Cryostress induces fragmentation and alters the abundance of sperm transcripts associated with fertilizing competence and reproductive processes in buffalo.

The study aimed to assess the influence of cryostress on RNA integrity and functional significance in sperm fertilizing ability. The fresh and post-thawed buffalo sperm (n = 6 each) samples were evaluated for their functional attributes, and sperm total RNA was subjected to transcriptome sequencing followed by validation using real-time PCR and dot blot. Overall, 6911 genes had an expression of FPKM > 1, and among these 431 genes were abundantly expressed (FPKM > 20) in buffalo sperm. These abundantly expressed genes regulate reproductive functions such as sperm motility (TEKT2, SPEM1, and PRM3, FDR = 1.10E-08), fertilization (EQTN, PLCZ1, and SPESP1, FDR = 7.25E-06) and the developmental process involved in reproduction (SPACA1, TNP1, and YBX2, FDR = 7.21E-06). Cryopreservation significantly (p < 0.05) affected the structural and functional membrane integrities of sperm. The expression levels of transcripts that regulate the metabolic activities and fertility-related functions were compromised during cryopreservation. Interestingly, cryostress induces the expression of genes involved (p < 0.05) in chemokine signaling (CX3CL1, CCL20, and CXCR4), G-protein coupled receptor binding (ADRB1, EDN1, and BRS3), translation (RPS28, MRPL28, and RPL18A), oxidative phosphorylation (ND1, ND2, and COX2), response to reactive oxygen species (GLRX2, HYAL2, and EDN1), and immune responses (CX3CL1, CCL26, and TBXA2R). These precociously expressed genes during cryopreservation alter the signaling mechanisms that govern sperm functional competence and can impact fertilization and early embryonic development.

Low-density lipoproteins protect sperm during cryopreservation in buffalo: Unraveling mechanism of action.

This study was carried out to reveal factors and the mechanism of action by which low-density lipoproteins (LDLs) protect sperm better than egg yolk (EY) during cryopreservation. We extracted LDL from EY and compared the amount of calcium, progesterone, and antioxidants in EY and LDL. We found a very high concentration of progesterone (1423.95 vs. 10.46 ng/ml) and calcium (29.19 vs. 0.47 mM) in EY as compared with LDL. Antioxidant assays like DPPH (2,2-diphenyl-1-picrylhydrazyl) and the ferric reducing antioxidants power assay revealed that the LDL extender had almost double ability to lose hydrogen than the EY extender. For sperm cryopreservation, 20 ejaculates from four Murrah buffalo bulls were collected. Each ejaculate was divided into four aliquots and extended in 10%, 12%, and 14% LDL (w/v) and EY-based extenders, followed by cryopreservation. The LDL-based extender prevented excessive cholesterol efflux, and its high content of antioxidants minimized reactive oxygen species generated during cryopreservation, resulting in a functional CatSper channel. The EY-based extender promoted excess cholesterol efflux due to the presence of high-density lipoprotein, resulting in a compromised CatSper channel. High intracellular calcium in a cryopreserved sperm in the EY group as compared with the LDL group indicates that progesterone present in EY activates the CatSper channel, resulting in a heavy calcium influx into the sperm. The greater tyrosine phosphorylation and increased number of F-pattern in the sperm cryopreserved in the EY extender indicate that high intracellular calcium triggers more capacitation-like changes in the sperm cryopreserved in EY than LDL extender. In conclusion, we demonstrated the new facts and understandings about LDL and EY for semen cryopreservation.

Inferring Relationship of Blood Metabolic Changes and Average Daily Gain With Feed Conversion Efficiency in Murrah Heifers: Machine Learning Approach.

Machine learning algorithms were employed for predicting the feed conversion efficiency (FCE), using the blood parameters and average daily gain (ADG) as predictor variables in buffalo heifers. It was observed that isotonic regression outperformed other machine learning algorithms used in study. Further, we also achieved the best performance evaluation metrics model with additive regression as the meta learner and isotonic regression as the base learner on 10-fold cross-validation and leaving-one-out cross-validation tests. Further, we created three separate partial least square regression (PLSR) models using all 14 parameters of blood and ADG as independent (explanatory) variables and FCE as the dependent variable, to understand the interactions of blood parameters, ADG with FCE each by inclusion of all FCE values (i), only higher FCE values (negative RFI) (ii), and inclusion of only lower FCE (positive RFI) values (iii). The PLSR model including only the higher FCE values was concluded the best, based on performance evaluation metrics as compared to PLSR models developed by inclusion of the lower FCE values and all types of FCE values. IGF1 and its interactions with the other blood parameters were found highly influential for higher FCE measures. The strength of the estimated interaction effects of the blood parameter in relation to FCE may facilitate understanding of intricate dynamics of blood parameters for growth.

A new role for RU486 (mifepristone): it protects sperm from premature capacitation during cryopreservation in buffalo.

The objective of this study was to determine the mechanism by which RU 486 (mifepristone) protects sperm to undergo premature capacitation during cryopreservation. For this, semen ejaculate (n = 20) was divided into four equal fractions and diluted using egg yolk-based extender supplemented with different concentrations of RU 486 (0, 5, 10 and 20 µM) and cryopreserved. We found that RU 486 did not impair the post-thaw sperm kinetics and motility but prevented cholesterol efflux, calcium influx, and protected CatSper channels during cryopreservation. The RU 486 protected sperm from premature capacitation which was confirmed by intracellular calcium level, expression of tyrosine phosphorylated proteins (75 and 80 kDa) and CTC (chlortetracycline) assay. Furthermore, antioxidant ability of RU 486 was reflected by the ferric reducing ability, lower production of sperm malondialdehyde and intracellular reactive oxygen species. Also, we demonstrated that RU 486 treated sperm underwent normal capacitation, zona pellucida binding and zygote cleavage indicating normal fertilizing ability of sperm. In conclusion, we report a new role of RU 486 in protecting buffalo sperm from premature capacitation during cryopreservation.

Deciphering foot-and-mouth disease (FMD) virus-host tropism.

The pattern of interactions between foot and mouth disease (FMD) viral protein 1 (VP1) with susceptible and resistant host integrins were deciphered. The putative effect of site-directed mutation on alteration of interaction is illustrated using predicted and validated 3D structures of VP1, mutated VP1 and integrins of Bos taurus, Gallus and Canis. Strong interactions were observed between FMDV-VP1 protein motifs at conserved tripeptide, Arg-Gly-Asp 143RGD145 and at domain 676SIPLQ680 in alpha-integrin of B. taurus. Notably, in-silico site-directed mutation in FMDV-VP1 protein led to complete loss of interaction between FMD-VP1 protein and B. taurus integrin, which confirmed the active role of arginine-glycine-aspartic acid (RGD) domain. Interestingly, in-vitro analysis demonstrates the persistence of the putative tropism site 'SIPLQ' in different cattle breeds undertaken. Thus, the attempt to decipher the tropism of FMDV at host receptor level interaction might be useful for future FMD control strategies through development of mimetic marker vaccines and/or host receptor manipulations. Communicated by Ramaswamy H. Sarma.

Structural Modeling and Analysis of Pregnancy-Associated Glycoprotein-1 of Buffalo (Bubalus bubalis).

The present study was conducted to design and analyze the structural model of buffalo pregnancy-associated glycoprotein-1 (PAG-1) using bioinformatics. Structural modeling of the deduced buffalo PAG-1 protein was done using PHYRE, CONSURF servers and its structure was subsequently constructed using MODELLER 9.9 and PyMOL softwares Buffalo PAG-1 structural conformity was analyzed using PROSA, WHATIF, and 3D-PSSM servers. Designed buffalo PAG-1 protein structure on BLAST analysis retrieved protein structures belonging to aspartic proteinase family. Moreover in silico analysis revealed buffalo PAG-1 protein retained bilobed structure with pepstatin-binding clefts near the active sites by docking studies with pepstatin A using PatchDock server. Structural studies revealed that the amino and carboxy terminal containing aspartic residues are highly conserved and buried within the protein structure. Structural conformity studies showed that more than 90% of the residues lie inside favored and allowed regions. It was also deduced that buffalo PAG-1 possesses low and high energy zones with a very low threshold for proteolysis ascertaining the stableness of the buffalo PAG-1 protein structure. This study depicts the structural conformity and stability of buffalo PAG-1 protein.