Digital infrared thermal imaging of udder skin surface temperature: a novel non-invasive technology to monitor calving process in Murrah buffalo (Bubalus bubalis).

Quantifiable decline in the maternal body temperature during the pre-calving offers the possibilities for predicting the calving that can improve the calving management. As infrared thermography (IRT) is a simple non-contact tool for precise measurement of surface temperature, we investigated the use of IRT to establish thermal signatures around calving in the Murrah buffalo. The IRT of eye, right lateral, left lateral and rear side of udder skin surface temperature (USST) were recorded at 6 h interval from 96 h before the expected date of calving, at the time of calving and 24 h post-calving in Murrah buffaloes (n = 28). In parallel, blood samples were collected for progesterone (P4) assay. The results revealed that the IRT of the eye, right and left lateral and rear side of USST showed a significant decrease in the temperature from 48 h pre-calving till the onset of calving with a ΔT (°C) of 0.56, 0.91, 0.70, and 0.90, respectively. Mean USST significantly declined from 48 h pre-calving with a ΔT of 0.85 °C. The residual temperature of both eye and various ROI of the udder also followed a similar and significant declining trend from 48 to 0 h of calving indicating that circadian influence on the USST was minimum. Plasma P4 concentration significantly decreased from 72 h pre-calving till calving. It is concluded that a marked reduction in the IRT of the USST at 6-12 h pre-calving would be useful in predicting the onset of calving in the Murrah buffalo.

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.

X chromosome-linked genes in the mature sperm influence semen quality and fertility of breeding bulls.

The role of sperm expressed X-linked genes on bull fertility has not been studied in detail. The objective of the present study was to assess the influence of X-linked genes on the sperm functional parameters and field fertility rate in the Holstein Friesian cattle (n = 12) and Murrah buffalo (n = 7) bulls. The enrichment analysis (cattle = 8; buffalo = 8) of the X-linked genes was carried out using retrospective RNA-seq data and mRNA expression levels of functionally relevant genes were validated using the RT-qPCR. The mRNA expression levels of these genes were functionally associated with sperm attributes and field fertility rate. The sperm transcriptome studies revealed that the total number of expressed genes and the transcript content of the X-linked genes in the mature sperm were very low in both species, and only 23.31% of these genes were commonly expressed between them. The transcript pool corresponding to the X-linked genes represents embryonic organ development (p = 0.03) and reproduction (p = 0.02) processes in cattle and buffalo sperm, respectively. The mRNA expression levels of X-linked genes, RPL10 and ZCCHC13 in cattle; AKAP4, TSPAN6, RPL10 and RPS4X in buffalo were significantly (p < 0.05) correlated with sperm kinematics. Importantly, the mRNA expression levels of the genes RPL10 (r = -0.68) and RPS4X (r = 0.81) had a significant correlation with the field fertility rate in cattle and buffalo, respectively. Multivariate regression models and receiver operating curve analysis suggest that the mRNA expression levels of X-linked genes may be useful in predicting bull fertility. The study indicates that sperm-expressed X-linked genes influence semen quality and field fertility rate in both cattle and buffalo.

Microenvironment of the male and female reproductive tracts regulate sperm fertility: Impact of viscosity, pH, and osmolality.

BACKGROUND: Terminally differentiated mammalian sperm are exposed to gradients of viscosity, pH, and osmolality both in the male and female reproductive tract during their perilous journey to quest the ovum. The complex physicochemical factors play an integral role in preparing sperm for the fertilization process. OBJECTIVES: To elucidate the influence of the reproductive tract microenvironment especially viscosity, pH, and osmolality in regulating sperm functional and fertilization competence. MATERIALS AND METHODS: The data used in this review were collected from the research papers and online databases focusing on the influence of viscosity, pH, and osmolality on sperm function. DISCUSSION: The gradients of viscosity, pH, and osmolality exist across various segments of the male and female reproductive tract. The changes in the viscosity create a physical barrier, pH aid in capacitation and hyperactivation, and the osmotic stress selects a progressive sperm subpopulation for accomplishing fertilization. The sperm function tests are developed based on the concept that the male genotype is the major contributor to the reproductive outcome. However, recent studies demonstrate the significance of sperm genotype-environment interactions that are essentially contributing to reproductive success. Hence, it is imperative to assess the impact of physicochemical stresses and the adaptive ability of the terminally differentiated sperm, which in turn would improve the outcome of the assisted reproductive technologies and male fertility assessment. CONCLUSION: Elucidating the influence of the reproductive tract microenvironment on sperm function provides newer insights into the procedures that need to be adopted for selecting fertile males for breeding, and ejaculates for the assisted reproductive technologies.

Sperm preparedness and adaptation to osmotic and pH stressors relate to functional competence of sperm in Bos taurus.

The adaptive ability of sperm in the female reproductive tract micromilieu signifies the successful fertilization process. The study aimed to analyze the preparedness of sperm to the prevailing osmotic and pH stressors in the female reproductive tract. Fresh bovine sperm were incubated in 290 (isosmotic-control), 355 (hyperosmotic-uterus and oviduct), and 420 (hyperosmotic-control) mOsm/kg and each with pH of 6.8 (uterus) and 7.4 (oviduct). During incubation, the changes in sperm functional attributes were studied. Sperm kinematics and head area decreased significantly (p < 0.05) immediately upon exposure to hyperosmotic stress at both pH. Proportion of sperm capacitated (%) in 355 mOsm/kg at 1 and 2 h of incubation were significantly (p < 0.05) higher than those in 290 mOsm media. The magnitude and duration of recovery of sperm progressive motility in 355 mOsm with pH 7.4 was correlated with the ejaculate rejection rate (R2 = 0.7). Using this information, the bulls were divided into good (n = 5) and poor (n = 5) osmo-adapters. The osmo-responsive genes such as NFAT5, HSP90AB1, SLC9C1, ADAM1B and GAPDH were upregulated (p < 0.05) in the sperm of good osmo-adapters. The study suggests that sperm are prepared for the osmotic and pH challenges in the female reproductive tract and the osmoadaptive ability is associated with ejaculate quality in bulls.

Supraphysiological concentration of urea affects the functional competence of Holstein-Friesian (Bos taurus) sperm.

To understand the effects of urea on sperm functional attributes, fresh bull semen (n = 12) was subjected to four different concentrations (mg/mL) of urea to mimic the physiological (0.04 and 0.13), supraphysiological (0.43) concentrations and control (0 mg/mL). Sperm membrane integrity, kinematics, chromatin integrity, and mitochondrial membrane potential were assessed at different time points (before incubation, 0, 1, 2, and 4 h) of incubation. The concentration of urea in serum and seminal plasma was estimated and correlated with the ejaculate rejection rate and sperm functional attributes. The relative expression of urea transporter gene transcripts (UT-A and UT-B) was assessed in sperm and testis (control) using real-time PCR. The supraphysiological concentration of urea affected sperm kinematics, viability, functional membrane integrity, and acrosome integrity within 1 h of incubation (p < 0.05). Sperm head area decreased (p < 0.05) at 0 h and subsequently increased at 1 h of incubation in all media except supraphysiological (0.43 mg/dL) concentration of urea. Seminal plasma urea concentration showed a significant negative correlation with sperm motility, membrane integrity, and mitochondrial membrane potential (p < 0.05), but had a positive correlation with the ejaculate rejection rate (r = 0.69). Relative expression of the urea transporter genes revealed that UT-A was expressed only in the testis. In contrast, UT-B was expressed in both the testis and sperm, suggesting UT-B's role in regulating urea transport in sperm. At a supraphysiological level, urea adversely affected sperm functional attributes, osmoadaptation and may affect fertility.

Establishment of bioinformatics pipeline for deciphering the biological complexities of fragmented sperm transcriptome.

Despite the development of several tools for the analysis of the transcriptome data, non-availability of a standard pipeline for analyzing the low quality and fragmented mRNA samples pose a major challenge to the computational molecular biologist for effective interpretation of the data. Hence the present study aimed to establish a bioinformatics pipeline for analyzing the biologically fragmented sperm RNA. Sperm transcriptome data (2 x 75 PE sequencing) generated from bulls (n = 8) of high-fertile (n = 4) and low-fertile (n = 4) classified based on the fertility rate (41.52 ± 1.07 vs 36.04 ± 1.04%) were analyzed with different bioinformatics tools for alignment, quantitation, and differential gene expression studies. TopHat2 was effectual compared to HISAT2 and STAR for sperm mRNA due to the higher exonic (6% vs 2%) mapping percentage and quantitating the low expressed genes. TopHat2 also had significantly strong correlation with STAR (0.871, p = 0.05) and HISAT2 (0.933, p = 0.01). TopHat2 and Cufflinks combo quantitated the number of genes higher than the other combinations. Among the tools (Cuffdiff, DESeq, DESeq2, edgeR, and limma) used for the differential gene expression analysis, edgeR and limma identified the largest number of significantly differentially expressed genes (p < 0.05) with biological relevance. The concordance analysis concurred that edgeR had an edge over the other tools. It also identified a higher number (9.5%) of fertility-related genes to be differentially expressed between the two groups. The present study established that TopHat2, Cufflinks, and edgeR as a suitable pipeline for the analysis of fragmented mRNA from bovine spermatozoa.

Orchestrating the expression levels of sperm mRNAs reveals CCDC174 as an important determinant of semen quality and bull fertility.

Bulls with acceptable semen quality vary in actual field fertility and this can be elucidated by studying the expression levels of mRNAs in the sperm. The present study aimed at assessing the variations in the sperm gene expression levels of PRM1, CCDC174, RPL36A, TMCO2, SWI5 and OIT3 in bulls differing in fertility status. Frozen semen samples from Holstein-Friesian bulls were classified into high-fertile (n = 8, average field conception rate = 46.1 ± 0.51, p < 0.001) and sub-fertile (n = 7, average field conception rate = 39.4 ± 0.69) groups. In the post-thaw semen samples, sperm kinematics, structural and functional membrane integrities, mitochondrial membrane potential and chromatin distribution were analyzed. The sperm total RNA was subjected to gene expression studies by Real-Time PCR. Multivariate regression analysis was performed using gene expression levels and conception rates. The sperm functional attributes did not differ significantly between the groups. The relative mRNA levels (fold change) of CCDC174 (6.20), RPL36A (4.66), SWI5 (1.86) and OIT3 (1.53) were higher in high-fertile bulls. Further, the expression level of the CCDC174 gene was significantly (p = 0.02) up-regulated in high-fertile bulls. The fertility prediction multivariate model with genes, CCDC174, RPL36A, TMCO2 and OIT3 had the maximum coefficient of determination (R2 = 0.68) with the field conception rate. This model had 93.3% bull fertility prediction accuracy with 100% sensitivity and 87.5% specificity. The study suggests that the expression level of CCDC174 can be used as a potential marker for assessing bull fertility.