Lyophilized Extender Supplemented with Rainbow Trout (Oncorhynchus mykiss) Seminal Plasma Improves Cryopreservation of Ram Sperm

This study aimed to investigate the effect of the Rainbow trout seminal plasma (RTSP) supplemented (control, 10% or 15%) lyophilized extender on freezability of ram semen. Collected semen was pooled and split into two aliquots, and each of the ejaculates was diluted with fresh or lyophilized extenders with RTSP (0%, FC and LC; 10% F10 and L10or 15% F15 and L15) using two-step dilution method. Semen was frozen using the programmable freezing machine. Semen samples were examined for sperm motility, defective acrosomes, plasma membrane integrity and DNA fragmentation at native and post-thaw stage. The highest percentages of post-thaw motility and plasma integrity were observed in the F10, F15, and L15 (P 0.05). There was no significant difference in the rate of post-thaw defective acrosomes when the other extender groups were compared with the control (P 0.05). The highest percentage of post-thaw DNA fragmentation values were observed in the FC group, while the lowest DNA fragmentation was obtained in the F15 but only significant different from FC and LC groups. In conclusion, the findings of this study show that the lyophilized extender with 15% RTSP added can be used successfully for freezing ram semen

Effects of triton X-100 pretreatment of lyophilized and frozen-thawed ram sperm on preimplantation embryo developmental competence

In this study, it was aimed to determine the effect of destruction of lyophilized and frozen-thawed ram sperm plasma and acrosomal membrane on development of embryos produced by intracytoplasmic sperm injection (ICSI). Semen samples were divided into two groups for lyophilization (L) and freezing (F). For the removal of the plasma membrane, L and F groups were incubated with Triton X-100 (LTX-100 and FTX-100, respectively). Integrities of the plasma membrane, acrosome and chromatin structure were evaluated. Oocytes were injected with these sperm groups. Although no plasma membrane and acrosome integrities of the L (0.0%) group were detected, the plasma membrane integrity of the F group (69.4%) was significantly higher than the FTX-100 group (23.6%) (p < 0.05). The acrosome integrity of the FTX-100 group (3.80%) was significantly lower than the F group (55.6%) (p < 0.05). The chromatin integrities of L and F groups were higher than the Triton X-100 treated groups (p < 0.05). ICSIs with L, LTX-100, F and FTX-100 sperm were produced similar cleavage and blastocyst rates. In conclusion, data presented here confirm that ram spermatozoa can effectively be lyophilized and injected into oocytes for initiation of embryonic development and Triton X-100 pretreatment is not necessary while using lyophilized and frozen semen.Scientific and Technological Research Council of Turkey (TUBTAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [113O593]This study was supported financially by the Scientific and Technological Research Council of Turkey (TUBTAK; project number: 113O593)

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

Liyofilizasyon Amaçlı Kullanılan Sulandırıcıların Koç Spermasının DNA Bütünlüğü Üzerine Etkisi

DNA hasarının belirlenebilmesi, sperma saklama yöntemlerinin geliştirilmesi açısından önemlidir. Biz çalışmamızda farklı liyofilizasyon medyumları kullanılarak saklanan sperm hücrelerinin DNA bütünlüğünün değerlendirilmesinde akridin oranj (AO) ve therminal deoxynucleotidyl transferase-medited dUDP nick and labelling (TUNEL) floresan boyama yöntemlerini kullandık. Koçlardan alınan sperma dört gruba bölündü ve her bir grup: I) %10 fötal buzağı serumu (FCS) içeren TCM 199 solüsyonu II) %10 FCS ve 0,2 mol/L trehaloz içeren TCM 199 solüsyonu III) 50 mmol/L NaCl ve EGTA [Ethylen glycol-bis (β-aminoethyl ether)-N, N, N, N,-tetraacetic acid] 10 mmol/L Tris solüsyonu ve IV) %20 yumurta sarısı ve %7 gliserol içeren Tris bazlı solüsyonlardan biri ile konsantrasyonu 10 x 106 spermatozoa/100 μL olacak şekilde sulandırıldı. Taze alınan spermada DNA fragmentasyon oranı AO ve TUNEL yöntemlerinde sırasıyla %2,0 ±1,2 ve %3,8±3,7 olarak tespit edildi (P&gt;0.05). AO ve TUNEL yöntemiyle değerlendirilen liyofilizasyon sonrası DNA bütünlüğüne sahip spermatozoa oranlarının, sulandırıcı farklılığına göre etkilenmediği tespit edildi (P&gt;0.05). Aynı şekilde her iki boyama yöntemi karşılaştırıldığında, liyofilizasyon sonrası elde edilen DNA bütünlüğünün değerlendirilmesinde AO ve TUNEL sonuçlarının istatistiksel olarak farklı olmadığı belirlendi (P&gt;0.05). Çalışmamızın sonuçları; sulandırıcı gruplarından her birinin koç spermasının liyofilizasyonu için uygun olduğunu ve her iki boyama yönteminin de tercih edilebileceğini göstermektedir. Anahtar Kelimeler: koç sperması, liyofilizasyon, DNA bütünlüğü, tunel, akridin oran

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)