High Fertility Bulls Research Articles

Comparative Transcriptomic Analysis of Spermatozoa From High- and Low-Fertile Crossbred Bulls: Implications for Fertility Prediction.

Crossbred bulls produced by crossing Bos taurus and Bos indicus suffer with high incidence of infertility/subfertility problems; however, the etiology remains poorly understood. The uncertain predictability and the inability of semen evaluation techniques to maintain constant correlation with fertility demand for alternate methods for bull fertility prediction. Therefore, in this study, the global differential gene expression between high- and low-fertile crossbred bull sperm was assessed using a high-throughput RNA sequencing technique with the aim to identify transcripts associated with crossbred bull fertility. Crossbred bull sperm contained transcripts for 13,563 genes, in which 2,093 were unique to high-fertile and 5,454 were unique to low-fertile bulls. After normalization of data, a total of 776 transcripts were detected, in which 84 and 168 transcripts were unique to high-fertile and low-fertile bulls, respectively. A total of 176 transcripts were upregulated (fold change > 1) and 209 were downregulated (<1) in low-fertile bulls. Gene ontology analysis identified that the sperm transcripts involved in the oxidative phosphorylation pathway and biological process such as multicellular organism development, spermatogenesis, and in utero embryonic development were downregulated in low-fertile crossbred bull sperm. Sperm transcripts upregulated and unique to low-fertile bulls were majorly involved in translation (biological process) and ribosomal pathway. With the use of RT-qPCR, selected sperm transcripts (n = 12) were validated in crossbred bulls (n = 12) with different fertility ratings and found that the transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes was significantly (p < 0.05) lower in low-fertile bulls than high-fertile bulls and was positively (p < 0.05) correlated with conception rate. It is inferred that impaired oxidative phosphorylation could be the predominant reason for low fertility in crossbred bulls and that transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes could serve as potential biomarkers for fertility in crossbred bulls.

Deciphering the complexity of sperm transcriptome reveals genes governing functional membrane and acrosome integrities potentially influence fertility

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 groupshad 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.

Spermatozoal transcripts associated with oxidative stress and mitochondrial membrane potential differ between high- and low-fertile crossbred bulls.

Thepresence of various forms of RNAs having roles in fertilisation and early embryonic developmentis welldocumentedin mammalian spermatozoa. In the present study,usingAgilent microarrayplatform, we compared sperm mRNA expression profiles between high- and low-fertile crossbred bulls with normal semen parameters.Microarray data acquisition and analysis were performed using GeneSpring GXversion software, wherein spermatozoa from high-fertile bulls were kept as control while spermatozoa fromlow-fertilebulls were considered as treatment group.A total of 6,238 transcripts were detected in crossbred bull spermatozoa; 559 transcripts (>1.5-fold) were differentially regulated between high- and low-fertile bulls. Functional annotation has categorised these transcripts into biological process, cellular, and molecular functions. It was observed that transcripts associated with oxidation reduction process (p=.003), mitochondrial membrane potential (p=.03), were significantly down-regulated while transcripts associated with apoptosis (p=.04) were up-regulated in low-fertile spermatozoa. The dysregulated genes were involved in important cellular pathways including oxidative phosphorylation (p=.002), oestrogen signalling (p=.002), Wnt signalling (p=.035), cGMP-PKG signalling (p=.007) and MAPK signalling (p=.032) pathways. Collectively, the present study discovered profound discrepancies in sperm mRNA expression between high- and low-fertile crossbred bulls, with potential possibilities for their use in fertility prediction.

Sperm binding to hyaluronan is an excellent predictor of Nili-Ravi buffalo bull fertility.

This study reports the first evaluation of sperm hyaluronan binding assay (HBA) for predicting the fertility of Nili-Ravi buffalo bulls in relation to standard parameters of sperm quality. Cryopreserved semen doses of low (n=6), medium (n=3) and high fertility (n=8) bulls based on their respective return rates were used. Significantly, more spermatozoa bound to hyaluronan from the most fertile bulls (57.15%±1.44) compared with medium (42.46%±1.08) and low fertility bulls (29.70%±0.78). A strongly positive correlation (r=.824, p<.01) was found between HBA and fertility that predicts a 67.9% variability (r2 =.679, p<.01) in fertility. HBA was also strongly positively correlated with sperm viability (r=.679, p<.01) followed by their live/dead ratio (r=.637, p<.01), uncapacitated spermatozoa (r=.631, p<.01), normal apical ridge (r=.459, p<.01), motility (r=.434, p<.01), mature spermatozoa with low residual histones (r=.364, p<.01), high plasma membrane integrity (r=.316, p<.01) and nonfragmented DNA levels (r=.236, p<.05). It was negatively correlated with spermatozoa having reacted acrosome (r=-.654, p<.01). A fertility model built using a combination of sperm HBA and either sperm livability or viability predicts, respectively, 86.1% (r2 =.861, p<.01) and 85.9% (r2 =.859, p<.01) variability in buffalo bull fertility. In conclusion, sperm HBA may prove to be a single robust predictor of Nili-Ravi buffalo bull fertility.

Herd manager attitudes and intentions regarding the selection of high-fertility EBV sires in Australia

Reproductive performance in dairy cattle has declined over the last 50 years as an unintended consequence of selection for high milk yield. Since the early 2000s, dairy geneticists have released successive versions of fertility estimated breeding values (EBV) to assist in reversing this trend. At the herd level, fertility EBV can help managers accelerate improvements in reproductive performance by acting as a second selection criteria when used in tandem with a breeding index. However, use of the fertility EBV in sire selection currently varies between herd managers. The aim of this study was to better understand the reasons why herd managers choose or do not choose to select high-fertility EBV sires, using the Theory of Planned Behavior (TPB) as a social research framework. Thirty-five Victorian dairy herd managers were recruited as part of a larger study investigating the daughter fertility Australian Breeding Value and interviewed using a series of questions examining TPB constructs. The interviews were recorded and transcribed using template analysis. A wide range of herd manager types were enrolled into the study, with representation from diverse systems. Out of the 35 herd managers, 27 included fertility in their list of high-priority breeding objectives. A wide variation in results was consistent with previous studies that have demonstrated marked heterogeneity in herd manager attitudes toward bull selection. Herd manager-perceived barriers to selection of sires with high daughter fertility EBV included a lack of high daughter fertility bulls with other desirable traits, a lack of trust in the fertility EBV or in the Australian EBV system, difficulty in interpreting international proofs, information overload, semen prices, low bull reliability, and difficulty in understanding bull catalogs. Not all herd managers found the process problematic, however, particularly if a breeding consultant was employed to select all or most of the sires. Herd manager-perceived barriers for choosing to select daughter fertility as a breeding objective include a lack of awareness of the EBV, a lack of interest in genetics in general, low confidence in the impact of genetic selection for fertility, and a feeling that fertility was not important for their production system. The results of this study suggest that animal geneticists and on-farm service providers need to work together to allow the opportunities arising from appropriate use of fertility EBV to be realized more broadly across the dairy industry.

Transcriptomic Profiling of Buffalo Spermatozoa Reveals Dysregulation of Functionally Relevant mRNAs in Low-Fertile Bulls.

Although, it is known that spermatozoa harbor a variety of RNAs that may influence embryonic development, little is understood about sperm transcriptomic differences in relation to fertility, especially in buffaloes. In the present study, we compared the differences in sperm functional attributes and transcriptomic profile between high- and low-fertile buffalo bulls. Sperm membrane and acrosomal integrity were lower (P < 0.05), while protamine deficiency and lipid peroxidation were higher (P < 0.05) in low- compared to high-fertile bulls. Transcriptomic analysis using mRNA microarray technology detected a total of 51,282 transcripts in buffalo spermatozoa, of which 4,050 transcripts were differentially expressed, and 709 transcripts were found to be significantly dysregulated (P < 0.05 and fold change >1) between high- and low-fertile bulls. Majority of the dysregulated transcripts were related to binding activity, transcription, translation, and metabolic processes with primary localization in the cell nucleus, nucleoplasm, and in cytosol. Pathways related to MAPK signaling, ribosome pathway, and oxidative phosphorylation were dysregulated in low-fertile bull spermatozoa. Using bioinformatics analysis, we observed that several genes related to sperm functional attributes were significantly downregulated in low-fertile bull spermatozoa. Validation of the results of microarray analysis was carried out using real-time qPCR expression analysis of selected genes (YBX1, ORAI3, and TFAP2C). The relative expression of these genes followed the same trend in both the techniques. Collectively, this is the first study to report the transcriptomic profile of buffalo spermatozoa and to demonstrate the dysregulation of functionally relevant transcripts in low-fertile bull spermatozoa. The results of the present study open up new avenues for understanding the etiology for poor fertility in buffalo bulls and to identify fertility biomarkers.

The transcriptomic response of bovine uterine tissue is altered in response to sperm from high and low fertility bulls

The transcriptomic response of bovine uterine tissue is altered in response to sperm from high and low fertility bulls

Bull seminal plasma stimulates in vitro production of TGF-β, IL-6 and IL-8 from bovine endometrial epithelial cells, depending on dose and bull fertility

Seminal plasma (SP) regulates immune responses in the female reproductive tract through specific cytokines. It is not known whether SP from high fertility bulls (H) differs from SP from low fertility bulls (L). In this study, the cytokine response of bovine endometrial epithelial cells (bEEC) in culture was investigated after challenge with SP from two bulls of below average (L) or three bulls of above average fertility (H). The bEECs were challenged with 1% or 4% SP from l- or H-fertility bulls (L1, L4, H1, H4, respectively) or 1% or 4% PBS as control (C1, C4) for 72 h. The culture media were analysed for concentrations (pg/million cells) of transforming growth factor beta (TGF-β1, TGF-β2 and TGF-β3) by Luminex, and Interleukin 6 and 8 (IL-6, IL-8) by ELISA. Challenge significantly affected production of TGF-ß1, TGF-ß2 and IL-8 compared to controls and was affected by bull fertility (p < 0.0001), SP concentration (p < 0.0001) and their interaction (p < 0.0001). A higher production of TGF-β1, TGF-β2 and IL-8 (p < 0.0001), and also IL-6 (p < 0.01), resulted from challenge with high doses of SP, being higher for L than H (p < 0.05). For TGF-β3, fertility of bull (p < 0.05). For TGF-B3, fertility of bull (p < 0.05) and the interaction between fertility and concentration of SP were significant (p < 0.01). In conclusion, 4% SP from L bulls stimulated more TGF-β1, TGF-β2, TGF-β3, IL-6 and IL-8 production than SP from H bulls, indicating that stimulation of the endometrium is relevant for fertility. Seminal plasma from high fertility bulls seems to affect cytokine production in utero positively in inseminated cows.

Heparin binding proteins in seminal plasma of breeding buffalo bulls and their relation with semen freezability and in vivo fertility

The present study was conducted to determine the concentrations of heparin binding proteins (HBP) in seminal fluid of breeding buffalo bulls and establish their relationship with frozen-thawed semen characteristics and fertility. Both fresh and frozen semen were collected from same ejaculate of 30 breeding buffalo bulls. Based on HBP concentrations in seminal plasma obtained from affinity chromatography, all the bulls were divided into 2 groups, viz. high fertility bulls (HFB, ≥ 6.5 mg/ml; n=15) and low fertility bulls (LFB, &lt; 6.5 mg/ml; n=15) for further comparisons. The frozen-thawed semen of same ejaculate was evaluated for per cent CASA-based sperm kinetics, viability, Hypo-osmotic swelling test (HOST), acrosome integrity, in vitro acrosome reaction and first service conception rate (FSCR). The overall HBP concentrations were significantly higher in seminal plasma (7.31±0.14 vs 5.33±0.19 mg/ml) and fresh (0.66±0.03 vs 0.59±0.02 mg/109 sperms) sperm extracts of HFB than LFB. The frozen-thawed semen characteristics, viz. total motility, per cent acrosome reaction and FSCR were also significantly higher in bulls with high fertility than in lower ones. No significant difference was observed in sperm viability, HOST and acrosome integrity in the 2 groups of bulls. Total 12 HBP bands in range of 11 to 135 kDa were recorded on SDS-PAGE. Higher levels of HBP in seminal plasma of HFB might be responsible for their better semen quality and fertility.

JUNO protein coated beads: A potential tool to predict bovine sperm fertilizing ability

Considerable variation in fertility exists between bulls in AI centres, despite passing minimum post-thaw quality control checks. The development of a reliable in vitro test to predict bull fertility could enable the identification and selection of high fertility bulls, without the need to resort to test inseminations. An in-depth knowledge of the molecular basis of fertilization is a prerequisite to the development of such a test or combination of tests. To date, JUNO is the only oocyte plasma membrane receptor described to be involved in gamete binding for which the partner in the sperm, IZUMO1, is known. Despite the fact that this interaction appears to be conserved among mammals, it has not been confirmed yet in some species including cattle. Furthermore, an association between binding and fertility has not been tested. Here, we propose a sperm-binding assay based on magnetic sepharose beads coated with bovine recombinant JUNO protein (BJUNO) to study sperm binding. Bull sperm bound specifically to BJUNO demonstrating that the JUNO-IZUMO1 interaction is conserved in cattle. Moreover, the assay was able to distinguish between epididymal and ejaculated sperm. Lastly, the number of sperm cells bound to BJUNO was significantly lower for frozen-thawed sperm from bulls of low vs high field fertility. In conclusion, our findings document a novel valid sperm-binding assay to predict mammalian sperm function and to investigate the role of specific proteins involved in gamete recognition and fusion.

Changes in Bull Semen Metabolome in Relation to Cryopreservation and Fertility.

Simple SummaryAlthough semen cryopreservation has facilitated the diffusion of artificial insemination and in vitro fertilization in cattle, it still represents a major factor affecting sperm fertility. It is known that cryopreservation induces the loss of fertility-associated proteins, while the effect on metabolites has not been evaluated, although several compounds affect sperm physiology and fertility. The aim of the present work was to study the metabolome in bovine sperm and seminal plasma after cryopreservation and to correlate the metabolic profile of high- and low-fertility bulls in order to identify fertility markers. The analysis, carried out by liquid chromatography–mass spectrometry methods, revealed differences in metabolite contents between fresh and cryopreserved semen, both at cellular and plasmatic levels. Interestingly, metabolites showing variation have important functions related to fertility. In addition, the study highlighted the differences in lipid profile between high- and low-fertility bulls. The identification of new potential fertility markers is of high economic impact. In addition, it opens the way for the development of corrective strategies to improve the fertility of low-fertility bulls.Semen cryopreservation determines several sperm damages, including the loss of fertility-associated proteins. The purpose of the study was to compare the metabolite contents in bovine sperm and seminal plasma before and after cryopreservation, and between high- and low-fertility bulls in vitro. Forty-eight ejaculates, collected from eight bulls (six per bull), were analyzed by liquid chromatography–mass spectrometry. Cryopreservation resulted in an over-expression of lysophosphatidylcholine (0:0/18:2(9Z,12Z)) in seminal plasma. In addition, higher levels of glycine betaine and pyro-l-glutaminyl-l-glutamine were observed in cryopreserved compared to fresh spermatozoa. The fresh seminal plasma of high-fertility bulls showed an over-expression of l-acetylcarnitine, glycerol tripropanoate, 2,3-diacetoxypropyl stearate and glycerophosphocholine, and an under-expression of lysophosphatidylcholine and butyrylcarnitine, compared to low-fertility bulls. Higher levels of glycerophosphocholine and lysophosphatidylcholine (16:0/0:0) were recorded in fresh spermatozoa from high-fertility bulls. In high-fertility bulls, a greater content of glycerophosphocholine and lower levels of butyrylcarnitine, glycine betaine and l-carnitine were found in cryopreserved seminal plasma, and lower levels of glycine betaine were detected in cryopreserved spermatozoa. In conclusion, cryopreservation affects bovine semen metabolome at both plasmatic and cellular compartments, and metabolic profile differs between high- and low-fertility bulls.

Whole-genome homozygosity mapping reveals candidate regions affecting bull fertility in US Holstein cattle

BackgroundAchieving rapid genetic progress while maintaining adequate genetic diversity is one of the main challenges facing the dairy industry. The increase in inbreeding can be used to monitor the loss of genetic diversity. Inbreeding tends to increase the proportion of homozygous loci, some of which cause homozygosity of recessive alleles that results in reduced performance. This phenomenon is known as inbreeding depression and tends to be most prominent on fitness-related traits, such as male fertility. Traditionally, inbreeding has been monitored using pedigree information, or more recently, genomic data. Alternatively, it can be quantified using runs of homozygosity (ROH), i.e., contiguous lengths of homozygous genotypes observed in an individual’s chromosome.ResultsThe objective of this study was to evaluate the association between ROH and sire conception rate. ROH were evaluated using 268 k genetic markers in 11,790 US Holstein bulls. Interestingly, either the sum, mean, or maximum length of ROH were negatively associated with bull fertility. The association analysis between ROH and sire fertility was performed comparing 300 high-fertility vs. 300 low-fertility bulls. Both the average and sum of ROH length were higher in the low-fertility group. The enrichment of ROH regions in bulls with low fertility was assessed using a Fisher’s exact test. Nine regions were significantly enriched in low-fertility compared to high-fertility bulls. Notably, these regions harbor genes that are closely related to sperm biology and male fertility, including genes exclusively or highly expressed in testis.ConclusionsThe results of this study can help not only to manage inbreeding in genomic selection programs by designing custom mating schemes, but also to better understand the mechanisms underlying male fertility in dairy cattle.

Evaluation of Friesian Holstein Bulls Fertility in Lembang and Singosari Artificial Insemination Center using West Java ISIKHNAS Data

Friesian Holstein bull (FH) is one of dairy cattle in Indonesia. It is important to increase the production of dairy cattle to meet the need for milk consumption. The way to develop is through artificial insemination (AI). This study was aimed to evaluate the FH bull fertility by calculating the percentage of first service conception rate (%FSCR) of FH bulls in Lembang and Singosari AI Center using ISIKHNAS data in West Java for the year 2017 until 2018. The data included in this study after editing consisted of AI data (n=141176) and service records as well as pregnancy diagnosis information (n=98120). The study showed that the FH bull semen spread in West Java mostly from Lembang and Singosari AI Center. The fertility rate can be grouped into high fertile (HF), and low fertile (LF) level and also divided into two groups based on the number of AI services. The %FSCR of FH bulls semen that used for AI <1000 services in Singosari 64.29% as HF level; 22.22% as LF level, while in Lembang had 74. 03% as HF; 21.15% as LF level. Then, the AI> 1000 services in Lembang had %FSCR is 61.61% as HF level; 35. 28% as LF level. While Singosari had 63.11% as HF level; 33.78% as LF level. In conclusion, the FH bulls in Lembang and Singosari AI center had the HF of the %FSCR is about 53.13 until 74.03%. It is needed the more accurate assessment through genomic analysis to get the biomarker of HF bulls as a suggestion to improve the FH breeding cattle.

148 Bull spermatozoa uptake of extracellular vesicles from bovine seminal plasma

Extracellular vesicles (EV) are important mediators of intercellular communication because they transfer microRNA (miRNA) that are able to repress translation of mRNA. Their presence in seminal plasma suggests a role in sperm fertility. It is known that bull seminal plasma contains fertility-associated proteins that are predictive of high and low fertility (Killian et al. 1993 Biol. Reprod. 49, 1202-1207). In addition, a difference in miRNA content between high and low spermatozoa motility has been observed in bulls, highlighting a potential role of EV on fertility (Capra et al. 2017 BMC Genom. 18, 14). We hypothesised that co-incubation of sperm of low-fertility bulls with EV isolated from the seminal plasma of high-fertility bulls could improve their fertility. Before testing this hypothesis, a preliminary study was carried out to investigate the presence and type of EV in bovine seminal plasma and their interaction with spermatozoa. Ejaculates of eight Holstein bulls collected weekly by artificial vagina were centrifuged at 1600×g for 10min to pellet spermatozoa and then centrifuged again at 2400×g for 30min to eliminate cell debris and large vesicles. After centrifugation, supernatants were collected and filtered twice (0.45 and 0.22µm) and stored at −80°C. A double ultracentrifugation at 100 000×g for 1h was performed, and pellets resuspended in a small volume of Tris buffer were kept at −80°C until used. Three ejaculates of the same bull were pooled to detect the concentration and size of EV by Nanosight Instruments. To trace the interaction with spermatozoa by fluorescence microscopy, EV were labeled with PKH26 dye and a dose-response curve in three replicates was performed. A suspension of 1×106 spermmL−1 was co-incubated with 200 or 400×106 EV labelled with pKH26 for 30, 60, 90, 120, 150, and 180min at 38.5°C. The end point of incubation was at 24h. Internalisation of EV was assessed using confocal microscopy. Our results showed that the size of EV ranged from 145.1 to 187.7nm, with an average of 166±29nm. For all seminal plasma samples, the number of EV ranged from 3.62 to 6.08×1013 particlesmL−1, with an average of 4.37±0.61×1013. Based on size, these EV can be categorised as shedding vesicles. Confocal microscopy was set to take fluorescent images at different planes scanned every 0.12µm from top to bottom of the spermatozoa. Our results showed that no fluorescence signal was detectable after co-incubation with 200×106 EV. At the concentration of 400×106 EV, up to 60min no signal was detectable, whereas at 90min spermatozoa showed a fine granular fluorescent pattern within the intermediate portion. At 120min, the signal was within the acrosome, and at 180min the spermatozoa were stained for the whole length, supposing a distribution of incorporated EV throughout all the cell. At 24h, the fluorescence signal decreased. In conclusion, this is the first study to demonstrate that bull spermatozoa incorporate EV from bull semen. We hypothesise that a transfer of molecules, such as miRNA and other noncoding RNA molecules, from EV to spermatozoa is probably involved in sperm fertility.

Sperm miR-15a and miR-29b are associated with bull fertility.

MicroRNAs modulate male fertility by regulating gene expression. In this study, dynamics of sperm miR-15a, miR-29b and miR-34a from high fertility (HF) and low fertility (LF) bulls using RT-qPCR were evaluated. Bioinformatic tools were employed to ascertain genes of interest of the sperm miRNAs. The expression levels of p53, BCL2, BAX and DNMT1 in bull spermatozoa were determined by immunoblotting. MicroRNA levels of miR-15a and miR-29 were higher in LF sires when compared with those present in HF bulls. Expression levels of miR-34a did not differ between the two groups. We found an inverse correlation between miR-15a and bull fertility. MiR29-b was also negatively associated with fertility scores. BCL2 and DNMT1 were higher in HF bulls while BAX was higher in the LF group. Our data showed a positive correlation between BCL2 and bull fertility. In addition, DNMT1 was positively associated with bull fertility. Furthermore, levels of BAX were negatively linked with bull fertility scores. Identification of miRNAs found in the spermatozoa of sires with different in vivo fertility helps understand the alterations in the fertilising capacity from cattle and other mammals. These potential biomarkers can be used in reproductive biotechnology as fertility markers to assess semen quality and predict male fertility.

Sperm cellular and nuclear dynamics associated with bull fertility

The objective of this study was to ascertain cellular characteristics and the dynamics of the sperm chromatin proteins protamine 1 (PRM1) and protamine 2 (PRM2) in the sperm of Holstein bulls having a different fertility status. Important sperm variables were analyzed using computer-assisted sperm analysis (CASA). Sperm membrane, acrosome status, DNA integrity were also assessed using propidium iodide (PI), fluorescein isothiocyanate conjugated to Arachis hypogaea (FITC-PNA), and acridine orange (AO) followed by flow cytometry. In addition, abundances of PRM1 and PRM2 were analyzed using flow cytometry experiments. Differences in sperm decondensation capacity were assessed in bulls of varying fertility using a decondensation assay. As determined using CASA, average pathway velocity, amplitude of lateral head displacement and straightness were different (P < 0.05) for sperm from high and low fertility bulls. There, however, were no differences between the high and low fertility bulls for characteristics of sperm plasma membrane, acrosome, and DNA integrity (P > 0.05). Relative abundances of PRM1 and PRM2 in sperm from the high and low fertility bulls were inversely related (P < 0.0001). Percentages of decondensed sperm were different between high and low fertility bulls (P < 0.0001) and total numbers of decondensed sperm were greater in low fertility bulls than high fertility bulls (R2 = 0.72). Results of the present study are significant because molecular and morphological phenotypes of sperm that were detected affect fertility in livestock species.

Uncovering sperm metabolome to discover biomarkers for bull fertility

BackgroundSubfertility decreases the efficiency of the cattle industry because artificial insemination employs spermatozoa from a single bull to inseminate thousands of cows. Variation in bull fertility has been demonstrated even among those animals exhibiting normal sperm numbers, motility, and morphology. Despite advances in research, molecular and cellular mechanisms underlying the causes of low fertility in some bulls have not been fully elucidated. In this study, we investigated the metabolic profile of bull spermatozoa using non-targeted metabolomics. Statistical analysis and bioinformatic tools were employed to evaluate the metabolic profiles high and low fertility groups. Metabolic pathways associated with the sperm metabolome were also reported.ResultsA total of 22 distinct metabolites were detected in spermatozoa from bulls with high fertility (HF) or low fertility (LF) phenotype. The major metabolite classes of bovine sperm were organic acids/derivatives and fatty acids/conjugates. We demonstrated that the abundance ratios of five sperm metabolites were statistically different between HF and LF groups including gamma-aminobutyric acid (GABA), carbamate, benzoic acid, lactic acid, and palmitic acid. Metabolites with different abundances in HF and LF bulls had also VIP scores of greater than 1.5 and AUC- ROC curves of more than 80%. In addition, four metabolic pathways associated with differential metabolites namely alanine, aspartate and glutamate metabolism, β-alanine metabolism, glycolysis or gluconeogenesis, and pyruvate metabolism were also explored.ConclusionsThis is the first study aimed at ascertaining the metabolome of spermatozoa from bulls with different fertility phenotype using gas chromatography-mass spectrometry. We identified five metabolites in the two groups of sires and such molecules can be used, in the future, as key indicators of bull fertility.

Characterization of antisperm antibody binding patterns in relation to sperm phenotypic attributes and field fertility in dairy bulls

To test our hypothesis that antisperm antibodies (ASA) might alter sperm phenotypic attributes thus leading to sub-fertility/infertility in bulls, ASA were generated in crossbred male calves by immunizing with sperm two times. Cryopreserved spermatozoa from crossbred bulls (n = 24) with different field fertility ratings were incubated with ASA and different patterns of ASA immunolocalization were studied. In addition, sperm membrane integrity, acrosomal integrity and cryo-capacitation status were also assessed. Immunolocalization of sperm antigens using antisperm antibody revealed three major patterns (Acrosomal-AR, apical-AP and, acrosome and tail-AT). The proportion of ASA reactive spermatozoa was significantly (P < 0.05) higher in low- and medium-fertile bulls compared to high-fertile bulls. Among the three patterns, the proportion of spermatozoa with AR pattern was significantly (P < 0.05) higher in low- and medium-fertile bulls compared to high-fertile bulls. The proportion of membrane and acrosome intact spermatozoa was significantly (P < 0.05) higher in high-fertile bulls compared to medium- and low-fertile bulls. There were no significant differences in the proportion of cryo-capacitated spermatozoa among high-, medium- and low-fertile bulls. The relationship between ASA reactive spermatozoa and conception rates (CR) of bulls was highly (P < 0.01) significant and negative. Similarly, AR and AT pattern were also significantly (P < 0.01) and negatively related to CR of bulls. The reactivity of spermatozoa with ASA was also significantly (P < 0.01) and negatively related to the membrane and acrosome integrity of spermatozoa. It was concluded that the proportion of spermatozoa responding to ASA was higher in low-compared to high-fertile bulls and ASA localization in sperm acrosomal area was negatively related to sperm membrane and acrosomal integrity and bull fertility.

Multicolor flow cytometric analysis of cryopreserved bovine sperm: A tool for the evaluation of bull fertility

The study aimed at the analysis of the functional status of cryopreserved bovine sperm using multicolor flow cytometry. The value of sperm functional traits as predictors of bull fertility was further evaluated through a retrospective fertility study. For this purpose, 20 Holstein-Friesian bulls serving as mature sperm donors in an artificial insemination (AI) center were selected based on their annual 56-d non-return rate (%) after at least 1,000 AI, and were accordingly classified as high (HF; nHF = 10 bulls) or low fertility bulls (LF; nLF = 10 bulls). Four to 5 cryopreserved ejaculates per bull (91 ejaculates in total) were examined immediately after thawing (0 h) and after a 3-h incubation at 38°C (3 h). A panel of 5 fluorochromes including calcein violet, propidium iodide, pycoerythrin-conjugated lectin of Arachis hypogea, Fluo-4, and cyanine dye DiIC1(5) was configured by means of a 3-laser flow cytometer, to simultaneously assess sperm esterase activity, plasma membrane integrity, acrosomal status, intracellular Ca2+ levels, and mitochondrial membrane potential, respectively. The % relative size of 18 sperm sub-populations showing 2 or more of a combination of the following features was determined: high esterase activity (Cpos), intact plasma membrane (PIneg), unstained acrosome (PNAneg), low intracellular Ca2+ levels (Fneg), and high mitochondrial membrane potential (Mpos). In both fertility groups, Mpos cells comprised more than 90 and 84% of PInegPNAneg sperm at 0 and 3 h, respectively. The percentage of CposPInegPNAnegFnegMpos sperm did not differ between HF and LF ejaculates; however, the percentage of Fneg cells within the PInegPNAneg and PInegMpos sperm populations at 0 h was higher in the HF than in the LF bulls. Applying the random forest ensemble learning method, approximately two-thirds of ejaculates could be correctly assigned to their fertility group. The fraction of Fneg sperm within the PInegMpos population at 0 h was the most important fertility predictor among the 18 defined sperm populations. In conclusion, multicolor flow cytometry offered an insight into the functional heterogeneity of cryopreserved bovine sperm. Indeed, the ability of viable sperm to retain low Ca2+ levels differed between bulls of diverse fertility. A classifier based on selected sperm populations assessed through multicolor flow cytometry could contribute to the prognosis of bull fertility after AI.

Efficiency of CellROX deep red® and CellROX orange® fluorescent probes in identifying reactive oxygen species in sperm samples from high and low fertility bulls

Detection of reactive oxygen species (ROS) is of great interest in semen analysis since their excess is detrimental to sperm function and male fertility. Fluorescence microscopy has achieved attention for providing broad possibilities of sperm evaluations and also for presenting substantial accessibility. In this context, this study investigated the efficiency of CellROX Deep Red® and Orange® probes in detecting ROS in bovine sperm cells and assessed their relationship with sperm fertility potential. First, 16 ejaculates were assigned in three treatments: T0 (no ROS production induced), T1x (ROS production induced once) and T2x (ROS production induced twice). Samples were incubated with Red and Orange probes and percentages of cells producing ROS were evaluated using fluorescence microscopy. Coefficient of determination was 0.61 for Red and 0.56 for Orange. Afterwards, frozen-thawed semen samples from high and low fertility bulls were evaluated regarding percentages of cells producing ROS detected by Red and Orange. Higher levels of ROS assessed by Red were detected in low fertility bovine samples. In conclusion, CellROX Red® and Orange® are both efficient in detecting ROS in bovine spermatozoa. Furthermore, higher sperm ROS detection by CellROX Red® might be associated with low fertility samples.