Browsing by Author "Memili E."

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Advances in Cryopreservation of Bull Sperm
(2019-08-27) Ugur M.R.; Saber Abdelrahman A.; Evans H.C.; Gilmore A.A.; Hitit M.; Arifiantini R.I.; Purwantara B.; Kaya A.; Memili E.
Cryopreservation of semen and artificial insemination have an important, positive impact on cattle production, and product quality. Through the use of cryopreserved semen and artificial insemination, sperm from the best breeding bulls can be used to inseminate thousands of cows around the world. Although cryopreservation of bull sperm has advanced beyond that of other species, there are still major gaps in the knowledge and technology bases. Post-thaw viability of sperm is still low and differs significantly among the breeding bulls. These weaknesses are important because they are preventing advances both in fundamental science of mammalian gametes and reproductive biotechnology. Various extenders have been developed and supplemented with chemicals to reduce cryodamage or oxidative stress with varying levels of success. More detailed insights on sperm morphology and function have been uncovered through application of advanced tools in modern molecular and cell biology. This article provides a concise review of progress in the cryopreservation of bull sperm, advances in extender development, and frontiers using diverse techniques of the study of sperm viability. This scientific resource is important in animal biotechnology because with the advances in discovery of sperm fertility markers, there is an urgent need to improve post-thaw viability and fertility of sperm through enhanced cryopreservation for precision agriculture to produce food animals to ensure food security on the global scale.
Advancing Semen Evaluation Using Lipidomics
(2021-04-16) Evans H.C.; Dinh T.T.N.; Hardcastle M.L.; Gilmore A.A.; Ugur M.R.; Hitit M.; Jousan F.D.; Nicodemus M.C.; Memili E.
Developing a deeper understanding of biological components of sperm is essential to improving cryopreservation techniques and reproductive technologies. To fully ascertain the functional determinants of fertility, lipidomic methods have come to the forefront. Lipidomics is the study of the lipid profile (lipidome) within a cell, tissue, or organism and provides a quantitative analysis of the lipid content in that sample. Sperm cells are composed of various lipids, each with their unique contribution to the overall function of the cell. Lipidomics has already been used to find new and exciting information regarding the fatty acid content of sperm cells from different species. While the applications of lipidomics are rapidly evolving, gaps in the knowledge base remain unresolved. Current limitations of lipidomics studies include the number of available samples to analyze and the total amount of cells within those samples needed to detect changes in the lipid profiles across different subjects. The information obtained through lipidomics research is essential to systems and cellular biology. This review provides a concise analysis of the most recent developments in lipidomic research. This scientific resource is important because these developments can be used to not only combat the reproductive challenges faced when using cryopreserved semen and artificial reproductive technologies in livestock such as cattle, but also other mammals, such as humans or endangered species.
Amino Acids of Seminal Plasma Associated With Freezability of Bull Sperm
(2020-01-14) Ugur M.; Dinh T.; Hitit M.; Kaya A.; Topper E.; Didion B.; Memili E.
Sperm cryopreservation is an important technique for fertility management, but post-thaw viability of sperm differs among breeding bulls. With metabolites being the end products of various metabolic pathways, the contributions of seminal plasma metabolites to sperm cryopreservation are still unknown. These gaps in the knowledge base are concerning because they prevent advances in the fundamental science of cryobiology and improvement of bull fertility. The objective of this study was to test the hypothesis that seminal plasma amino acids are associated with freezability of bull sperm. To accomplish this objective, amino acid concentrations in seminal plasma from seven bulls of good freezability (GF) and six bulls of poor freezability (PF) were quantified using gas chromatography–mass spectrometry (GC–MS). Multivariate and univariate analyses were performed to identify potential freezability biomarkers. Pathways and networks analyses of identified amino acids were performed using bioinformatic tools. By analyzing and interpreting the results we demonstrated that glutamic acid was the most abundant amino acid in bull seminal plasma with average concentration of 3,366 ± 547.3 nM, which accounts for about 53% of total amino acids. The other most predominant amino acids were alanine, glycine, and aspartic acid with the mean concentrations of 1,053 ± 187.9, 429.8 ± 57.94, and 427 ± 101.3 nM. Pearson’s correlation analysis suggested that phenylalanine concentration was significantly associated with post-thaw viability (r = 0.57, P-value = 0.043). Significant correlations were also found among other amino acids. In addition, partial least squares-discriminant analysis (PLS-DA) bi-plot indicated a distinct separation between GF and PF groups. Phenylalanine had the highest VIP score and was more abundant in the GF groups than in the PF groups. Moreover, pathway and network analysis indicated that phenylalanine contributes to oxidoreductase and antioxidant reactions. Although univariate analysis did not yield significant differences in amino acid concentration between the two groups, these findings are significant that they indicate the potentially important roles of amino acids in seminal plasma, thereby building a foundation for the fundamental science of cryobiology and reproductive biotechnology.
Cellular and Functional Physiopathology of Bull Sperm With Altered Sperm Freezability
(2020-10-23) Hitit M.; Ugur M.R.; Dinh T.T.N.; Sajeev D.; Kaya A.; Topper E.; Tan W.; Memili E.
The objective of this study was to ascertain the cellular and functional parameters as well as ROS related changes in sperm from bulls with varied sperm freezability phenotypes. Using principal component analysis (PCA), the variables were reduced to two principal components, of which PC1 explained 48% of the variance, and PC2 explained 24% of the variance, and clustered animals into two distinct groups of good freezability (GF) and poor freezability (PF). In ROS associated pathophysiology, there were more dead superoxide anion positive (Dead SO+) sperm in GF bulls than those in PF (15.72 and 12.00%; P = 0.024), and that Dead SO+ and live hydrogen positive cells (live H2O2+) were positively correlated with freezability, respectively (R2 = 0.55, P < 0.0130) and (rs = 0.63, P = 0.0498). Related to sperm functional integrity, sperm from PF bulls had greater dead intact acrosome (DIAC) than those from GF bulls (26.29 and 16.10%; P = 0.028) whereas sperm from GF bulls tended to have greater live intact acrosome (LIAC) than those from PF bulls (64.47 and 50.05%; P = 0.084). Sperm with dead reacted acrosome (DRAC) in PF bulls were greater compared to those in GF (19.27 and 11.48%; P = 0.007). While DIAC (R2 = 0.56, P = 0.0124) and DRAC (R2 = 0.57, P < 0.0111) were negatively correlated with freezability phenotype, LIAC (R2 = 0.36, P = 0.0628) was positively correlated. Protamine deficiency (PRM) was similar between sperm from GF and PF bulls (7.20 and 0.64%; P = 0.206) and (rs = 0.70, P = 0.0251) was correlated with freezability. Sperm characteristics associated with cryotolerance are important for advancing both fundamental andrology and assisted reproductive technologies across mammals.
Expression profile of Toll-like receptor 4 in rat testis and epididymis throughout postnatal development
(2020-04-01) Özbek M.; Hitit M.; Ergün E.; Ergün L.; Beyaz F.; Erhan F.; Yıldırım N.; Kandil B.; Özgenç Ö.; Memili E.
Toll-like receptors (TLRs) belonging to pattern recognition receptors are involved in maintaining testicular and epididymal immune homeostasis. The purpose of the current study was to investigate TLR4 expression in rat testis and epididymis throughout postnatal development. Weak staining was detected in peritubular myoid cells and immature Sertoli cells while no staining was observed in gonocytes during prepubertal period. However, TLR4 expression began to appear in spermatocytes in pubertal period and gradually increased in spermatids. An intense staining was observed in steps 5–19 spermatids in post pubertal and mature periods. Similarly, TLR4 expression in the testes steadily increased from pubertal period to mature period. Puberty also caused a significant increase in TLR4 expression in epididymis. TLR4 expression in cauda epididymis was lower as compared to those of other epididymal segments. The majority of epididymal epithelial cells exhibited apical TLR4 expression, whereas basal cells showed intense intracytoplasmic immunoreaction. We detected an intense staining in epididymal smooth muscle cells. The expression levels of TLR4 showed dynamic changes in both spermatogenic cells, and entire testicular and epididymal tissues during postnatal development. These results suggest that TLR4 expression contributes not only to inflammation but also to the development of spermatogenic cells.
Functional variables of bull sperm associated with cryotolerance
(2021-01-01) Gilmore A.; Hitit M.; Ugur M.R.; Dinh T.T.N.; Tan W.; Jousan D.; Nicodemus M.; Topper E.; Kaya A.; Memili E.
The objective of this study was to ascertain sperm population and cellular characteristics as well as total antioxidant capacity in spermatozoa from Holstein bulls with Good (11 bulls) and Poor (5 bulls) cryotolerance. Post-thaw sperm kinetics were evaluated using CASA, membrane integrity was assessed via HOS test, and DNA fragmentation was measured using the HaloSperm kit. Data were analyzed using principal component analysis. The spermatozoa from Good bulls had a higher number of cells with intact membranes (P=0.029), non-fragmented DNA (P=0.018), and post-thaw viability (P<0.001) compared to sperm cells from Poor cryotolerance bulls. Sperm cells from Good bulls also had a faster average path velocity (P=0.017) and straight-line velocity (P=0.036), along with a greater distance average path (P=0.006) and distance straight line (P=0.011). However, total antioxidant capacity, number of live cells, and other kinetic parameters between spermatozoa from Good and Poor groups were not different. There is no one specific sperm function variable alone that can accurately predict cryotolerance of bull spermatozoa, and thus, a combination of sperm cell attributes and kinematics needs to be utilized by the AI industry in differentiating between freezability of spermatozoa between bulls.
Lipidomic markers of sperm cryotolerance in cattle
(2020-12-01) Evans H.C.; Dinh T.T.N.; Ugur M.R.; Hitit M.; Sajeev D.; Kaya A.; Topper E.; Nicodemus M.C.; Smith G.D.; Memili E.
The objective of the current study was to determine the fatty acid composition of sperm from Holstein bulls with different freezability (Good and Poor; n = 12). Fatty acids were extracted from frozen sperm in 1:2 (v/v) chloroform–methanol solvent, fractionated into neutral and polar fractions, and composition determined by gas chromatography–mass spectrometry. Thirty-four fatty acids were quantified and their concentrations and percentages within each lipid fraction were calculated. Overall, saturated fatty acids (SFA) were predominant, accounting for 71 to 80% of fatty acids in neutral and polar lipid factions. There were marked differences in fatty acid composition between the lipid fractions (P < 0.001). The branched chain fatty acid (BCFA) concentration (15 to 18 µg) was almost twice as much as polyunsaturated fatty acids (PUFA) concentration found in the polar lipid fraction (8 to 9 µg; P < 0.001). Sperm with different freezability phenotypes only had a few differences in 22:0, 18:1 cis 9, and 14:0 13-methyl fatty acids (P ≤ 0.011). These results are significant because they reveal key understandings of fatty acid composition of sperm membrane and lay a foundation for the manipulation of membrane integrity, fluidity, and stability to advance the assisted reproductive technologies.
Proteomic fertility markers in ram sperm
(2021-12-01) Hitit M.; Özbek M.; Ayaz-Guner S.; Guner H.; Oztug M.; Bodu M.; Kirbas M.; Bulbul B.; Bucak M.N.; Ataman M.B.; Memili E.; Kaya A.
Precise estimation of ram fertility is important for sheep farming to sustain reproduction efficiency and profitability of production. There, however, is no conventional method to accurately predict ram fertility. The objective of this study, therefore, was to ascertain proteomic profiles of ram sperm having contrasting fertility phenotypes. Mature rams (n = 66) having greater pregnancy rates than average (89.4 ± 7.2%) were assigned into relatively-greater fertility (GF; n = 31; 94.5 ± 2.8%) whereas those with less-than-average pregnancy rates were assigned into a lesser-fertility (LF; n = 25; 83.1 ± 5.73%; P = 0.028) group. Sperm samples from the outlier greatest- and least-fertility rams (n = 6, pregnancy rate; 98.4 ± 1.8% and 76.1 ± 3.9%) were used for proteomics assessments utilizing Label-free LC-MS/MS. A total of 997 proteins were identified, and among these, 840 were shared by both groups, and 57 and 93 were unique to GF and LF, respectively. Furthermore, 190 differentially abundant proteins were identified; the abundance of 124 was larger in GF while 66 was larger in LF rams. The GF ram sperm had 79 GO/pathway terms in ten major biological networks while there were 47 GO/pathway terms in six biological networks in sperm of LF rams. Accordingly, differential abundances of sperm proteins between sperm of GF and LF rams were indicative of functional implications of sperm proteome on male fertility. The results of this study emphasize there are potential protein markers for evaluation of semen quality and estimation of ram sperm fertilizing capacity.
Sperm Functional Genome Associated With Bull Fertility
(2021-06-22) Özbek M.; Hitit M.; Kaya A.; Jousan F.D.; Memili E.
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.
Sperm signatures of fertility and freezability
(2022-12-01) Hitit M.; Memili E.
Apart from traditional semen examination parameters, there is not yet a set of functional markers for accurate determination of bull fertility and sperm freezability or cryopreservability, which are vital for production of food animals to feed the world. Therefore, reliable biomarkers are needed to objectively analyze semen quality and predict male fertility. Rapid developments in animal biotechnology have led to significant progress in developing science-based solutions for global problems in food animal production. Although andrology studies employing genomic and functional genomics (transcriptomics, proteomics, and metabolomics) approaches have elucidated some molecular aspects of sperm, there is also a need for additional mechanistic studies to ascertain the functional underpinnings. Biomarkers discovered through applying various -omics technologies using sperm from bulls with varying fertility phenotypes are valuable for semen evaluation and fertility prediction.