%) ve toplam morfolojik (%) bozukluk oranları sırasıyla 3,92, 84,0, 1,96, 93,8, 5,60, 0,4 ve 6,0 bulundu. Sulandırılan sperma örnekleri bir saat içinde 5ºC'a indirildi ve %6 gliserol eklendikten sonra iki saat boyunca ekilibrasyona tabi tutuldu. Sperma, payette (0,25ml) 200x106 spermatozoon olacak şekilde çekildi ve dondurma cihazıyla dondurularak (-120 oC) analize kadar sıvı azotun içinde saklandı. Çözdürme sonrası motilite, HOST, akrozomal bozukluk, diğer ve toplam morfolojik bozukluk muayeneleri yapıldı. Motilite ve HOST oranları, kontrol ile tüm antioksidan grupları arasında fark (P<0,05) ve pozitif korelasyon tespit edildi (r=0,880). Akrozomal bozukluk oranlarında kontrol ile 2,5mM, 5mM metiyonin ve 1mM, 2mM sistein grupları arasında fark saptandı (P<0,05). Ayrıca metiyonin ve sistein grupları kendi dozları arasında da farklıydı (P<0,05). Diğer morfolojik bozukluk oranlarında ise fark yalnızca 1mM sistein ile 2mM BHT grupları arasında tespit edildi (P<0,05). Toplam morfolojik bozukluk oranlarında ise 1mM, 2mM BHT ve 1mM sistein grupları hariç, kontrol ve diğer deneme grupları arasında fark görüldü (P<0,05). Sonuç olarak, koç sperma sulandırıcısına çeşitli dozlarda eklenen antioksidanların çözüm sonrası incelenen spermatolojik parametreler üzerine koruyucu etkilerde bulunduğu kanaatine varıldı.The aim of this research is to study the effects and comparison of the antioxidants [2,5mM, 5mM methionine, 1mM, 2mM cysteine, 1mM, 2mM butylhyroxytoluene (BHT)] for semen parameters on freeze-thawed ram sperm that diluted in TRIS-citric acid and egg yolk (%20) sperm extender. Sperm was collected from five Kivircik rams twice a week with electro-ejaculator in breeding season and pooled. After pooling the mean of the volume (ml), motility (%), sperm number (x109/ml), plasma membrane integrity (HOST, %), acrosomal, other morphologic and total morphologic defects (%) was found 3,92, 84,0, 1,96, 93,8, 5,60, 0,4 ve 6,0 respectively. The diluted sperm samples were cooled to 5ºC within an hour and equlibrated for another two hours after adding %6 of glycerol. 200x106 sperm cells were placed into 0,25ml french straws and freezed with a programmable freezing device (-120 oC) and plunged into liquid nitrogen tanks until spermatological analyses. After thawing of the sperm straws, the motility, HOST also acrosomal, other morphologic and total morphologic defects parameters were investigated. All antioxidant added study groups were statistically different (P<0,05) from antioxidant-free control group in terms of motility and HOST parameters and also were in a positive correlation between these parameters (r=0,880). In terms of acrosomal defects, there was a statistical difference among control group with the 2,5mM, 5mM methionine and 1mM, 2mM cysteine groups (P<0,05). Also there was a difference between the doses of methionine and cysteine groups (P<0,05). The difference between 1mM cysteine and 2mM BHT groups was statistically significant (P<0,05). The statistical difference for the total morphologic defects was found among control group and 2mM cysteine, 2,5mM and 5mM methionine groups (P<0,05). The study had reached the conclusion that the effects of antioxidants that added to ram sperm diluents with different doses had protective effects on spermatological parameters

Sığır Serum Albumini İlave Edilmiş Lesitin Bazlı Sulandırıcı İle Sulandırılan Koç Spermasının Dondurma-Çözdürme Sonrası Uzun Süreli İnkübasyon Direnci]

The objective of the study was to determine the optimum concentration of BSA in lecithin-based extender for post-thawing quality and incubation resilience (0 h, 6 h and 10 h) of ram sperm. Ejaculates were collected from five rams via electro ejaculation. Ejaculates were mixed to obtain pooled semen.Then, pooled semen was diluted with soybean lecithin-based extender without BSA (control) or supplemented with different concentrations of BSA (2.5 mg/mL, 5 mg/mL, 7.5 mg/mL and 10 mg/mL), at a final concentration of 150x106 spermatozoon/mL. Sperm motility, plasma membrane functional integrity (HOST), mitochondrial activity (rhodamine123), capacitation status (CTC), and DNA integrity (TUNEL) were evaluated. At the 10 h incubation, motility, plasma membrane functional integrity and mitochondria! function were better preserved in the BSA5 group compared to the control group. It was determined that high doses of BSA (5 mg/mL, 7.5 mg/mL and 10 mg/mL) affected acrosome reaction.The highest acrosome reaction rates were obtained in BSA10 groups in 6 h and 10 h incubation (P<0.05). TUNEL assay demonstrated that there were no differences among groups for DNA fragmentation at post-thaw and during incubation periods. The study shows that BSA supplemented extenders may have beneficial effect on ram semen parameters at 0 h, 6 h and 10 h of incubation. The results of the present study demonstrated a remarkable advantage of using 5 mg/mL of BSA in 1% lecithin-based extender.Uludag University Scientific Research Projects Unit, Bursa, Turkey, (BAP)Uludag University [KUAP (V)-2015/55]This work was supported by the Uludag University Scientific Research Projects Unit, Bursa, Turkey, (BAP) (Project number: KUAP (V)-2015/5

Administration time of misoprostol affects fertility rate in artificially inseminated Kivircik ewes with frozen-thawed ram semen

The aim of this study was to determine the effects of the administration time of misoprostol (11 h (Miso11) and 6 h (Miso6) before artificial insemination) on fertility rates in Kivircik ewes (control: n = 41, Miso11: n = 32 and Miso6: n = 33) during breeding season. Artificial insemination (AI) was performed 48 h after sponge removal using frozen-thawed semen (150 million sperm per dose in 0.25 ml straws). Estrus synchronization parameters (onset and duration) and lambing rate were evaluated. No significant difference was observed among groups for the estrus onset and duration hours (P > 0.05). The lambing rates in the control, Miso11 and Miso6 groups were 39.0, 62.5 and 54.5%, respectively. There were significant differences among the control, Miso11 and Miso6 groups according to lambing rates (P < 0.05). In conclusion, misoprostol treatment significantly improved fertility in ewes when using frozen-thawed semen in AI. Administration of misoprostol 11 h before AI resulted in a higher lambing rate than that at 6 h before AI; therefore, treatment of misoprostol 11 h before AI can effectively be used

Brain thromboxane A(2) via arachidonic acid cascade induces the hypothalamic-pituitary-gonadal axis activation in rats

The current study was designed to determine the effect of centrally administrated arachidonic acid (AA) on plasma gonadotropin hormone-releasing hormone (GnRH), follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone level, and sperm parameters, and to show the mediation of the central cyclooxygenase (COX) to thromboxane A(2) (TXA(2)) signaling pathway in AA-induced hormonal and sperm parameter effects. Studies were performed in male Sprague-Dawley rats. A total of 150 or 300 mu l/5 mu l doses of AA were injected intracerebroventricularly (icv). AA significantly caused dose- and time-dependent increases in plasma FSH, LH and testosterone levels of animals, but not plasma GnRH level. AA also significantly increased sperm motility of the rats without change sperm number. Pretreated with ibuprofen, a nonselective COX inhibitor (250 mu g/5 mu l; icv), and furegrelate, a TXA(2) synthesis inhibitor (250 mu g/5 mu l; icv), prevented AA-evoked increase in plasma FSH, LH and testosterone levels, and sperm motility

Intracerebroventricular injection of histamine induces the hypothalamic-pituitary-gonadal axis activation in male rats

Brain histamine holds a key position in the regulation of behavioral states, biological rhythms, body weight, energy metabolism, thermoregulation, fluid balance, stress and reproduction in female animals. However, it is not clear whether central histamine exerts any effect on hypothalamic-pituitary-testicular in male rats and if so, the involvement of type of central histamine receptors. The current study was designed to determine the effect of centrally administrated histamine on plasma gonadotropin hormone-releasing hormone (GnRH), luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone level, and sperm parameters, and to show the mediation of the central histaminergic H1, H2 and H3/H4 receptors on histamine-evoked hormonal and sperm parameters' effects. Studies were performed in male Sprague-Dawley rats. A total of 50 or 100 nmol doses of histamine were injected intracerebroventricularly (icy). 100 nmol dose of histamine significantly caused increases in plasma GnRH, LH, FSH and testosterone levels of animals, but not 50 nmol dose of histamine. Moreover, central pretreatment with chlorpheniramine, histaminergic H1 receptor antagonist (100 nmol), ranitidine and histaminergic H2 receptor antagonist (100 nmol) completely prevented histamine evoked increase in plasma GnRH, LH, FSH and testosterone levels, while thioperamide, histaminergic H3/H4 receptor antagonist (100 nmol) pretreatment failed to reverse sex hormones responses to histamine. Both central histamine treatment alone and central histamine treatment after central histaminergic receptors antagonists' pretreatments did not alter any sperm parameters in rats. In conclusion, our findings show that centrally administered histamine increases plasma GnRH, LH, FSH and testosterone levels of conscious male rats without change any sperm parameters. Moreover, according to our findings, central histaminergic H1, and H2 receptors mediate these histamine-induced effects.Commission of Scientific Research Projects of Uludag UniversityUludag University [OUAP(V)2015/26]This study was supported by Commission of Scientific Research Projects of Uludag University (OUAP(V)2015/26)