Expression of dna methyltransferases (dnmts) at mrna level in ovine endometrium during estrus cycle and early pregnancy

Aim: To elucidate mRNA expression of DNA methyltransferase enzyme genes (DNMT1, DNMT3A, and DNMT3B) in ovine endometrium whether they are modulated during the estrus cycle and early pregnancy. Materials and Methods: The endometrial samples including intercaruncular sites was obtained from a total of 24 ewes on days of 12 (P12, n = 4), 16 (P16, n = 4) and 22 (P22, n = 4) of pregnancy following mating and cyclic days of 12 (C12, n = 4), 16 (C16, n = 4) and 22 (C22, n = 4) of the estrous cycle. The relative mRNA level of DNMTs were evaluated through real-time quantitative RT-qPCR. Results: Abundances of DNMTs including DNMT1, DNMT3A, and DNMT3B were detected during the estrous cycle and early pregnancy in the ovine endometrium by this study. DNMT1 mRNA steady-state level was greater in P16 than in C16 whereas they did not change within the rest of the groups. The level of DNMT3A mRNA, during early pregnancy, had the highest expression levels on P12 compared to P22 and P16 (p<0.01). However, the level of DNMT3A mRNA was lower in P16 than in C16 (p<0.01) and had a similar decrease in P22 compared to C22 (p<0.01). DNMT3B mRNA level did not differ (p>0.01) during the estrus cycle and early pregnancy. Conclusion: DNA methylation related DNMTs may be required to control of gene expression in the ovine endometrium during the estrus cycle and early pregnancy

Pentoxifylline May Restore Kanamycin-Induced Renal Damage in Rats

Background: Kidney damage can be caused by many factors, such as using certain drugs in high doses or over the longterm. The use of one such group of drugs, aminoglycosides, which act as Gram-negative antibacterial therapeutic agents,can lead to nephrotoxicity. It has been hypothesized that aminoglycoside-induced nephrotoxicity might be prevented byusing pentoxifylline, which has antioxidant and anti-inflammatory effects and improves microcirculation. The objectiveof this present research was to determine the protective effects of pentoxifylline on kanamycin-induced kidney damage.Materials, Methods &amp; Results: Thirty-two male Wistar rats were divided into four groups as follows: control, pentoxifylline,kanamycin, and kanamycin + pentoxifylline. The control group received intraperitoneal (IP) injections of 0.5 mL normalsaline solution once a day (d) (SID) for 20 d; the pentoxifylline group received IP injections of 50 mg/kg pentoxifyllinetwice a day (BID) for 20 d, the kanamycin group received subcutaneous (SC) injections of 500 mg/kg kanamycin SID for20 d, and the kanamycin + pentoxifylline group received both SC injections of 500 mg/kg kanamycin SID and IP injectionsof 50 mg/kg pentoxifylline BID for 20 d. At the end of 20 d, blood samples were taken from the heart by cardiac punctureunder general anesthesia. After euthanizing the rats by cervical dislocation under anesthesia, the kidneys were immediatelyremoved, relative kidney weights were calculated, and routine pathologic evaluations were conducted. Hemogramparameters were measured using a blood cell count apparatus and serum biochemical parameters were measured usingan autoanalyzer. Kanamycin also caused (P &lt; 0.05) tubular degeneration and tubular dilatation. Although pentoxifyllinesignificantly reduced the level of kanamycin-induced tubular degeneration (P &lt; 0.05), it did not significantly reduce tubulardilatation. Increases in relative kidney weights (P &lt; 0.05) and in interstitial mononuclear cell (MNC) infiltrates wereobserved in the kanamycin and kanamycin + pentoxifylline groups compared to those in the control and pentoxifyllinegroups. Statistically significant changes were determined in the levels of some hemogram and biochemical parameterswithin reference ranges (P &lt; 0.05).Discussion: In this study, both tubular degeneration and dilatation were observed in the kanamycin group. Pentoxifyllineinhibited (P &lt; 0.05) kanamycin-induced tubular degeneration and appeared to also reduce tubular dilatation, although thisreduction was not significant. Tubular necrosis, epithelial edema of proximal tubules, tubular fibrosis, and perivascularinflammation might also be observed in aminoglycoside-induced nephrotoxicity. In current research, pentoxifylline preventedtubular damage induced by kanamycin, but did not inhibit infiltration by MNCs. Pentoxifylline also amelioratedamikacin- or gentamycin-induced histopathologic changes, especially those associated with tubular structures. The protectiveeffects of pentoxifylline on kanamycin-induced tubular nephrotoxicity in this research might be a result of its stimulatingthe production of prostaglandin, a vasodilator, and of its improving microcirculation. Although the anti-inflammatoryeffects of pentoxifylline have been reported, these did not inhibit kanamycin-induced infiltration by interstitial MNCs inthe present study. These results could indicate that the anti-inflammatory effects of pentoxifylline are not obvious and/orare dose dependent. Statistically significantly changes were determined in the levels of some hemogram and biochemicalparameters in reference ranges. However, these changes were within the reference ranges for rats. These results suggestedthat kanamycin-induced tubular degeneration and dilatation might be prevented by administering pentoxifylline

Extender development for optimal cryopreservation of buck sperm to increase reproductive efficiency of goats

Preservation of sperm significantly contributes to the advancement of assisted reproductive technologies, genetic conservation and improvement efforts, and precision breeding of livestock. This review distills knowledge from the existing information and emerging patterns in the field of buck sperm cryopreservation. The primary focus is on the challenges and opportunities associated with improving extender formulations and freezing techniques in order to enhance the vitality of sperm after thawing and to increase the potential for conception. This review assesses the efficacy and limitations of conventional extenders derived from egg yolk or soybean lecithin, and the adverse impacts of seminal plasma enzymes on sperm quality during the processes of chilling and cryopreservation. Significant progress has been made in the fields of molecular biology namely lipidomics, proteomics, metabolomics, DNA methylation providing valuable knowledge regarding the unique reactions of sperm to cryopreservation. The utilization of the “omics” technologies has shown intricate molecular transformation that occur in sperm during freezing and thawing. Moreover, detection of molecular biomarkers that indicate the quality of sperm and their ability to withstand freezing provides opportunities to choose the best sperm samples for cryopreservation. This, in turn, enhances the results of artificial insemination and genetic conservation endeavors. This review emphasizes the necessity for adopting a comprehensive approach that combines molecular and cellular knowledge with practical methods in the field of sperm cryopreservation to ensure production of goats as major food animals in the global scale

Comparison of reverse-transcriptase polymerase chain reaction (rt-pcr) and rapid test for the detection of bovine rotavirus and bovine coronavirus in anatolian water

Aim: Coronaviruses and Rotaviruses are important virologic factors for both animal and human health in Turkey and the world. Bovine Rotavirus (BRV) and Bovine Coronavirus (BCoV) in cattle cause significant economic losses. The aim of this study was to determine the presence of BRV and BCoV in Anatolian buffaloes which were on the same farms with cattle. For this purpose, presence of these two viruses were investigated by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and BRV-BCoV Rapid tests and sensitivity and specificity ratios of these two tests were compared. Materials and Methods: In this study, 230 Anatolian buffaloes were clinically evaluated in cattle farms in Afyonkarahisar region. Fecal samples were collected from 27 buffaloes which had clinical signs (weakness, dehydration, vomiting, watery consistency and yellow stool). The fecal samples were evaluated by Rapid Test and RT-PCR for Bovine Rotavirus and Bovine Coronavirus. The analyzes were performed according to the procedure of the commercial RT-PCR and rapid kits. Results: The RT-PCR results were positive as 22.2% (6/27) for BRV and 3.7% (1/27 27/1) for BcoV while Rota-Corona Rapid test results were negative in all samples. When compared with RT-PCR results for both viruses, the rapid test sensitivity and specificity was determined as 0% and 100%, respectively. In addition, positive rates of BRV was statistically important as BCoV rate in analyzed samples (p Conclusion: In conclusion, low sensitivity of rapid test may be due to the change in the amount of virus scattered throughout the course of enteric infections

The expression of steroidogenic genes in ovine corpus luteum during early pregnancy

Aim: The goal of this study was to investigate the expression of steroidogenic genes in ovine corpus luteum during early pregnancy. Materials and Methods: The animal model was designed as pregnancy; ewes were divided into three sub-groups, pregnancy 12th day: P12, pregnancy 16th day: P16, pregnancy 22th day: P22, and cyclic day 16 (C16). The expression of steroidogenic genes (steroidogenic acute regulatory protein; StAR, cytochrome P450 side-chain cleavage; P450sscc, and 3b-hydroxysteroid dehydrogenase/ delta5 delta4-isomerase; 3?HSD) was evaluated using qPCR, and mRNA localization of StAR was detected on P16 against C16 through in-situ hybridization. Results: The expression of StAR mRNA was higher on day P22 and P16 compared to P12 (p<0.05), while it was at a steady-state level on day P22 vs P16 (p>0,05). mRNA expression of P450scc was greater on day P22 and P16 than day P12 (p<0.05), however on day P22 vs P16, it was at a steady-state level (p>0,05). Also, mRNA expression of 3?HSD had a similar trend; it was higher on day P22 and P16 compared to P12 (p<0.05), while it was at a steady-state level on day P22 vs P16 (p>0,05). In in-situ hybridization, we did not detect StAR mRNA on cyclic day 16 (C16) while abundantly expressed in luteal cells in P16. Conclusion: The mRNA expression of steroidogenic genes may appear to play a critical role during early pregnancy in ewes. Accordingly, it can be suggested that the steroidogenic pathway in the corpus luteum of ewe may transcriptionally regulate progesterone synthesis required for maintenance of early pregnancy

Melatonin protects bovine embryos from heat stress and oxygen tension and improves embryo production in vitro

The objective of this study was to determine melatonin"s ameliorating effects against heat stress and oxygen tension in developing bovine embryos in vitro. The oocytes were collected from ovaries obtained from a local abattoir, followed by in vitro maturation, fertilization, and embryo culture. During in vitro culture, embryos were exposed to 5% (Group I) and 20% (Group II) oxygen tension with 10-3, 10-6, and 10-9 molar (M) melatonin, along with the control group without melatonin (Group III). Compared to the control group, melatonin at 10-6 and 10-9 concentrations increased in vitro development rates and decreased caspase 3/7 activity at 5% and 20% oxygen tension (P<0.01). Onehalf of the zygotes were cultured under normal temperature (38.5ºC) during the culture period, and the other half of the zygotes were heat stressed at 41ºC for six hours. Then they transferred into the normal culture conditions for the rest of the period using 0, 10- 6, and 10-9 M of melatonin (Group IV). Under normal temperature (38.5ºC), melatonin at 10-9 M was beneficial for in vitro development and DNA integrity. Under heat stress at 41ºC, melatonin at 10-6 and 10-9 M was useful for in vitro development and DNA integrity (P<0.05). Supplementation of melatonin to embryo culture medium did not alter the caspase 3 and 7 activities (P>0.05). In conclusion, melatonin prevents the adverse effects of heat stress and O2 tension on preimplantation bovine embryos in vitro

Sperm Functional Genome Associated With Bull Fertility

Bull fertility is an important economic trait in sustainable cattle production, as infertile or subfertile bulls give rise to large economic losses. Current methods to assess bull fertility are tedious and not totally accurate. The massive collection of functional data analyses, including genomics, proteomics, metabolomics, transcriptomics, and epigenomics, helps researchers generate extensive knowledge to better understand the unraveling physiological mechanisms underlying subpar male fertility. This review focuses on the sperm phenomes of the functional genome and epigenome that are associated with bull fertility. Findings from multiple sources were integrated to generate new knowledge that is transferable to applied andrology. Diverse methods encompassing analyses of molecular and cellular dynamics in the fertility-associated molecules and conventional sperm parameters can be considered an effective approach to determine bull fertility for efficient and sustainable cattle production. In addition to gene expression information, we also provide methodological information, which is important for the rigor and reliability of the studies. Fertility is a complex trait influenced by several factors and has low heritability, although heritability of scrotal circumference is high and that it is a known fertility maker. There is a need for new knowledge on the expression levels and functions of sperm RNA, proteins, and metabolites. The new knowledge can shed light on additional fertility markers that can be used in combination with scrotal circumference to predict the fertility of breeding bulls. This review provides a comprehensive review of sperm functional characteristics or phenotypes associated with bull fertility

Investigation of liver X receptor pathway in luteal regression

Kolesterol steroid hormonları, safra asidi ve vitamin D'nin öncüsü olan nötral bir lipittir. Liver X reseptörleri kolesterolün reverz taşımında (efluks) önemli rol üstlenir ve makrofaj gibi periferal hücrelerden kolesterolün dışarı atılmasını sağlar. LXR'nin karaciğer ve diğer organlardaki rolü açıklanmış olsada, LXR' nin üreme organlarındaki rolü moleküler düzeyde tam olarak bilinmemektedir. Korpus luteumda (KL) gebelik gerçekleşmemişse progesteron üretimi durur, hücre bütünlüğü kaybolur, doku kitlesi azalarak apoptozis ve otofaji yolakları vasıtasıyla regresyon başlar. Regresyon süresince meydana gelen progesteron azalışında kolesterolün luteal dokudan efluksunun rolü olup olmadığı netlik kazanmamıştır. Bu çalışmanın amacı, kolesterolün reverz olarak tranpsortunun luteal regresyona katkısının araştırılmasıdır. Bu amaç için iki araştırma yapıldı: 1) Birinci araştırmada, siklik (S12 ve S16-doğal luteolizis) ve gebelik (G12, G16 ve G22) gruplarından, 2) İkinci araştırmada siklusun 12. günde luteolizis PGF2α enjeksiyonu ile uyarılıp 1 saat (PG1, n=4), 4 saat (PG4, n=4), 16 saat (PG16, n=4) ve kontrol (n=4) olmak üzere KL doku örnekleri toplandı. LXR yolağı (LXRα, LXRβ, ABCA1, ABCG1, APOE ve APOA1), steroidogenik progesteron sentez yolağı (StAR, 3βHSD ve P450scc) ile kolesterol alım reseptörlerinin (SRBI ve LDLR) gen ifadeleri mRNA düzeyinde RT-qPZ ve LXR yolağının genlerinin protein seviyesi western blot ile araştırıldı. Ayrıca, in-situ hibridizasyon ile StAR (S16 vs G16), SR-BI (S12 vs PG1 vs PG4 vs PG16), LXRα, LXRβ, ABCA1 genlerinin mRNA lokalizasyonları saptandı. Doğal luteolizisde LXRα ve ABCA1 mRNA ekspresyonu değişmezken (P>0,05), LXRβ, ABCG1, APOE ve ApoA-1 mRNA ekspresyon seviyesi S16'da S12'ye göre artmıştır (P0,05). PG4'de S12'ye göre LXRα, LXRβ, APOE, ApoA-I, LDLR, SR-BI, StAR, P450scc ve 3βHSD mRNA ekspresyonu azalırken (P0,05). PG16'da S12'ye göre StAR, P450scc, 3βHSD, LDLR, SR-BI ve ApoA-I azalmış (P0,05). In-situ hibridizasyonda StAR mRNA'sının ifadesi G16'da S16'ya göre daha fazladır (P0,05), expressions of LXRβ, ABCG1, APOE, and ApoA-1 were increased in S16 compared to S12 (P0,05). While, mRNA expressions of LXRα, LXRβ, APOE, ApoA-I, LDLR, SR-BI, StAR, P450scc, and 3βHSD were decreased, expressions of ABCA1, and ABCG1 were not changed (P>0,05) in PG4 compared to S12. Expressions of StAR, P450scc, 3βHSD, LDLR, SR-BI, and ApoA-I were decreased (P<0,05), while ABCG1 increased (P<0,05) in PG16 compared to S12. Protein level of LXRβ was increased in S16 (P<0,05) compared to S12 while others was at stead state levels in both experimental models. In in-situ hybridization, StAR mRNA was higher in G16 that S16 (P<0,05). SR-BI levels of mRNA was not changed in PG1 and S12 (P<0,05), while decreased in both PG4, and PG16 (P<0,05). In conclusion, it is suggested that decrease of lipoprotein receptor-mediated cholesterol uptake may be the reason of lutel regression rather than the reverse transport of cholesterol.Bu tez Selçuk Üniversitesi Öğretim Üyesi Yetiştirme Programı Koordinatörlüğü tarafından 2013-ÖYP-090 proje numarası ile desteklenmiştir

Supplementary files

The results of this study emphasize there are potential protein markers for evaluation of semen quality and estimation of ram sperm fertilizing capacit

Östrus siklusu ve erken gebelikte koyun endometriyumunda DNA metiltransferazların (DNMT) mRNA düzeyinde ekspresyonu

Aim: To elucidate mRNA expression of DNA methyltransferase enzyme genes (DNMT1, DNMT3A, and DNMT3B) in ovine endometrium whether they are modulated during the estrus cycle and early pregnancy. Materials and Methods: The endometrial samples including intercaruncular sites was obtained from a total of 24 ewes on days of 12 (P12, n = 4), 16 (P16, n = 4) and 22 (P22, n = 4) of pregnancy following mating and cyclic days of 12 (C12, n = 4), 16 (C16, n = 4) and 22 (C22, n = 4) of the estrous cycle. The relative mRNA level of DNMTs were evaluated through real-time quantitative RT-qPCR. Results: Abundances of DNMTs including DNMT1, DNMT3A, and DNMT3B were detected during the estrous cycle and early pregnancy in the ovine endometrium by this study. DNMT1 mRNA steady-state level was greater in P16 than in C16 whereas they did not change within the rest of the groups. The level of DNMT3A mRNA, during early pregnancy, had the highest expression levels on P12 compared to P22 and P16 (p0.01) during the estrus cycle and early pregnancy. Conclusion: DNA methylation related DNMTs may be required to control of gene expression in the ovine endometrium during the estrus cycle and early pregnancy.Amaç: Bu çalışmanın amacı, östrus döngüsü ve erken gebelik sırasında DNA metiltransferaz enzim genlerinin (DNMT1, DNMT3A ve DNMT3B) ekspresyonunun koyun endometriyumunda düzenlenip düzenlenmediğini açıklamaktır. Gereç ve Yöntem: İnterkarunkular bölgeleri içeren endometriyal örnekler çiftleşmeyi takiben gebeliğin 12 (P12, n = 4), 16 (P16, n = 4) ve 22. (P22, n = 4) günlerinde ve östrus döngüsününe karşılık gelen 12. (C12, n = 4), 16. (C16, n = 4) ve 22. (C22, n = 4) günlerde toplam 24 koyundan toplandı. DNMT'lerin nispi mRNA ekspresyon düzeyleri gerçek zamanlı polimeraz zincir reaksiyonu (RT-qPCR) kullanılarak değerlendirildi. Bulgular: Tüm DNMT'lerin ekspresyonları östrus döngüsü ve erken gebelik sırasında koyun endometriyumda tespit edildi. DNMT1 mRNA ekspresyonunun kararlı durum seviyeleri P16'da C16'dan daha yüksekti, ancak grupların geri kalanında değişmedi. Bununla birlikte, gebeliğin erken döneminde DNMT3A mRNA seviyesi P22 ve P16'ya kıyasla P12'de en yüksek ekspresyon seviyelerine sahipti (p<0.01). Öte yandan, DNMT3A mRNA seviyesi P16'da C16'dan daha düşüktü (p<0.01), ayrıca P22'de C22'ye kıyasla benzer bir düşüş bulundu (p<0.01). DNMT3B mRNA ekspresyon seviyesi ise hem östrus döngüsü ve hem de erken gebelik sırasında değişmedi. Öneri: Östrus siklusu ve erken gebelik sırasında koyun endometriyumundaki gen ekspresyonunun kontrolü için DNA metilasyonu ile ilgili DNMT'ler gerekli olabileceği kanısına varıldı