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Sericin is a water-soluble protein produced by silk cocoons and known to have antioxidant activity. This study is aimed to analyze the nuclear maturation and the quality of bovine oocytes in the collection and in vitro maturation (IVM) medium supplemented with sericin. Bovine oocytes were collected using a collection medium supplemented with sericin in 0 (control) concentrations, 0.1%, 0.5%, and 1%. Selected oocytes were then matured for 24 h at 38.5 oC in 5% CO₂ and evaluated for nuclear maturation. In the subsequent experiment, oocytes were collected and matured with or without 0.1% sericin at 38.5 oC in 5% CO2. Matured oocytes were counterstained with terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) and Hoechst 33342. Matured oocytes were characterized by oocytes that reached the MII stage. The results showed that supplementation of 0.1% sericin in the collection medium increased the number of oocytes reaching the metaphase II (MII) stage compared to the control group (p

Abstract Background Subfertility decreases the efficiency of the cattle industry because artificial insemination employs spermatozoa from a single bull to inseminate thousands of cows. Variation in bull fertility has been demonstrated even among those animals exhibiting normal sperm numbers, motility, and morphology. Despite advances in research, molecular and cellular mechanisms underlying the causes of low fertility in some bulls have not been fully elucidated. In this study, we investigated the metabolic profile of bull spermatozoa using non-targeted metabolomics. Statistical analysis and bioinformatic tools were employed to evaluate the metabolic profiles high and low fertility groups. Metabolic pathways associated with the sperm metabolome were also reported. Results A total of 22 distinct metabolites were detected in spermatozoa from bulls with high fertility (HF) or low fertility (LF) phenotype. The major metabolite classes of bovine sperm were organic acids/derivatives and fatty acids/conjugates. We demonstrated that the abundance ratios of five sperm metabolites were statistically different between HF and LF groups including gamma-aminobutyric acid (GABA), carbamate, benzoic acid, lactic acid, and palmitic acid. Metabolites with different abundances in HF and LF bulls had also VIP scores of greater than 1.5 and AUC- ROC curves of more than 80%. In addition, four metabolic pathways associated with differential metabolites namely alanine, aspartate and glutamate metabolism, β-alanine metabolism, glycolysis or gluconeogenesis, and pyruvate metabolism were also explored. Conclusions This is the first study aimed at ascertaining the metabolome of spermatozoa from bulls with different fertility phenotype using gas chromatography-mass spectrometry. We identified five metabolites in the two groups of sires and such molecules can be used, in the future, as key indicators of bull fertility.

MicroRNA sequences (miRNA) are small RNAs 19 to 22 nucleotides in length that play a crucial role during mammalian development and disease. They are found in plants and animals and regulate the expression of protein-coding genes. Mechanisms regulating gene expression during gamete and embryo development are critical for setting the stage in later development; however, very little is known about the roles of miRNAs in male gametes (spermatozoa). Therefore, the objective of this study was to profile miRNA populations in spermatozoa collected from high and low fertility bulls. Frozen–thawed straws containing spermatozoa from one low and one high fertility bulls were purified using a Percoll gradient (which removes somatic cells) followed by 3 washing steps in PBS. Total RNA were isolated from pelleted spermatozoa using Trizol (3 independent isolations for each bull, total of 6 samples). RNA samples were quantified (NanoDrop Spectrophotometer, Thermo Scientific, Wilmington, DE) and verified for potential somatic cells (Agilent BioAnalyzer, Agilent, Foster City, CA) and DNA (No-RT-PCR) contaminations. Samples devoid of any contaminations went through an enrichment process of miRNA, consisting of dephosphorylation (CIP) and labeling (pCp-Biotin) of the 3′-end of miRNA, followed by their purification (BioSpin6, Bio-Rad, Hercules, CA). The purified miRNAs were analyzed by a miRNA microarray containing approximately 30 000 probes from human, rat, mouse, and other organisms (Ambion/Affymetrix DiscovArray, Asuragen, Austin, TX). The results showed that roughly 48% of total miRNA probe sets (14 215) were successfully analyzed (P2 and t-test P< 0.001). The roles of these miRNAs are not yet identified because the annotations are unavailable. Nevertheless, our preliminary results suggest that bull spermatozoa are rich in miRNAs, which could play important roles during fertilization and embryo development. Future studies will be aimed at identifying potential molecular mechanisms by which these 7 miRNAs might regulate bovine embryonic development.This study was funded by the Mississippi Agricultural and Forestry Experiment Station and Alta Genetics Inc. [ABSTRACT FROM AUTHOR]

The reproductive performance of a herd is the biggest factor affecting production and product quality of livestock. Thus, a decline of male fertility represents a dramatic economic loss in beef and dairy industries. Caused by molecular defects in the spermatozoa, uncompensatory infertility is a current challenge for the cattle industry, because even with normal sperm morphology, motility, and number, fertility of bulls is still sub-par. Thus, the objective of this study was to determine the global proteome of spermatozoa collected from bulls with different fertility and study the proteins playing a role in uncompensatory infertility. We performed difference gel electrophoresis (2D-DIGE) using cryopreserved sperm from a total of 6 bulls. Spermatozoa were thawed, purified by percoll gradient, and washed in PBS solution. For each bull, a pellet of 100 million sperm cells was resuspended in the 2D-DIGE labeling buffer, and the total protein was quantified. Cell lysates were separately labeled with CyDye DIGE Fluor dyes (and reciprocally with different dyes) and multiplexed in pairs on 3 gels. The samples were focused according to their isoelectric point through an Immobiline DryStrip, pH4-7, using a IPGphor 2, followed by separation on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (21.5–200 kDa) using a DALTsix. The resulting gels were scanned, and their topographic digital maps were used for algorithmic spot matching, background normalization, spot differences quantification, and elimination of artifacts (DeCyder 2-D Differential Analysis Software, GE Healthcare Life Sciences, Piscataway, NJ, USA). The results showed between 2600 and 2800 proteins with high confidence. Compared to the high-fertile bulls, 30 proteins were increased, and 27 were decreased in the low-fertility bulls within a 2-fold range. The largest significant increase and decrease were 3.97- and 2.4-fold, respectively. The identification of these differentially represented proteins is in progress. However, our results provided a panoramic view of sperm proteome from bulls of different fertility and thus paved the way for research on mechanisms of uncompensatory bull infertility. [ABSTRACT FROM AUTHOR]

Molecular-based biomarkers are believed to be more accurate in determining bulls' fertility and sperm's various fertility characteristics. The sperm acrosome-associated 1 (SPACA1) molecule, located in the anterior acrosomal and equatorial segments of the sperm head, is thought to be related to the function of binding and fusion between sperm and oocyte. This study aims to analyze the association of SPACA1 mRNA and protein with the fertility rate and semen quality of Bali bulls (Bos sondaicus) and assess its potential as a potential molecular biomarker determining bull fertility and sperm quality. Frozen semen from 20 Bali bulls was used in the research, which was then divided into two groups: high (HF) and low fertility (LF). SPACA1 mRNA abundance was analyzed using qRT-PCR, and SPACA1 protein abundance was analyzed using EIA. The semen quality parameters analyzed were motility (CASA), plasma membrane integrity (HOS test), sperm head morphology abnormalities (William staining), sperm viability (SYBR 14-PI), sperm capacitation (CTC assay), and sperm acrosome integrity (FITC-PNA). The results showed that there was a significant difference (P < 0.05) between high fertility (HF) and low fertility (LF) bulls, both in the abundance of SPACA1 at the mRNA, protein, and semen quality levels. Overall, the correlation test results showed a close relationship (P < 0.01) between the abundance of SPACA1 mRNA and protein with field fertility and various semen quality parameters tested in the study. A low level of SPACA1 molecules indicates low fertility and semen quality in Bali bulls. The SPACA1 molecule has the potential to be developed through further research to become a biomarker for determining fertility and semen quality in bulls., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In Brief: Bovine sperm and seminal plasma (SP) proteoform atlas was characterized using top-down proteomics. Specific post-translational modifications and protein truncations correlated with semen freezability, with potential links to sperm functional processes., Abstract: Top-down proteomics was employed to construct a proteoform atlas of sperm and SP from bulls with low semen freezability (LF) and high semen freezability (HF). Sperm and seminal proteins were fractionated by tandem size exclusion chromatography (<30 kDa) and analyzed by reversed-phase liquid chromatography-tandem mass spectrometry. This approach enabled identification of 299 SP (from 46 families) and 267 sperm proteoforms (from 139 families). Seventy proteoforms belonging to beta-defensin 10, c-type natriuretic peptide (NPPC), caltrin, seminal ribonuclease, osteopontin and binder of sperm protein (BSP) 3 families were unique to HF bulls' SP. LF seminal proteins had 77 unique proteoforms, including caltrin, NPPC, osteopontin, BSP3, serpin family A member 5 and β-NGF families. Proteoform families of SP in HF and LF bulls were related to Ca2+ uptake, capacitation, acrosome reaction, sperm protection, fertilization and proteolytic processes. Thirty-three proteoforms of NPPC, caltrin and cylicin-2 families were upregulated in HF sperm. Twenty-two proteoforms of caltrin, cylicin-2, adenosine triphosphate (ATP) synthases and malate dehydrogenase families were among those upregulated in LF sperm. Truncated and acetylated histone H2A and non-truncated and acetylated c-Myc-binding protein were prevalent in LF sperm. Cylicin-2 proteoforms were observed in HF and LF sperm, and truncated glyceraldehyde-3-phosphate dehydrogenases were observed only in HF sperm. In silico analyses indicated the enrichment of mitochondrial metabolic pathways in HF sperm, including fatty acid metabolism and TCA cycle. Our study provides an unprecedented description of the bovine SP and sperm proteoforms. Post-translational processing appears to define the bioproperties of semen proteins and their associations with sperm cryoresistance.

Efficient and sustainable reproduction and production of cattle and goats are vitally important for ensuring global food security. There is a need for potent biomarkers to accurately evaluate semen quality and predict male fertility. Although there is a reasonable set of biomarkers identified in bull sperm, there is a significant lack of such information in buck sperm along with a lack of transfer of proven technologies in goat reproductive biotechnology. These gaps are important problems because they are preventing advances in fundamental andrology and applied science of goat production. Both cattle and goats are ruminants, and they share significant similarities in their genetics and physiology although subtle differences do exist. This review harnesses the power of utilizing the knowledge developed in bull sperm to generate information on buck sperm fertility markers. These include genomic, functional genomic, epigenomic fertility markers. Revealing molecular underpinnings of such similarity and diversity using systems biology is expected to advance both fundamental and applied andrology of livestock and endangered species., Competing Interests: Declaration of Competing Interest The authors of this manuscript declare that they have no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

It is critical in sheep farming to accurately estimate ram fertility for maintaining reproductive effectiveness and for production profitability. However, there is currently a lack of reliable biomarkers to estimate semen quality and ram fertility, which is hindering advances in animal science and technology. The objective of this study was to uncover long non-coding RNAs (lncRNAs) in sperm from rams with distinct fertility phenotypes. Mature rams were allocated into two groups: high and low fertility (HF; n = 31; 94.5 ± 2.8%, LF; n = 25; 83.1 ± 5.73%; P = 0.028) according to the pregnancy rates sired by the rams (average pregnancy rate; 89.4 ± 7.2%). Total RNAs were isolated from sperm of the highest- and lowest-fertility rams ( n = 4, pregnancy rate; 99.2 ± 1.6%, and 73.6 ± 4.4%, respectively) followed by next-generation sequencing of the transcripts. We uncovered 11,209 lncRNAs from the sperm of rams with HF and LF. In comparison to each other, there were 93 differentially expressed (DE) lncRNAs in sperm from the two distinct fertility phenotypes. Of these, 141 mRNAs were upregulated and 134 were downregulated between HF and LF, respectively. Genes commonly enriched for 9 + 2 motile cilium and sperm flagellum were ABHD2, AK1, CABS1, ROPN1, SEPTIN2, SLIRP , and TEKT3 . Moreover, CABS1, CCDC39, CFAP97D1, ROPN1, SLIRP, TEKT3 , and TTC12 were commonly enriched in flagellated sperm motility and sperm motility. Differentially expressed mRNAs were enriched in the top 16 KEGG pathways. Targets of the differentially expressed lncRNAs elucidate functions in cis and trans manner using the genetic context of the lncRNA locus, and lncRNA sequences revealed 471 mRNAs targets of 10 lncRNAs. This study illustrates the existence of potential lncRNA biomarkers that can be implemented in analyzing the quality of ram sperm and determining the sperm fertility and is used in breeding soundness exams for precision livestock farming to ensure food security on a global scale., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hitit, Kaya and Memili.)

The objectives of this study were to evaluate fixed risk factors associated with PWM and to estimate genetic parameters for PWM. A total of 927 birth records from a mixed population of purebred and crossbred Boer and Spanish goats born between 2016 and 2023 at the International Goat Research Center (IGRC) were used for this study. Four binary traits were studied: D0-3 (death within 3 days after birth), D4-60 (death between 4 and 60 days), D61-90 (death between 61 and 90 days), and D0-90 (death within 90 days). Logistic regression models were used to evaluate the risk factors associated with PWM traits. Bayesian threshold models and Gibbs sampling were used to estimate the genetic parameters. Birth weight, season, litter size, sex, dam age, breed, and heterosis were found to be significantly associated with at least one of the PWM traits. Heritability estimates were 0.263, 0.124, 0.080, and 0.207, for D0-3, D4-60, D61-90, and D0-90, respectively. The genetic correlations between the studied traits ranged from 0.892 (D0-3 and D0-90) to 0.999 (D0-3 and D61-90). These results suggest that PWM in goats is influenced by both non-genetic and genetic factors and can be reduced by management, genetic selection, and crossbreeding approaches.

Infertility is a global health concern inflicting a considerable burden on the global economy and a severe socio-psychological impact. Approximately 15% of couples suffer from infertility globally, with a male factor contribution of approximately 50%. However, male infertility remains largely unexplored, as the burden of infertility is mostly assigned to female people. Endocrine-disrupting chemicals (EDCs) have been proposed as one of the factors causing male infertility. Pyrethroids represent an important class of EDCs, and numerous studies have associated pyrethroid exposure with impaired male reproductive function and development. Therefore, the present study investigated the potentially toxic effects of two common pyrethroids, cypermethrin and deltamethrin, on androgen receptor (AR) signaling. The structural binding characterization of cypermethrin and deltamethrin against the AR ligand-binding pocket was performed using Schrodinger's induced fit docking (IFD) approach. Various parameters were estimated, such as binding interactions, binding energy, docking score, and IFD score. Furthermore, the AR native ligand, testosterone, was subjected to similar experiments against the AR ligand-binding pocket. The results revealed commonality in the amino acid-binding interactions and overlap in other structural parameters between the AR native ligand, testosterone, and the ligands, cypermethrin and deltamethrin. The estimated binding energy values of cypermethrin and deltamethrin were very high and close to those calculated for AR native ligand, testosterone. Taken together, the results of this study suggested potential disruption of AR signaling by cypermethrin and deltamethrin, which may result in androgen dysfunction and subsequent male infertility., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sheikh, Beg, Hamoda, Mandourah and Memili.)

Polybrominated diphenyl ethers (PBDEs) are a common class of anthropogenic organobromine chemicals with fire-retardant properties and are extensively used in consumer products, such as electrical and electronic equipment, furniture, textiles, and foams. Due to their extensive use, PBDEs have wide eco-chemical dissemination and tend to bioaccumulate in wildlife and humans with many potential adverse health effects in humans, such as neurodevelopmental deficits, cancer, thyroid hormone disruption, dysfunction of reproductive system, and infertility. Many PBDEs have been listed as chemicals of international concern under the Stockholm Convention on Persistent Organic Pollutants. In this study, the aim was to investigate the structural interactions of PBDEs against thyroid hormone receptor (TRα) with potential implications in reproductive function. Structural binding of four PBDEs, i.e., BDE-28, BDE-100, BDE-153 and BDE-154 was investigated against the ligand binding pocket of TRα using Schrodinger's induced fit docking, followed by molecular interaction analysis and the binding energy estimation. The results indicated the stable and tight binding of all four PDBE ligands and similarity in the binding interaction pattern to that of TRα native ligand, triiodothyronine (T3). The estimated binding energy value for BDE-153 was the highest among four PBDEs and was more than that of T3. This was followed by BDE-154, which is approximately the same as that of TRα native ligand, T3. Furthermore, the value estimated for BDE-28 was the lowest; however, the binding energy value for BDE-100 was more than BDE-28 and close to that of TRα native ligand, T3. In conclusion, the results of our study suggested the thyroid signaling disruption potential of indicated ligands according to their binding energy order, which can possibly lead to disruption of reproductive function and infertility.

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., Competing Interests: Conflict of interest The authors of this manuscript declare that they have no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Seminal plasma is a dynamic, intricate combination of fluids from the testicles, epididymides, seminal vesicles, bulbourethral glands, and prostate, containing molecules that modulate sperm functions, post-fertilization events, and the female reproductive tract physiology. Significant variations in sperm parameters and fertility status of bulls relate to differences in the seminal plasma proteome. In this framework, a meta-analytical study was conducted examining 29 studies (published between 1990 and 2021) to ascertain the effects of seminal fluid proteins on parameters associated with bull fertility and the influence of distinct methodologies on such effects. Our results revealed that seminal proteins ameliorate sperm parameters, such as motility, integrity, capacitation, and fertilizing ability, and favours sperm protection. Seminal binder of sperm proteins and beta-defensin 126 highly favoured sperm protection when cells were collected from the epididymis by retrograde flux and analysed under room temperature conditions. Furthermore, seminal proteins improved the motility and quality of Bos taurus sperm collected by artificial vagina, mainly in the presence of heparin-binding proteins. The key limitations faced by this meta-analysis were the paucity of studies evaluating the effects of whole seminal fluid proteins and the limited number of studies conducted in vivo. In conclusion, the present meta-analytical study confirms that seminal proteins improve fertility-related parameters in the bovine species. However, methodological strategies used by authors are diverse, with distinct endpoints and methods. Thus, the translational aspects of seminal plasma research should be taken into consideration to precisely define how seminal proteins can be harnessed to advance reproductive biotechnology., (© 2022 Wiley-VCH GmbH.)

The human population is ever increasing while the quality and quantity of natural resources used for livestock production decline. This calls for improved product efficiency and the development of improved and sustainable cattle production methods to produce higher quality products to satisfy the demands of both the modern and transient world. The goal of this review was to summarize the interactions, challenges, and opportunities in cattle production relating to their endocrine system, and how reproductive hormones and others impact economically important traits, animal welfare, and human health. A comprehensive literature search was conducted with a focus on analysis of natural hormones and the use of exogenous hormone administration for reproduction, growth, and development of beef and dairy cattle. Hormones regulate homeostasis and enhance important traits in cattle, including fertility, growth and development, health, and the production of both meat and milk products. Reproductive hormones such as testosterone, estradiol, progesterone, and related synthetics like trenbolone acetate and zeranol can be strategically utilized in both beef and dairy cattle production systems to enhance their most valuable traits, but the impact of these substances must account for the welfare of the animal as well as the health of the consumer. This scientific review provides a comprehensive analysis of the bovine endocrine system's impact on food animals and product quality which is vital for students, researchers, livestock producers, and consumers. Although important advances have been made in animal science and related technological fields, major gaps still exist in the knowledge base regarding the influence of hormones on the production and welfare of food animals as well as in the public perception of hormone use in food-producing animals. Filling these gaps through transformative and translational research will enhance both fundamental and applied animal science to feed a growing population., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Heat shock protein 70 (HSP70) is one of the most abundant chaperone proteins. Their function is well documented in facilitating the protein synthesis, translocation, de novo folding, and ordering of multiprotein complexes. HSP70 in bovine consists of four genes: HSP70-1, HSP70-2, HSP70-3, and HSP70-4. HSP70-2 was found to be involved in fertility. Current knowledge implicates HSP70-2 in sperm quality, sperm capacitation, sperm-egg recognition, and fertilization essential for bull reproduction. HSP70-2 is also involved in the biological processes of spermatogenesis, as it protects cells from the effects of apoptosis and oxidative stress. Fertilization success is not only determined by the amount of sperm found in the female reproductive tract but also by the functional ability of the sperm. However, subfertility is more likely to be associated with changes in sperm molecular dynamics not detectable using conventional methods. As such, molecular analyses and omics methods have been developed to monitor crucial aspects of sperm molecular morphology that are important for sperm functions, which are the objectives of this review., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Rosyada, et al.)

Prediction of bull fertility is critical for the sustainability of both dairy and beef cattle production. Even though bulls produce ample amounts of sperm with normal parameters, some bulls may still suffer from subpar fertility. This causes major economic losses in the cattle industry because using artificial insemination, semen from one single bull can be used to inseminate hundreds of thousands of cows. Although there are several traditional methods to estimate bull fertility, such methods are not sufficient to explain and accurately predict the subfertility of individual bulls. Since fertility is a complex trait influenced by a number of factors including genetics, epigenetics, and environment, there is an urgent need for a comprehensive methodological approach to clarify uncertainty in male subfertility. The present review focuses on molecular and functional signatures of bull sperm associated with fertility. Potential roles of functional genomics (proteome, small noncoding RNAs, lipidome, metabolome) on determining male fertility and its potential as a fertility biomarker are discussed. This review provides a better understanding of the molecular signatures of viable and fertile sperm cells and their potential to be used as fertility biomarkers. This information will help uncover the underlying reasons for idiopathic subfertility., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare.

Low fertility is the single most important factor limiting livestock reproductive performance, adversely affecting the cattle industry and causing millions of dollars of economic loss. In the livestock industry, male fertility is of crucial importance for the reproductive performance of livestock. However, there is a lack of reliable biomarkers to predict bull fertility in artificial insemination service. The objective of this study was to identify sperm proteins as biomarkers for bull fertility. To discover candidate sperm quality biomarkers, sperm proteome profiling was conducted in extreme high- and extreme low-fertile bulls selected from a pool of 1000 AI sires with varied fertility. Thirty-two differentially expressed proteins were identified. Among them, high levels of sperm outer dense fiber of sperm tails 2 (ODF2) and post-acrosomal assembly of sperm head protein (PAWP/WBP2NL) represented the most extreme differences in quantity between high- and low-fertility bulls. Protein immunodetection and flow cytometry used to validate these putative fertility markers in a combined cohort of 154 AI sires. Both ODF2 and PAWP correlated significantly with fertility. In conclusion, ODF2 and PAWP can be used to assess semen quality and predict sire fertility., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Pre- and post-transcriptional modifications of gene expression are emerging as foci of disease studies, with some studies revealing the importance of non-coding transcripts, like long non-coding RNAs (lncRNAs) and microRNAs (miRNAs). We hypothesize that transcription factors (TFs), lncRNAs and miRNAs modulate immune response in bovine mastitis and could potentially serve as disease biomarkers and/or drug targets. With computational analyses, we identified candidate genes potentially regulated by miRNAs and lncRNAs base pair complementation and thermodynamic stability of binding regions. Remarkably, we found six miRNAs, two being bta-miR-223 and bta-miR-24-3p, to bind to several targets. LncRNAs NONBTAT027932.1 and XR&#95;003029725.1, were identified to target several genes. Functional and pathway analyses revealed lipopolysaccharide-mediated signaling pathway, regulation of chemokine (C-X-C motif) ligand 2 production and regulation of IL-23 production among others. The overarching interactome deserves further in vitro/in vivo explication for specific molecular regulatory mechanisms during bovine mastitis immune response and could lay the foundation for development of disease markers and therapeutic intervention., (© 2021. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Özbek, Hitit, Kaya, Jousan and Memili.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Evans, Dinh, Hardcastle, Gilmore, Ugur, Hitit, Jousan, Nicodemus and Memili.)

Proteomic approaches have been widely used in reproductive studies to uncover protein biomarkers of bull fertility. Seminal plasma is one of the most relevant sources of these proteins that may influence sperm physiology. Nonetheless, there are still gaps in existing knowledge in the functional attributes of seminal proteins. Thus, we reviewed the relationships between seminal plasma proteins and bull fertility by conducting a systematic review with data obtained from 71 studies. This review showed that the associations related to fertility improvement with the use of total seminal plasma proteins are still controversial. None of the studies explored the sperm fertilizing ability following these interactions. By contrast, the exposure to a single protein, such as osteopontin, binder of sperm proteins, and heparin binding proteins, can increment sperm motility, capacitation, and fertilizing ability by modulating intracellular calcium concentrations, removing lipids from sperm membranes, and regulating the acrosome reaction. Variations in protein analyses and the protein contents and their abundances between animals contributed to the difficulty of establishing protein biomarkers of fertilizing potential of the bull sperm. Indeed, the heterogenicity of methodologies was a limitation of this review. Standardized methods of seminal protein analyses, as well as sperm endpoints, may minimize such discrepancies. In conclusion, potential biomarkers of sperm parameters are still to be established. Future studies should evaluate protein isoforms and how they interact with sperm to ascertain their biological functions.

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.

Background and Aim: Indonesia has two National Artificial Insemination centers and 17 Regional Artificial Insemination Centers. The frozen semen production techniques differed between the centers, including the type of diluent and semen dilution technique. The aim of the research was to compare the quality of frozen Limousin bull semen diluted using different techniques., Materials and Methods: Semen was collected from three sexually mature Limousin bulls using an artificial vagina. Immediately after collection, the semen was evaluated macroscopically and microscopically. Semen that had >70% motile sperm and <20% sperm abnormality was divided into three tubes and diluted with skim milk-egg yolk (SMEY) using three different dilution techniques: One-step dilution (100% SMEY with 8% glycerol) at room temperature ([RT] 20°C until 25°C) two-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol after 1 h stored at 5°C); and three-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol added twice at 1 h and 1.5 h after being stored at 5°C). The diluted semen was loaded into 0.25 mL mini straws, equilibrated, and frozen using a freezing machine. Sperm motility, viability, membranes, DNA integrity, and concentrations of malondialdehyde (MDA) and aspartate aminotransferase (AST) enzymes were evaluated after thawing., Results: The results showed that there were no significant differences in sperm motility and DNA integrity between dilutions (p > 0.05). However, sperm viability and membrane intactness of one-step dilutions were higher than those of three-step dilutions. The concentrations of MDA and AST enzymes of sperm in one-step dilutions were lower than those of three-step dilutions (p < 0.05)., Conclusion: It was concluded that the one-step-dilution technique was better than three-step dilution for cryopreservation of Limousin bull semen., (Copyright: © Arif, et al.)

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 H 2 O 2 +) were positively correlated with freezability, respectively ( R 2 = 0.55, P < 0.0130) and (r s = 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 ( R 2 = 0.56, P = 0.0124) and DRAC ( R 2 = 0.57, P < 0.0111) were negatively correlated with freezability phenotype, LIAC ( R 2 = 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 (r s = 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., (Copyright © 2020 Hitit, Ugur, Dinh, Sajeev, Kaya, Topper, Tan and Memili.)

The present study investigated the seminal plasma proteome of Holstein bulls with low (LF; n = 6) and high (HF; n = 8) sperm freezability. The percentage of viable frozen-thawed sperm (%ViableSperm) determined by flow cytometry varied from -2.2 in LF to + 7.8 in HF bulls, as compared to the average %ViableSperm (54.7%) measured in an 860-sire population. Seminal proteins were analyzed by label free mass spectrometry, with the support of statistical and bioinformatics analyses. This approach identified 1,445 proteins, associated with protein folding, cell-cell adhesion, NADH dehydrogenase activity, ATP-binding, proteasome complex, among other processes. There were 338 seminal proteins differentially expressed (p < 0.05) in LF and HF bulls. Based on multivariate analysis, BSP5 and seminal ribonuclease defined the HF phenotype, while spermadhesin-1, gelsolin, tubulins, glyceraldehyde-3-phosphate dehydrogenase, calmodulin, ATP synthase, sperm equatorial segment protein 1, peroxiredoxin-5, secretoglobin family 1D and glucose-6-phosphate isomerase characterized the LF phenotype. Regression models indicated that %ViableSperm of bulls was related to seminal plasma peroxiredoxin-5, spermadhesin-1 and the spermadhesin-1 × BSP5 interaction (R 2  = 0.84 and 0.79; p < 0.05). This report is the largest dataset of bovine seminal plasma proteins. Specific proteins of the non-cellular microenvironment of semen are potential markers of sperm cryotolerance.

Bull fertility is crucial for efficient, profitable, and sustainable agriculture of cattle. Despite the fact that the uses of sperm from low fertility bulls cause millions of dollars economic losses, conventional methods available to accurately predict bull fertility are still of limited use. Although breeding bulls produce billions of sperm mostly with normal motility and morphology, some bulls are afflicted with poor fertility due to molecular or cellular defects in the sperm. Sperm functional genome including transcriptome, proteome, and metabolome influence bull fertility. Through high throughput screening methods of metabolomics, specific small molecules have been described both for seminal plasma and sperm. Compared to sperm, seminal plasma contains much higher numbers and levels of metabolites. Although the identities and functions of many of these metabolites are known, such knowledge is still yet to be generated for a greater number of metabolites of sperm and seminal plasma. Once validated as fertility markers, sperm, and seminal plasma metabolites can be used to evaluate semen quality and predict bull fertility, and/or used in assisted reproductive technologies. This review describes the possibility to use small molecules (in the review called metabolites) as fertility predictors., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest that could affect our objectivity in this study., (Copyright © 2020. Published by Elsevier B.V.)

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., (© 2020 Blackwell Verlag GmbH.)

Seminal plasma is a critical and complex fluid that carries sperm to eggs to initiate the fertilization process. Here, we present a top-down mass spectrometry (TDMS) strategy for identifying proteins and posttranslational modifications (PTMs) in bovine seminal plasma. In this study, proteins were separated using sheathless capillary zone electrophoresis (CZE)-MS and reversed-phase liquid chromatography (LC)-MS, and then fragmented using electron-transfer/higher-energy collisional dissociation (EThcD) and 213 nm ultraviolet photodissociation (213 nm UVPD) to provide more comprehensive information about the proteomic landscape of this biological fluid. Four hundred and seventeen proteoforms were identified by sheathless CZE-MS, and one hundred and seventy-two species were unique to this method. LC-MS identified 3090 proteoforms, including 1707 unique species. All identifications were within ±10 ppm (mass error) and with a P-Score ≤1 × 10 -04 . Pooling results (triplicate measurements) from sheathless CZE-MS and LC-MS resulted in the identification of 1433 proteoforms (EThcD) and 2151 proteoforms (213 nm UVPD) with 612 species unique for EThcD and 1021 for 213 nm UVPD. The average sequence coverage was found to be higher for EThcD (28%) than for 213 nm UVPD (23%). The use of sheathless CZE-MS and LC-MS with EThcD and 213 nm UVPD provided complementary protein profiling and proteoform data that were more comprehensive than those of either method alone.

MicroRNAs modulate male fertility by regulating gene expression. In this study, dynamics of sperm miR-15a, miR-29b and miR-34a from high fertility (HF) and low fertility (LF) bulls using RT-qPCR were evaluated. Bioinformatic tools were employed to ascertain genes of interest of the sperm miRNAs. The expression levels of p53, BCL2, BAX and DNMT1 in bull spermatozoa were determined by immunoblotting. MicroRNA levels of miR-15a and miR-29 were higher in LF sires when compared with those present in HF bulls. Expression levels of miR-34a did not differ between the two groups. We found an inverse correlation between miR-15a and bull fertility. MiR29-b was also negatively associated with fertility scores. BCL2 and DNMT1 were higher in HF bulls while BAX was higher in the LF group. Our data showed a positive correlation between BCL2 and bull fertility. In addition, DNMT1 was positively associated with bull fertility. Furthermore, levels of BAX were negatively linked with bull fertility scores. Identification of miRNAs found in the spermatozoa of sires with different in vivo fertility helps understand the alterations in the fertilising capacity from cattle and other mammals. These potential biomarkers can be used in reproductive biotechnology as fertility markers to assess semen quality and predict male fertility., (© 2019 Blackwell Verlag GmbH.)

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., (Copyright © 2020 Ugur, Dinh, Hitit, Kaya, Topper, Didion and Memili.)

The objective of this study was to ascertain cellular characteristics and the dynamics of the sperm chromatin proteins protamine 1 (PRM1) and protamine 2 (PRM2) in the sperm of Holstein bulls having a different fertility status. Important sperm variables were analyzed using computer-assisted sperm analysis (CASA). Sperm membrane, acrosome status, DNA integrity were also assessed using propidium iodide (PI), fluorescein isothiocyanate conjugated to Arachis hypogaea (FITC-PNA), and acridine orange (AO) followed by flow cytometry. In addition, abundances of PRM1 and PRM2 were analyzed using flow cytometry experiments. Differences in sperm decondensation capacity were assessed in bulls of varying fertility using a decondensation assay. As determined using CASA, average pathway velocity, amplitude of lateral head displacement and straightness were different (P < 0.05) for sperm from high and low fertility bulls. There, however, were no differences between the high and low fertility bulls for characteristics of sperm plasma membrane, acrosome, and DNA integrity (P >  0.05). Relative abundances of PRM1 and PRM2 in sperm from the high and low fertility bulls were inversely related (P <  0.0001). Percentages of decondensed sperm were different between high and low fertility bulls (P < 0.0001) and total numbers of decondensed sperm were greater in low fertility bulls than high fertility bulls (R 2  = 0.72). Results of the present study are significant because molecular and morphological phenotypes of sperm that were detected affect fertility in livestock species., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

Integrins have been shown to act as signalling receptors, and they primarily recognise extracellular matrix ligands on the oocyte surface. However, their possible roles in oocyte activation and embryo development are not clearly understood. The objectives of this study were to evaluate expression of Integrin Subunit Beta 5 (ITGβ5) in bovine sperm, oocytes, and early embryos and to ascertain the evolutionary conservation of ITGβ5. To accomplish these objectives, we used western blotting to study expression levels of ITGβ5 protein in sperm and RT-qPCR to determine expression levels of ITGβ5 transcripts in oocytes and embryos. We have also used bioinformatic analysis to determine the evolutionary conservation of the ITGβ5 protein among various species. Western blotting showed that ITGβ5 protein was detectable in bull sperm. Moreover, results of RT-qPCR showed that levels of ITGβ5 were significantly higher in the two-cell embryos, followed by the 8-16-cell embryos. However, no significant difference in expression levels were noted for the morula and blastocyst stages as compared to MII oocytes. Bioinformatic analysis revealed that ITGβ5 is conserved among various species. We conclude that expression of ITGβ5 in bovine gametes and embryos implies an important role in fertilisation and embryogenesis., (© 2019 Blackwell Verlag GmbH.)

Bull fertility, ability of the sperm to fertilize and activate the egg and support embryo development, is vital for cattle reproduction and production. Even though majority of histones are replaced by protamines, some histones are retained in sperm. It is known that chromatin remodeling during spermatogenesis results in dynamic changes in sperm chromatin structure through post-translational modifications (PTM) of sperm histones, which are important for regulation of gene expression. However, amounts of sperm Histone 4 (H4), its acetylated form (H4 acetyl), and to what extent these molecular attributes influence sperm chromatin structure and bull fertility are unknown. These gaps in the knowledge base are important because they are preventing advances in the fundamental science of bovine male gamete and improvement of bull fertility. The objective of this study was to test the hypothesis that expression dynamics as well as PTM of sperm H4 are associated with bull fertility. Flow cytometry was utilized to quantify H4 and H4 acetylated form in sperm from seven high and seven low fertility Holstein bulls. The results indicated that the average number of cells with H4 or H4 acetyl expression in high and low fertility bull sperm were 34.6 ± 20.4, 1.88 ± 1.8, 15.2 ± 20.8, and 1.4 ± 1.2, respectively. However, the sperm enriched in both H4 and H4 acetyl were different between high and low fertility groups (3.5 ± 0.6; 1.8 ± 0.8; P = 0.043). The localization and detection of H4 and H4 acetylation were measured by immunocytochemistry which revealed that H4 and H4 acetylation were equally distributed in the sperm head of high and low fertility sires. Western blotting results confirmed the presence of the H4 and its acetylated form in the sperm. Bioinformatics studies demonstrated that H4 is highly conserved among mammalians, and have significant gene ontology on spermatogenesis, early embryo implantation, and sperm capacitation. The results are significant because it demonstrates the replacement of canonical histone H4 into modified H4 acetylation in sperm and regulate its dynamics which is crucial for bull fertility and reproductive biotechnology. These findings advance fundamental science of mammalian early development and reproductive biotechnology.

While the size of the Weimaraner may assist in the breed performing the tasks of a sporting dog, the large size coupled with these tasks may also make the breed more susceptible to orthopedic issues. The understanding of the normal gait mechanics of the Weimaraner can be a useful tool in examining for gait abnormalities associated with these orthopedic issues, and yet, research concerning breed-specific gaits in the canine is limited. Therefore, study objectives were to define the normal Weimaraner trotting kinematics and determine the influence of speed on these parameters. Markers were attached to palpation points on the limbs and head of American Kennel Club registered Weimaraners. Dogs were tracked while performing a slow (1.2-1.7 m/s) and fast (1.9-2.3 m/s) trotting speed. Frame-by-frame analysis was performed. Paw ground contact and lift-off was documented and marker displacement was tracked. At both speeds, the trot had a diagonal footfall sequence with diagonal limb pairing alternating between diagonal bipedal support and suspension. The faster speed was achieved with significant increases in stride length and displacements of the head, withers, and fore and hind paws (P < .05). Range of motion of the elbow and hip significantly increased as the dog transitioned from a slow to fast speed (P < .05). Through gait analysis, the Weimaraner trot was defined as a 2-beat diagonal rhythm gait with suspension. Speed did not change these characteristics, but did influence stride length and linear and angular displacements, and thus, should be a consideration in clinical examination., (Published by Elsevier Inc.)

Male fertility is the ability of sperm to fertilize the egg and sustain embryo development. Several factors determine the fertilizing capacity of mammalian sperm, including those intrinsic to sperm and components of the seminal plasma. The present study analyzed the seminal fluid proteome of Bos taurus and potential associations between proteins and fertility scores. Mass spectrometry coupled with nano HPLC allowed the identification of 1,159 proteins in the dairy bull seminal plasma. There were 50 and 29 seminal proteins more abundant in high (HF) low fertility (LF) bulls, respectively. Based on multivariate analysis, C-type natriuretic peptide, TIMP-2, BSP5 and sulfhydryl oxidase indicated relationship with HF bulls. Clusterin, tissue factor pathway inhibitor 2, galectin-3-binding protein and 5'-nucleotidase were associated with LF bulls. Abundance of NAD(P)(+)-arginine ADP-ribosyltransferase, prosaposin and transmembrane protein 2 proteins had the highest positive correlations with fertility ranking. Quantities of vitamin D-binding protein, nucleotide exchange factor SIL1 and galectin-3-binding protein showed the highest negative correlations with fertility ranking. A fertility ranking score was calculated and the relationship with these proteins was significant (Spearman's rho = 0.94). The present findings represent a major and novel contribution to the study of bovine seminal proteins. Indicators of fertility can be used to improve reproductive biotechnologies.

Molecular components of sperm and in the media surrounding them influence male fertility. In this regard, seminal plasma proteins and metabolites modulate various reproductive events, including sperm motility and capacitation, cell protection, acrosome reaction, fertilization and embryonic development. Empirical associations between seminal proteins and metabolites and fertility indicate that these molecules are potential molecular markers of male reproductive status in cattle and other species.

Metabolites play essential roles in biological systems, but detailed identities and significance of the seminal plasma metabolome related to bull fertility are still unknown. The objectives of this study were to determine the comprehensive metabolome of seminal plasma from Holstein bulls and to ascertain the potential of metabolites as biomarkers of bull fertility. The seminal plasma metabolome from 16 Holstein bulls with two fertility rates were determined by gas chromatography-mass spectrometry (GC-MS). Multivariate and univariate analyses of the data were performed, and the pathways associated with the seminal plasma metabolome were identified using bioinformatics approaches. Sixty-three metabolites were identified in the seminal plasma of all bulls. Fructose was the most abundant metabolite in the seminal fluid, followed for citric acid, lactic acid, urea and phosphoric acid. Androstenedione, 4-ketoglucose, D-xylofuranose, 2-oxoglutaric acid and erythronic acid represented the least predominant metabolites. Partial-Least Squares Discriminant Analysis (PLSDA) revealed a distinct separation between high and low fertility bulls. The metabolites with the greatest Variable Importance in Projection score (VIP > 2) were 2-oxoglutaric acid and fructose. Heat-map analysis, based on VIP score, and univariate analysis indicated that 2-oxoglutaric acid was less (P = 0.02); whereas fructose was greater (P = 0.02) in high fertility than in low fertility bulls. The current study is the first to describe the metabolome of bull seminal plasma using GC-MS and presented metabolites such as 2-oxoglutaric acid and fructose as potential biomarkers of bull fertility.

Epigenetic modifications in histones are crucial for proper sperm physiology, egg activation and reproductive development of males. The objectives of this study were to determine the conservation and interactomes of histone three (H3) and ascertain the expression dynamics of acetylated and methylated H3 lysine 27 (H3K27ac and H3K27me3) in spermatozoa from Holstein bulls with different fertility. Methods in immunocytochemistry and flow cytometry were used to evaluate the expression dynamics of H3K27ac and H3K27me3 in spermatozoa from 10 bulls with different in vivo fertility. Computational biology methods including Clustal Omega and Cytoscape were performed to determine the evolutionary conservation and interactome of H3. The post-translational modifications (PTM) of H3 (H3K27ac and H3K27me3) had different spatiotemporal dynamics in the sperm head. Intensities of methylation were higher than those of acetylation and inversely correlated between the two fertility groups (p = .0032). The interacting proteins of H3 are involved in critical subcellular processes such as regulation of methylation, nucleosome assembly, regulation of DNA replication and chromatin assembly. These results are significant because they help advance fundamental science and biotechnology of mammalian reproduction., (© 2017 Blackwell Verlag GmbH.)

Background: Bull fertility is the degree of sperm's ability to fertilize and activate the egg and support embryo development, and this is critical for herd reproductive performance. We used the bull as a unique model organism for the study of male fertility because cattle genetics and physiology is similar to those of other mammals including humans. Moreover, reliable fertility data along with well-established in vitro systems are available for bovine. The objective of this original study was to ascertain evolutionary diversification and expression dynamics of Testis Specific Histone 2B (TH2B) in sperm from Holstein bulls with different fertility scores., Methods: The intensity of TH2B was determined by using flow cytometry in sperm from 13 high and 13 low fertility bulls. Expression levels of TH2B were measured using immunofluorescence and Western blotting in sperm from five high and five low fertility bulls. Sequence identity, evolutionary distance and interactome of TH2B were evaluated by dotmatcher, STRING and Cytoscape. Data were analyzed using linear mixed effects model and regression plots were drawn., Results: The intensity of TH2B as measured by flow cytometry was significantly affected by an interaction between fertility group and fertility score (P = 0.0182). The intensity of TH2B in sperm from the high fertility group decreased (P = 0.0055) as fertility increased. TH2B was constantly detectable in sperm and expression levels of TH2B decreased in relation to fertility in sperm from the high fertility group (P = 0.018). TH2B biological functions include male gamete generation, chromosome organization, DNA packaging, DNA conformation change, chromatin organization, nucleosome organization, chromatin disassembly, spermatid nucleus elongation, spermatid nucleus differentiation, sperm motility, chromatin organization, chromatin condensation, chromatin silencing, nucleus organization, and chromatin remodeling (P < 0.05)., Conclusions: We elucidated the cellular localization and molecular physiology of TH2B using both computational and cell biology approaches. In addition to advancing the fundamental science of mammalian male gamete, the present findings can be potentially used to evaluate semen quality and predict male fertility in the future., Trial Registration: This study did not involve any live animals. We did not perform any anesthesia, euthanasia, or any kind of animal sacrifice. The cryopreserved semen samples were obtained from Alta Genetics, Inc., Watertown, WI, USA. All samples were preserved in liquid nitrogen.

The present study was aimed at evaluating the seminal plasma proteins and sperm parameters of Curraleiro Pé-Duro bulls. Semen was collected from 10 bulls by electroejaculation, and sperm parameters were evaluated in fresh and frozen-thawed semen. Seminal plasma proteins were analyzed by 2-D SDS-PAGE and mass spectrophotometry. Tools in computational biology were used to generate bioinformatic knowledge and evaluate gene ontology, protein-protein interactions, phylogenetic trees and multiple sequence alignments. Sperm motility in fresh and frozen-thawed semen was 78.8±1.8% and 21.2±1.6%, respectively. Pearson's correlations were evaluated (p<0.05). Sperm motility and vigor in fresh semen were correlated with clusterin, TIMP2 and cathepsin S (r=0.64-0.71) and sperm defects were related to inhibitor of carbonic anhydrase and BSP 5 (r=0.78-0.80). Clusterin, BSP 5, alpha-enolase, creatine kinase M-type, glyceraldehyde-3-phosphate dehydrogenase, BSP 3, albumin, and 5'-nucleotidase and legumain were correlated with acrosome intact live sperm (r=0.80-0.64). Associations were detected between sperm vigor and spermadhesin 1 (r=-0.89), and between sperm defects in fresh semen and spermadhesin 1 and clusterin (r=-0.81). Sperm motility in frozen-thawed semen was associated with BSP 1, spermadhesin 1, clusterin and spermadhesin Z13 (r=0.64-0.85). The percent of motile sperm after freeze-thawing was negatively correlated (r=-0.64) with the amount of spermadhesin 1 in the seminal plasma. Based on in silico analysis, TIMP2 interacted with BSP1, BSP3, BSP5 and metalloproteinases. Molecular functions of proteins associated with sperm parameters were binding, catalytic activity and enzymatic regulation. Amino acid sequences of spermadhesin 1 and BSP 1 from Bos taurus, and other domestic species were similar. Phylogenetic tree analysis demonstrated that clusterin from Bos taurus was related to Ovis aries and domains of clusterin, spermadhesin 1, BSP 1 and inhibitor of carbonic anhydrase were conserved as well. In summary, specific seminal proteins are associated with sperm parameters of locally-adapted bulls. Use of the endangered mammalian as a model may assist in understanding aspects of evolutionary adaptations and could improve assisted reproductive biotechnologies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bull fertility, ability of the sperm to fertilize and activate the egg that sustain embryo development, is vitally important for effective and efficient production of cattle. Fertility is a complex trait with low heritability. Despite recent advances in genomic selection and possibility of enormous paternal benefits to profitable cattle production, there exist no reliable tests for evaluating semen quality and predicting bull fertility. This review focuses on sperm macromolecules such as transcripts, proteins and the epigenome, i.e., the functional genome that are associated with bull fertility. Generating new information in these systems is important beyond agriculture because such progress advances the fundamental science of the mammalian male gamete while at the same time introduces biotechnology into livestock production. Sperm macromolecules and epigenome markers associated with bull fertility can be used alone or in combination with the current SNP microarrays to determine sperm quality and to indicate bull fertility., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Decreasing mammalian fertility and sperm quality have created an urgent need to find effective methods to distinguish non-viable from viable fertilising spermatozoa. The aims of the present study were to evaluate expression levels of ?-tubulin 2C (TUBB2C), heat shock protein 10 (HSP10), hexokinase 1 (HXK1) and superoxide dismutase 1 (SOD1) in spermatozoa from Holstein bulls with varying fertility using western blotting and to analyse the biological networks of these key sperm proteins using a bioinformatics software (Metacore; Thomson-Reuters, Philadelphia, PA, USA). The rationales behind this study were that the sperm proteins play crucial roles in fertilisation and early embryonic development in mammals and ascertaining the biological networks of the proteins helps us better understand sperm physiology and early mammalian development. The results showed that expression of SOD1 was higher in spermatozoa from high fertility bulls (PPin vivo bull fertility. The findings are important because they illuminate molecular and cellular determinants of sperm viability and the identified protein markers can be used to determine bull fertility.