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Deciphering sperm transcriptome is the key to understanding the molecular mechanisms governing peri-fertilization, embryonic development, and pregnancy establishment. This study aimed to profile sperm transcriptome to identify signature transcripts regulating male fertility. Semen samples were collected from 47 bulls with varied fertility rates. The sperm total RNA was isolated (n = 8) and subjected to transcriptome sequencing. Based on the expression pattern obtained from RNA profiling, the bulls were grouped (p = 0.03) into high-fertile and sub-fertile, and signature transcripts controlling sperm functions and fertility were identified. The results were validated using the OMIM database, qPCR, and sperm function tests. The sperm contains 1100 to 1700 intact transcripts, of which BCL2L11 and CAPZA3 were abundant and associated (p < 0.05) with spermatogenesis and post-embryonic organ morphogenesis. The upregulated genes in the acrosome integrity and functional membrane integrity groups had a close association with the fertility rate. The biological functions of these upregulated genes (p < 0.05) in the high-fertile bulls were associated with spermatogenesis (AFF4 and BRIP1), sperm motility (AK6 and ATP6V1G3), capacitation and zona binding (AGFG1), embryo development (TCF7 and AKIRIN2), and placental development (KRT19). The transcripts involved in pathways regulating embryonic development such as translation (EEF1B2 and MTIF3, p = 8.87E-05) and nonsense-mediated decay (RPL23 and RPL7A, p = 5.01E-27) were upregulated in high-fertile bulls. The identified transcripts may significantly impact oocyte function, embryogenesis, trophectoderm development, and pregnancy establishment. In addition, the study also reveals that the genes governing sperm functional membrane integrity and acrosome integrity have a prospective effect on male fertility., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

With artificial insemination (AI) and other precision dependent assisted reproductive technologies (ART) being followed in large scale in human and animal reproduction, assessing semen quality and fertilizability is under continuous scrutiny. Various tests have been developed to predict semen quality, but so far no single, highly reliable test is available. In this regard, transcriptomic profiling of spermatozoa assumes significance as it carries the information about spermatogenesis, sperm function, and paternal roles in post-fertilization events. Human spermatozoal transcriptome profiling has been carried out on a large number of individuals to predict the semen quality. A study in human indicated that the outcome of some idiopathic couples seeking reproductive care could be helped using transcriptomic profiling of spermatozoa. Such studies have a direct impact on the bovine dairy industry, wherein AI is practiced. Limited studies in bovine spermatozoal transcriptome profiling have revealed that the spermatozoa contain various classes of RNA, like in human. Approximately 13,000 bovine genes yield a series of spermatozoal transcripts, of which most are fragmented in nature. Their abundance is indicative of the timing of events associated with spermatogenesis, e.g., PRM1, IGF1, BMP2; sperm function, TSSK6, CRISP, HSFY2; fertility, UBE2D3, Integrin-β, LDC-1; and embryonic development, miR34c-5p, BCL2L11, BRCA1. The most abundant translated bovine transcripts are BSP3 and SPATA18, and are involved in regulation of germ cell development and the maintenance of chromatin integrity during spermatogenesis respectively. The presence of transcripts associated with placental development, e.g., placental associated glycoproteins (PAGs) have suggested their possible influence beyond early embryonic development. Changes in transcript levels like RPL31 and PRKCE that increase, and PRM1 that decreases, during cryopreservation need to be defined in order to optimize cryopreservation and fertility yield. Spermatozoal transcriptome profiling with validation studies are warranted in large numbers of animals to elucidate their significance for selecting fertile bulls for the breeding program. Abbreviations: AI: artificial insemination; BSE: breeding soundness evaluation; cfs-mRNA: cell-free seminal mRNA; piRNA: PIWI-interacting RNA; tRNA: transfer RNA; fg: femtogram; TPM: transcripts per million reads; RPKM: reads per kilobase million; rRNA: ribosomal RNA; mt-RNA: mitochondrial RNA; lncRNA: long non-coding RNA; sncRNA: small noncoding RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; miRNA: microRNA; snaR: small NF90-associated RNAs; SINES: short interspersed nuclear elements; LINES: long interspersed nuclear elements; MER: medium reiterated sequence; F1 offspring: filial 1 offspring; PAGs: placental associated glycoproteins; TCP: Transcription factor T complex protein; BSP3: bovine seminal plasma protein 3; SCNT: somatic cell nuclear transfer; qPCR: quantitative (real-time) polymerase chain reaction; SSH: suppression subtractive hybridization; SNP: single nucleotide polymorphism; 2-DE: 2 dimensional gel electrophoresis; LC-MS/MS: liquid chromatography-tandem mass spectrometry.

Mammalian spermatozoa deliver various classes of RNAs to the oocyte during fertilization, and many of them may regulate fertility. The objective of the present study was to determine the composition and abundance of spermatozoal transcripts in fresh bull semen. The entire transcriptome of the spermatozoa from bulls (n = 3) was sequenced using two different platforms (Ion Proton and Illumina) to identify the maximum number of genes present in the spermatozoa. The bovine spermatozoa contained transcripts for 13,833 genes (transcripts per million, TPM > 10). Both intact and fragmented transcripts were found. These spermatozoal transcripts were associated with various stages of spermatogenesis, spermatozoal function, fertilization, and embryo development. The presence of intact transcripts of pregnancy-associated glycoproteins (PAGs) in the spermatozoa suggest a possible influence of sperm transcripts beyond early embryonic development. The specific regions (exon, intron, and exon-intron) of the particular spermatozoal transcripts might help regulate fertilization. This study demonstrates that the use of two different RNA-seq platforms provides a comprehensive profile of bovine spermatozoal RNA. Spermatozoal RNA profiling may be useful as a non-invasive method to delineate possible causes of male infertility and to predict fertility in a manner that is more effective than the conventional methods.

Sperm RNA can be used to understand the past spermatogenic process, future successful fertilization, and embryo development. To study the sperm RNA composition and function, isolation of good quality RNA with sufficient quantity is essential. The objective of this study was to assess the influence of sperm input concentrations and RNA isolation methods on RNA yield and quality in bull sperm. The fresh semen samples from bulls (n = 6) were snap-frozen in liquid nitrogen and stored at -80 °C. The sperm RNA was isolated using membrane-based methods combined with TRIzol (RNeasy+TRIzol and PureLink+TRIzol) and conventional methods (TRIzol, Double TRIzol, and RNAzol RT). Based on fluorometric quantification, combined methods resulted in significantly (P < 0.05) higher total RNA yields (800-900 ng/30-40 × 10(6)) as compared with other methods and yielded 20 to 30 fg of RNA/spermatozoon. The quality of RNA isolated by membrane-based methods was superior to that isolated by conventional methods. The sperm RNA was observed to be intact as well as fragmented (50-2000 bp). The study revealed that the membrane-based methods with a cocktail of lysis solution and an optimal input concentration of 30 to 40 million sperm were optimal for maximum recovery of RNA from bull spermatozoa., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The current study aimed to assess the effect of different seasons (summer, monsoon and winter) on the quality, preservability and fertilizing ability of spermatozoa in Bannur rams. The level of heat stress was significantly greater during the summer than monsoon or winter seasons. Spermatozoa concentration (×106/ml) in semen was found significantly (P < 0.05) lesser during the summer (2446 ± 80) as compared to monsoon (2759 ± 56) or winter (2769 ± 56). The progressive motility (%) of spermatozoa at the 72 h post-preservation was significantly (P < 0.01) lesser during the summer (47.5 ± 2.5) than monsoon (62.5 ± 2.5) and winter (57.5 ± 2.5). Significantly (P < 0.01 or P < 0.05) lesser liveability and acrosomal integrity, but greater total morphological abnormalities of spermatozoa were recorded at all the preservation time points during the summer as compared to monsoon or winter. The cleavage rate (%) was found significantly (P < 0.01) greater during the monsoon (56.6 ± 2.24) as compared to summer (41.7 ± 1.26) and winter (41.4 ± 0.99). The results indicate a definite seasonal pattern in the quality, preservability and fertilizing ability of spermatozoa in Bannur rams, which were found superior during the monsoon. [ABSTRACT FROM AUTHOR]