| Project ID | IMP_100017 |
| Title | Abundance of specific lipid molecules in sperm plasma membrane regulates fertility in zebu cattle bulls |
| Uploaded by | Fanny |
| Research Area | Animal Sciences |
| Funding Source | National Dairy Research Institute |
| Contributors | Dr. Rakesh Kumar |
| Description | Spermatozoa are remarkable cells comprising a specialized plasma membrane (PM) that renders the functional capability for successful fertilization. As the PM contains complex lipids, including phospholipids, glycolipids, and sterols, any subtle alterations in these lipids disturb cellular homeostasis, leading to defective fertilizing ability. The study aims to profile lipids in the PM of spermatozoa from zebu cattle bulls. In this study, LC-MS/MS analysis, fluorescence microscopy, flow cytometry, and CASA were performed to identify and correlate the PM lipid molecules to bulls with contrasting fertility. We identified a total of 2074 lipid molecules in zebu cattle bulls. From the phospholipid class, molecular species of phosphatidylethanoleamine were most abundant in the HF group, followed by phosphatidyglycerol and lysophosphatidylethanoleamin. In the LF group, phosphatidylcholine, phosphatidylinositol, phosphatidic acid, and phosphatidylserine were the majorly identified lipid species. Glycolipids disialoganglioside and glucosylceramide were also differentially abundant between the two fertility groups. On the assessment of sperm viability, acrosome integrity, cryo-induced capacitation, lipid peroxidation, and reactive oxygen species levels, a significant difference (p<0.05) was observed exhibiting better functionality in the HF group. Furthermore, computer assisted semen analysis revealed significant differences in various sperm kinematic parameters between distinct fertility groups. In this study, we identified differentially abundant lipid molecules (DALMs) in HF and LF bull spermatozoa with their potential role in the regulation of various sperm functional parametrs crucial to male fertility. |
| Study ID | IMS_100013 |
| Title | Abundance of specific lipid molecules in sperm plasma membrane regulates fertility in zebu cattle bulls |
| Uploaded by | Fanny |
| Study Type | Mass Spectrometry (MS) |
| Data Type | Untargeted |
| Release Date | Dec. 12, 2024 |
| IEC/IBSC No. | |
| Publication | |
| Summary | In this study, 31 zebu cattle bulls with more than 50 artificial insemination field fertility records were categorized into high-fertility (HF) and low-fertility (LF) groups based on their conception rates.The 31 bulls included in this study had their conception rates (CRs) tested for normal distribution using the Shapiro-Wilk normality test. The bulls with CRs lying between 52.36% and 36.60 % were considered as average fertile. Ten bulls, five each in high-fertility (HF, n = 5, CR between 52.94% and 59.59%) and low-fertility (LF, n = 5, CR between 26.92% and 33.33%) chosen for the study had their CRs above. The semen straws from both HF (n=5) and LF (n=5) bulls were thawed by submerging them in a water bath for 30 s, and the extender was removed completely after three wash using 1X Sp-TALP by centrifuging for 10 min in Sp-TALP. Aproximately 4x108 spermatozoa/ml were taken for sperm plasma membrane (PM) isolation, and then the sample was transferred to an ice-cold Dounce homogenizer.Homogenization was done after adding Working Buffer Solution (WBS). The samples were sonicated using a probe sonicator. Afterward, centrifugation was done. The supernatant, i.e., PNS (post-nuclear fraction) was re-sonicated.One ml of the sample was placed on the bottom of a clean, fresh, pre-cooled ultracentrifuge tube after mixing it with gradient working solution (GWS) provided with the PM isolation kit. On top of it, gradient solution was layered. The tubes were then ultracentrifuged. The PM fractions were then collected and stored at -80 °C in a glass tube.For lipid extraction, Bligh and Dyer method was used with certain modifications.The organic layer was removed carefully and transferred to a fresh glass vial. This stored organic layer was used for lipidomic analysis.All LC/MS experiments were carried out using the DionexUltiMate 3000 UHPLC (Thermo Scientific, MA) system equipped with a binary solvent delivery manager and a sample manager, coupled with a Thermo Q-EXACTIVE PLUS operating in positive (ESI +) and negative (ESI−) electrospray ionization mode (Thermo Fisher Scientific, Sunnyvale, CA, USA), separately.The injection volume was 20 μl, and the analysis time was 15 min. The mass spectrometric data were collected using a Thermo Q-EXACTIVE PLUS mass spectrometer.All the samples were processed, and RAW files generated were analyzed using LipidSearch 4.2.13(Thermo Fisher Scientific Inc) to identify the lipid molecular species within each lipid fraction. Both positive and negative mode analysis was performed separately.Statistical analysis was performed on redundant list of lipid molecules. Intensity values were log2 transformed with normalized z-score. |
| # | Sample ID | Sample Name | Organism | Source | Sample Type | Experimental Condition | Gender | Age | Replicates | Storage | Files/Sample |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | IMSM_100843 | SW_HF1_N | Bos indicus | Spermatozoa | Animal Sample(01) | Abundance of specific lipid molecules in sperm plasma membrane. | NA | NA | NA | -80oC | |
| 2 | IMSM_100844 | SW_HF1_P | Bos indicus | Spermatozoa | Animal Sample(02) | Abundance of specific lipid molecules in sperm plasma membrane. | NA | NA | NA | -80oC | |
| 3 | IMSM_100845 | SW_HF2_N | Bos indicus | Spermatozoa | Animal Sample(03) | Abundance of specific lipid molecules in sperm plasma membrane. | NA | NA | NA | -80oC | |
| 4 | IMSM_100846 | SW_HF2_P | Bos indicus | Spermatozoa | Animal Sample(04) | Abundance of specific lipid molecules in sperm plasma membrane. | NA | NA | NA | -80oC | |
| 5 | IMSM_100847 | SW_HF3_N | Bos indicus | Spermatozoa | Animal Sample(05) | Abundance of specific lipid molecules in sperm plasma membrane. | NA | NA | NA | -80oC | |
| 6 | IMSM_100848 | SW_HF3_P | Bos indicus | Spermatozoa | Animal Sample(06) | Abundance of specific lipid molecules in sperm plasma membrane. | NA | NA | NA | -80oC | |
| 7 | IMSM_100849 | SW_LF1_N | Bos indicus | Spermatozoa | Animal Sample(07) | Abundance of specific lipid molecules in sperm plasma membrane. | NA | NA | NA | -80oC | |
| 8 | IMSM_100850 | SW_LF1_P | Bos indicus | Spermatozoa | Animal Sample(08) | Abundance of specific lipid molecules in sperm plasma membrane. | NA | NA | NA | -80oC | |
| 9 | IMSM_100851 | SW_LF2_N | Bos indicus | Spermatozoa | Animal Sample(09) | Abundance of specific lipid molecules in sperm plasma membrane. | NA | NA | NA | -80oC | |
| 10 | IMSM_100852 | SW_LF2_P | Bos indicus | Spermatozoa | Animal Sample(10) | Abundance of specific lipid molecules in sperm plasma membrane. | NA | NA | NA | -80oC | |
| 11 | IMSM_100853 | SW_LF3_N | Bos indicus | Spermatozoa | Animal Sample(11) | Abundance of specific lipid molecules in sperm plasma membrane. | NA | NA | NA | -80oC | |
| 12 | IMSM_100854 | SW_LF3_P | Bos indicus | Spermatozoa | Animal Sample(12) | Abundance of specific lipid molecules in sperm plasma membrane. | NA | NA | NA | -80oC |
| # | MS Exp ID | Sample Name / ID | MS Type | Instrument Name | Instrument Type | Ionization Method | Ion Mode | Software Used |
|---|---|---|---|---|---|---|---|---|
| 1 | IME_100492 | SW_HF1_P / IMSM_100844 | LCMS (Liquid Chromatography- Mass Spectrometry) | Others | Orbitrap | Others | Positive | Compound Discoverer |
| 2 | IME_100493 | SW_HF1_N / IMSM_100843 | LCMS (Liquid Chromatography- Mass Spectrometry) | Others | Orbitrap | Others | Negative | Compound Discoverer |
| 3 | IME_100494 | SW_HF2_P / IMSM_100846 | LCMS (Liquid Chromatography- Mass Spectrometry) | Others | Orbitrap | Others | Positive | Compound Discoverer |
| 4 | IME_100495 | SW_HF2_N / IMSM_100845 | LCMS (Liquid Chromatography- Mass Spectrometry) | Others | Orbitrap | Others | Negative | Compound Discoverer |
| 5 | IME_100496 | SW_HF3_P / IMSM_100848 | LCMS (Liquid Chromatography- Mass Spectrometry) | Others | Orbitrap | Others | Positive | Compound Discoverer |
| 6 | IME_100497 | SW_HF3_N / IMSM_100847 | LCMS (Liquid Chromatography- Mass Spectrometry) | Others | Orbitrap | Others | Negative | Compound Discoverer |
| 7 | IME_100498 | SW_LF1_P / IMSM_100850 | LCMS (Liquid Chromatography- Mass Spectrometry) | Others | Orbitrap | Others | Positive | Compound Discoverer |
| 8 | IME_100499 | SW_LF1_N / IMSM_100849 | LCMS (Liquid Chromatography- Mass Spectrometry) | Others | Orbitrap | Others | Negative | Compound Discoverer |
| 9 | IME_100500 | SW_LF2_P / IMSM_100852 | LCMS (Liquid Chromatography- Mass Spectrometry) | Others | Orbitrap | Others | Positive | Compound Discoverer |
| 10 | IME_100501 | SW_LF2_N / IMSM_100851 | LCMS (Liquid Chromatography- Mass Spectrometry) | Others | Orbitrap | Others | Negative | Compound Discoverer |
| 11 | IME_100503 | SW_LF3_N / IMSM_100853 | LCMS (Liquid Chromatography- Mass Spectrometry) | Others | Orbitrap | Others | Negative | Compound Discoverer |
| 12 | IME_100502 | SW_LF3_P / IMSM_100854 | LCMS (Liquid Chromatography- Mass Spectrometry) | Others | Orbitrap | Others | Positive | Compound Discoverer |
| # | First Name | Last Name | Organization | Designation | |
|---|---|---|---|---|---|
| 1 | Rakesh | Kumar | rakeshcift@gmail.com | National Dairy Research Institute | scientist |
| # | FTP Run ID | MS Exp ID | MS Data File |
|---|---|---|---|
| 1 | IMR_101091 | IME_100492 | SW_HF1_P.raw |
| 2 | IMR_101092 | IME_100493 | SW_HF1_N.raw |
| 3 | IMR_101093 | IME_100494 | SW_HF2_P.raw |
| 4 | IMR_101094 | IME_100495 | SW_HF2_N.raw |
| 5 | IMR_101095 | IME_100496 | SW_HF3_P.raw |
| 6 | IMR_101096 | IME_100497 | SW_HF3_N.raw |
| 7 | IMR_101097 | IME_100498 | SW_LF1_P.raw |
| 8 | IMR_101098 | IME_100499 | SW_LF1_N.raw |
| 9 | IMR_101099 | IME_100500 | SW_LF2_P.raw |
| 10 | IMR_101100 | IME_100501 | SW_LF2_N.raw |
| 11 | IMR_101101 | IME_100502 | SW_LF3_P.raw |
| 12 | IMR_101102 | IME_100503 | SW_LF3_N.raw |