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Summary: The association between sperm morphology characteristics and DNA conformation and integrity is still controversial. In bulls, major morphological sperm abnormalities have been associated with reduced fertility, and morphological assessment is used to provide an indication of potential fertility of the individual. Sperm DNA fragmentation and damage has a negative effect on embryo development and subsequently fertility, with bull spermatozoa generally displaying low levels of DNA damage and tight chromatin. However, sensitive methods for detecting chromatin damage may reveal associations with morphological defects. The objective was to determine whether morphological sperm abnormalities and variables expressing sperm DNA integrity and protamination are correlated in bulls, using the sperm chromatin structure assay (SCSA) and the sperm protamine deficiency assay (SPDA). Electroejaculated samples (n = 1009) from two‐year‐old tropically adapted bulls were split and fixed and submitted to microscopic sperm morphology assessment, and snap‐frozen for sperm nuclear integrity assessments by SPDA and SCSA. For SPDA, the variables were defective (MCB) and deprotaminated (HCB), and for SCSA, the variables were DNA fragmentation index (DFI) and high DNA stainability (HDS). HCB correlated with DFI; τKen2 = 0.317 and HDS; 0.098, and MCB correlated with DFI; 0.183 (p < 0.001). The percentage of morphological normal spermatozoa was correlated negatively to DFI; τKen2 = −0.168, MCB; −0.116 and HCB; −0.137 (p < 0.001). HCB and DFI were both positively correlated to head defects, proximal droplets, and spermatogenic immaturity, but not to distal droplets, vacuoles, or diadems. Sperm DNA integrity and protamination, using the SCSA and SPDA, respectively, in bulls show associations with morphological parameters, particularly with head shape abnormalities and indicators of spermatogenic immaturity, including proximal droplets. The vacuoles and diadem defects were not correlated with sperm nuclear integrity, and hence, these are likely physiological features that may not directly affect sperm chromatin configuration. [ABSTRACT FROM AUTHOR]

Summary: Progesterone signaling and uterine function are crucial in terms of pregnancy establishment. To investigate how the uterine tissue and its secretion changes in relation to puberty, we sampled tissue and uterine fluid from six pre‐ and six post‐pubertal Brahman heifers. Post‐pubertal heifers were sampled in the luteal phase. Gene expression of the uterine tissue was investigated with RNA‐sequencing, whereas the uterine fluid was used for protein profiling with mass spectrometry. A total of 4034 genes were differentially expressed (DE) at a nominal P‐value of 0.05, and 26 genes were significantly DE after Bonferroni correction (P < 3.1 × 10−6). We also identified 79 proteins (out of 230 proteins) that were DE (P < 1 × 10−5) in the uterine fluid. When we compared proteomics and transcriptome results, four DE proteins were identified as being encoded by DE genes: OVGP1, GRP, CAP1 and HBA. Except for CAP1, the other three had lower expression post‐puberty. The function of these four genes hypothetically related to preparation of the uterus for a potential pregnancy is discussed in the context of puberty. All DE genes and proteins were also used in pathway and ontology enrichment analyses to investigate overall function. The DE genes were enriched for terms related to ribosomal activity. Transcription factors that were deemed key regulators of DE genes are also reported. Transcription factors ZNF567,ZNF775,RELA,PIAS2,LHX4,SOX2,MEF2C,ZNF354C,HMG20A,TCF7L2,ZNF420,HIC1,GTF3A and two novel genes had the highest regulatory impact factor scores. These data can help to understand how puberty influences uterine function. [ABSTRACT FROM AUTHOR]

Contents: Increasing use of fixed‐time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN2) in a Tris‐egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post‐thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen. [ABSTRACT FROM AUTHOR]

Puberty onset is a developmental process influenced by genetic determinants, environment, and nutrition. Mutations and regulatory gene networks constitute the molecular basis for the genetic determinants of puberty onset. The emerging knowledge of these genetic determinants presents opportunities for innovation in the breeding of early pubertal cattle. This paper presents new data on hypothalamic gene expression related to puberty in Bos indicus (Brahman) in age- and weight-matched heifers. Six postpubertal heifers were compared with 6 prepubertal heifers using whole-genome RNA sequencing methodology for quantification of global gene expression in the hypothalamus. Five transcription factors (TF) with potential regulatory roles in the hypothalamus were identified in this experiment: E2F8, NFAT5, SIX5, ZBTB38, and ZNF605. These TF genes were significantly differentially expressed in the hypothalamus of postpubertal versus prepubertal heifers and were also identified as significant according to the applied regulatory impact factor metric (P < 0.05). Two of these 5 TF, ZBTB38 and ZNF605, were zinc fingers, belonging to a gene family previously reported to have a central regulatory role in mammalian puberty. The SIX5 gene belongs to the family of homologues of Drosophila sine oculis (SIX) genes implicated in transcriptional regulation of gonadotrope gene expression. Tumor-related genes such as E2F8 and NFAT5 are known to affect basic cellular processes that are relevant in both cancer and developmental processes. Mutations in NFAT5 were associated with puberty in humans. Mutations in these TF, together with other genetic determinants previously discovered, could be used in genomic selection to predict the genetic merit of cattle (i.e., the likelihood of the offspring presenting earlier than average puberty for Brahman). Knowledge of key mutations involved in genetic traits is an advantage for genomic prediction because it can increase its accuracy. [ABSTRACT FROM AUTHOR]

The primary purpose of spermatozoa is to deliver the paternal DNA to the oocyte at fertilization. During the complex events of fertilization, if the spermatozoon penetrating the oocyte contains compromised or damaged sperm chromatin, the subsequent progression of embryogenesis and foetal development may be affected. Variation in sperm DNA damage and protamine content in ejaculated spermatozoa was reported in the cattle, with potential consequences to bull fertility. Protamines are sperm-specific nuclear proteins that are essential to packaging of the condensed paternal genome in spermatozoa. Sperm DNA damage is thought to be repaired during the process of protamination. This study investigates the potential correlation between sperm protamine content, sperm DNA damage and the subsequent relationships between sperm chromatin and commonly measured reproductive phenotypes. Bos indicus sperm samples ( n = 133) were assessed by two flow cytometric methods: the sperm chromatin structure assay ( SCSA) and an optimized sperm protamine deficiency assay ( SPDA). To verify the SPDA assay for bovine sperm protamine content, samples collected from testis, caput and cauda epididymidis were analyzed. As expected, mature spermatozoa in the cauda epididymidis had higher protamine content when compared with sperm samples from testis and caput epididymidis ( p < 0.01). The DNA fragmentation index ( DFI), determined by SCSA, was positively correlated ( r = 0.33 ± 0.08, p < 0.05) with the percentage of spermatozoa that showed low protamine content using SPDA. Also, DFI was negatively correlated ( r = −0.21 ± 0.09, p < 0.05) with the percentage of spermatozoa with high protamine content. Larger scrotal circumference contributes to higher sperm protamine content and lower content of sperm DNA damage ( p < 0.05). In conclusion, sperm protamine content and sperm DNA damage are closely associated. Protamine deficiency is likely to be one of the contributing factors to DNA instability and damage, which can affect bull fertility. [ABSTRACT FROM AUTHOR]

Harsh tropical environments impose serious challenges on poorly adapted species. In beef cattle, tropical adaptation in the form of temperature and disease resistance, coupled with acclimatization to seasonal and limited forage, comes at a cost to production efficiency. Prominent among these costs is delayed onset of puberty, a challenging phenotype to manipulate through traditional breeding mechanisms. Recently, system biology approaches, including gene networks, have been applied to the genetic dissection of complex phenotypes. We aimed at developing and studying gene networks underlying cattle puberty. Our starting material comprises the association results of ~50,000 SNP on 22 traits, including age at puberty, and 2 cattle breed populations: Brahman (n = 843) and Tropical Composite (n = 866). We defined age at puberty as the age at first corpus luteum (AGECL). By capturing the genes harboring mutations minimally associated (P < 0.05) to AGECL or to a set of traits related with AGECL, we derived a gene network for each breed separately and a third network for the combined data set. At the intersection of the 3 networks, we identified candidate genes and pathways that were common to both breeds. Resulting from these analyses, we identified an enrichment of genes involved in axon guidance, cell adhesion, ErbB signaling, and glutamate activity, pathways that are known to affect pulsatile release of GnRH, which is necessary for the onset of puberty. Furthermore, we employed network connectivity and centrality parameters along with a regulatory impact factor metric to identify the key transcription factors (TF) responsible for the molecular regulation of puberty. As a novel finding, we report 5 TF (HIVEP3, TOX, EYA1, NCOA2, and ZFHX4) located in the network intersecting both breeds and interacting with other TF, forming a regulatory network that harmonizes with the recent literature of puberty. Finally, we support our network predictions with evidence derived from gene expression in hypothalamic tissue of adult cows. [ABSTRACT FROM AUTHOR]