Metabolomic fingerprinting of pig seminal plasma identifies in vivo fertility biomarkers

Abstract Background Metabolomic approaches, which include the study of low molecular weight molecules, are an emerging -omics technology useful for identification of biomarkers. In this field, nuclear magnetic resonance (NMR) spectroscopy has already been used to uncover (in) fertility biomarkers in the seminal plasma (SP) of several mammalian species. However, NMR studies profiling the porcine SP metabolome to uncover in vivo fertility biomarkers are yet to be carried out. Thus, this study aimed to evaluate the putative relationship between SP-metabolites and in vivo fertility outcomes. To this end, 24 entire ejaculates (three ejaculates per boar) were collected from artificial insemination (AI)-boars throughout a year (one ejaculate every 4 months). Immediately after collection, ejaculates were centrifuged to obtain SP-samples, which were stored for subsequent metabolomic analysis by NMR spectroscopy. Fertility outcomes from 1525 inseminations were recorded over a year, including farrowing rate, litter size, stillbirths per litter and the duration of pregnancy. Results A total of 24 metabolites were identified and quantified in all SP-samples. Receiver operating characteristic (ROC) curve analysis showed that lactate levels in SP had discriminative capacity for farrowing rate (area under the curve [AUC] = 0.764) while carnitine (AUC = 0.847), hypotaurine (AUC = 0.819), sn-glycero-3-phosphocholine (AUC = 0.833), glutamate (AUC = 0.799) and glucose (AUC = 0.750) showed it for litter size. Similarly, citrate (AUC = 0.743), creatine (AUC = 0.812), phenylalanine (AUC = 0.750), tyrosine (AUC = 0.753) and malonate (AUC = 0.868) levels had discriminative capacity for stillbirths per litter; and malonate (AUC = 0.767) and fumarate (AUC = 0.868) levels for gestation length. Conclusions The assessment of selected SP-metabolites in ejaculates through NMR spectroscopy could be considered as a promising non-invasive tool to predict in vivo fertility outcomes in pigs. Moreover, supplementing AI-doses with specific metabolites should also be envisaged as a way to improve their fertility potential.

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Metabolomic fingerprinting of pig seminal plasma identifies in vivo fertility biomarkers

10.21203/rs.3.rs-510687/v1 ◽

2021 ◽

Author(s):

Yentel Mateo-Otero ◽

Pol Fernández-López ◽

Ariadna Delgado-Bermúdez ◽

Pau Nolis ◽

Jordi Roca ◽

...

Keyword(s):

Nmr Spectroscopy ◽

Litter Size ◽

Seminal Plasma ◽

Mammalian Species ◽

Area Under The Curve ◽

Gestation Length ◽

Roc Curve Analysis ◽

Nmr Studies ◽

Farrowing Rate

Abstract Background Metabolomic approaches, which include the study of low molecular weight molecules, is an emerging -omics technology useful for the identification of biomarkers. In this field, nuclear magnetic resonance (NMR) spectroscopy approach has already been used to uncover (in)fertility biomarkers in the seminal plasma (SP) of several mammalian species. However, NMR studies profiling SP metabolome to uncover in vivo fertility biomarkers are yet to be carried out in pigs. Thus, this study aimed to evaluate the putative relationship between the presence/concentration of SP-metabolites and in vivo fertility outcomes. To this end, 24 entire ejaculates (three ejaculates per boar) were collected from artificial insemination (AI)-boars throughout a year (one ejaculate every four months). Immediately after collection, ejaculates were centrifuged (1,500×g for 10 min twice) to obtain SP-samples and were stored (− 80°C) for subsequent metabolomic analysis by NMR spectroscopy. Fertility outcomes from 1,525 inseminations were recorded over a year, including farrowing rate, litter size, stillbirths per litter and the duration of pregnancy. These data were corrected to isolate the direct boar effect on each in vivo fertility parameter using a multivariate statistical model. Results A total of 24 metabolites were identified and quantified in all SP-samples. ROC curve analysis showed that lactate levels in SP had discriminative capacity for farrowing rate (area under the curve (AUC) = 0.764; P < 0.05) while carnitine (AUC = 0.847), hypotaurine (AUC = 0.819), sn-glycero-3-phosphocholine (AUC = 0.833), glutamate (AUC = 0.799) and glucose (AUC = 0.750) had it for litter size (P < 0.05). Similarly, citrate (AUC = 0.743), creatine (AUC = 0.812), phenylalanine (AUC = 0.750), tyrosine (AUC = 0.753) and malonate (AUC = 0.868) levels had discriminative capacity for stillbirths per litter (P < 0.05); and malonate (AUC = 0.767) and fumarate (AUC = 0.868) concentrations for gestation length (P < 0.05). Conclusions Considering these results, the assessment of selected SP-metabolites in ejaculates through NMR spectroscopy could be considered as a promising non-invasive tool to predict in vivo fertility outcomes in pigs. Moreover, supplementing AI-doses with specific metabolites should also be contemplated as a way to improve their fertility potential.

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Measurement of Oxidative Stress Index in Seminal Plasma Can Predict In Vivo Fertility of Liquid-Stored Porcine Artificial Insemination Semen Doses

Antioxidants ◽

10.3390/antiox10081203 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1203

Author(s):

Isabel Barranco ◽

Camila P. Rubio ◽

Asta Tvarijonaviciute ◽

Heriberto Rodriguez-Martinez ◽

Jordi Roca

Keyword(s):

Oxidative Stress ◽

Litter Size ◽

Sperm Motility ◽

Seminal Plasma ◽

Artificial Insemination ◽

Stress Index ◽

Trolox Equivalent Antioxidant Capacity ◽

Oxidative Stress Index ◽

Farrowing Rate

The study evaluated the relation between the oxidative stress index (OSI) in porcine seminal plasma (n = 76) with sperm resilience and in vivo fertility (farrowing rate and litter size of 3137 inseminated sows) of liquid-stored artificial insemination (AI) semen doses. The OSI was assessed as the ratio of advanced oxidation protein products to Trolox-equivalent antioxidant capacity, both measured using an automated analyzer. Sperm motility (computer-assisted sperm analyzer) and viability (flow cytometry) were evaluated in semen AI-doses at 0 and 72 h of storage at 17 °C. Sperm resilience was defined as the difference between storage intervals. Semen AI-doses were hierarchically clustered as having high, medium and low seminal OSI (p < 0.001) with those of low displaying higher resilience (p < 0.01). Boars were hierarchically clustered into two groups (p < 0.001) as having either positive or negative farrowing rate and litter size deviation; the negative one showing higher seminal OSI (p < 0.05). In sum, seminal OSI was negatively related to sperm motility and the in vivo fertility of liquid-stored boar semen AI-doses, with the receiver operating characteristic curve presenting seminal OSI as a good predictive biomarker of in vivo fertility of AI-boars (area under the curve: 0.815, p < 0.05).

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Oxytocin in pig seminal plasma is positively related with in vivo fertility of inseminated sows

Journal of Animal Science and Biotechnology ◽

10.1186/s40104-021-00620-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lorena Padilla ◽

Marina López-Arjona ◽

Silvia Martinez-Subiela ◽

Heriberto Rodriguez-Martinez ◽

Jordi Roca ◽

...

Keyword(s):

Seminal Plasma ◽

Sperm Quality ◽

Sperm Count ◽

Sperm Concentration ◽

Reproductive Function ◽

Peptide Hormone ◽

Large White ◽

Male Reproductive Function ◽

Farrowing Rate

Abstract Background Identification of relevant in vivo biomarkers for fertility remains a challenge for the livestock industry. Concentrations of the small peptide hormone oxytocin (OXT), involved in male reproductive function and present in the seminal plasma (SP) of several species could be a robust one. This study characterized concentrations of SP-OXT in ejaculates from boars used in artificial insemination (AI) programs aiming to evaluate its relationship with sperm quality variables and in vivo fertility of their liquid-stored AI-semen. Seminal OXT concentrations (ng/mL) were measured in 169 ejaculates from 61 boars of the Duroc, Pietrain, Landrace and Large White breeds using a direct competitive immunoassay test based on AlphaLISA® technology. Ejaculate (ejaculate volume, sperm concentration, total sperm count) and sperm parameters (motility, viability, intracellular generation of reactive oxygen species, plasma membrane fluidity) were assessed at 0 h and 72 h in AI-semen samples stored at 17 °C. In vivo fertility included only 18 Large White and Landrace boars whose AI-semen was used to inseminated > 100 sows and evaluated both farrowing rate and litter size of 3,167 sows. Results The results showed that SP-OXT differed between boars and between ejaculates within boar (P < 0.05) but not between breeds (Duroc, Pietrain, Landrace and Large White). Ejaculates with higher SP-OXT concentration/mL (hierarchically grouped; P < 0.001) had larger volume and came from younger boars (P < 0.05). Ejaculates of boars showing positive farrowing rate deviation exhibited higher (P < 0.05) SP-OXT concentration/mL than those with negative farrowing rate deviation. Conclusion The SP concentrations of OXT are boar, ejaculate and age dependent, and positively related with ejaculate volume and farrowing rates of liquid-stored semen AI-doses.

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Investigation of exponential RNA amplification by the Qβ replicase complex

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314083971 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1602-C1602

Author(s):

Heidi Olesen ◽

Charlotte Knudsen ◽

Paulina Seweryn ◽

Ditlev Brodersen ◽

Zarina Kutlubaeva ◽

...

Keyword(s):

Amino Acids ◽

Nmr Spectroscopy ◽

Rna Virus ◽

Β Subunit ◽

Rna Amplification ◽

Nmr Studies ◽

Basic Amino Acids ◽

Rna Backbone

Positive-stranded RNA viruses are common among human pathogenic viruses, which often cooperate with host proteins to fulfill essential functions during infection. One function is replication of the viral genome. The Qβ phage is a positive-stranded RNA virus that infects E.coli. The Qβ replicase holo enzyme comprises the phage-encoded RNA-dependent RNA polymerase (β-subunit) and the host-encoded translation elongation factors, EF-Ts and EF-Tu as well as the ribosomal protein S1. The Qβ replicase has an extraordinary ability to exponentially amplify RNA in vivo and in vitro. A prerequisite for this is release of product and template RNA as single strands that can serve as new templates in subsequent rounds of replication. The role of S1 in the Qβ replicase is not clear. Recently, S1 was found to promote release of single-stranded product in Qβ replicase–mediated RNA synthesis. We have undertaken NMR spectroscopy and crystallization trials to improve our understanding of distinct S1 domains in solution as well as the ribosome- and replicase-binding properties of S1. Expression of distinct S1 domains for NMR spectroscopy has been optimized by use of autoinduction and results in high yields of [13C15N]-labelled protein fragments. These have proven very suitable for NMR studies and spectra revealed both ordered and disordered regions in the protein. Studies are ongoing. The structure of the Qβ core complex was recently determined at 2.5Å resolution. Thus, co-crystallization of the Qβ core in complex with S1 domains was undertaken and different crystal forms were obtained. These initial crystals diffracted to 3.2Å resolution and data processing as well as further optimization of the crystals is ongoing. S1 is thought to bind the β-subunit close to a region lined with basic amino acids, which potentially could facilitate interactions with the template RNA backbone and split it from the product strand. We demonstrate that neutralization of these basic amino acids indeed decrease or abolish infectivity of the Qβ phage. However, only one mutation, R503A affects the exponential replication in vitro. Crystallization of the Qβ holo enzyme bound to a truncated legitimate RNA template will be the next step for investigation of the mechanism of exponential RNA amplification by Qβ replicase.

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Fertility of boars - what is important to know

Biotechnology in Animal Husbandry ◽

10.2298/bah1702135s ◽

2017 ◽

Vol 33 (2) ◽

pp. 135-149 ◽

Cited By ~ 3

Author(s):

Radomir Savic ◽

Raquel Marcos ◽

Milica Petrovic ◽

Dragan Radojkovic ◽

Cedomir Radovic ◽

...

Keyword(s):

Litter Size ◽

Return Rate ◽

Low Fertility ◽

Reproductive Efficiency ◽

Reproductive Management ◽

Fertility Traits ◽

Farrowing Rate ◽

Sperm Traits

The most important part in reproductive management is the control of boar fertility. A common division of fertility traits is on the: in vitro (sperm traits) and in vivo (return rate, farrowing rate and litter size traits) fertility. In many studies were found differences between breed in the both groups of fertility traits. Variability of sperm traits of boars during the reproductive exploitation is influenced by various genetic (boar, breed) and paragenetic factors/effects (age, season, intensity of use). Good libido is desirable characteristics in boars, but the knowlegde of the correlation of libido and boar fertility traits are limited. Also, there is no standardised procedure or methods for the estimation of libido of the boars. The permanent ranking of boars according to the reproductive efficiency should be performing. Good reproductive management implies the timely identification of boars with the low fertility (or close to the average).

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Application of a scheimpflug-based biomechanical analyser and tomography in the early detection of subclinical keratoconus in chinese patients

BMC Ophthalmology ◽

10.1186/s12886-021-02102-2 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yan Liu ◽

Yu Zhang ◽

Yueguo Chen

Keyword(s):

Sensitivity And Specificity ◽

Area Under The Curve ◽

High Sensitivity ◽

Biomechanical Properties ◽

Chinese Patients ◽

Youden Index ◽

Roc Curve Analysis ◽

Surgical Safety ◽

Morphological Index

Abstract Background In vivo corneal biomechanics evaluation has been used to help screen early keratoconus in recent years. This study is to evaluate the value of a Scheimpflug-based biomechanical analyser combined with tomography in detecting subclinical keratoconus by distinguishing normal eyes from frank keratoconus (KC) and forme frusta keratoconus (FFKC) eyes in Chinese patients. Methods Study design: diagnostic test. This study included 31 bilateral frank keratoconus patients, 27 unilateral clinically manifesting keratoconus patients with very asymmetric eyes, and 79 control subjects with normal corneas. Corneal morphological and biomechanical parameters were measured using a Pentacam HR and a Corvis ST (OCULUS, Wetzlar, Germany). The diagnostic ability of computed parameters reflecting corneal biomechanical and morphological traits [including the Belin-Ambrósio deviation index (BAD_D), the Corvis biomechanical index (CBI) and the tomographic and biomechanical index (TBI)] was determined using receiver operating characteristic (ROC) curve analysis and compared by the DeLong test. Additionally, the area under the curve (AUC), the best cut-off values, and the Youden index for each parameter were reported. A novel corneal stiffness parameter, the stress-strain index (SSI), was also compared between KC, FFKC and normal eyes. Results Every morphological and biomechanical index analysed in this study was significantly different among KC, FFKC and normal eyes (P = 0.000). The TBI was most valuable in detecting subclinical keratoconus (FFKC eyes), with an AUC of 0.928 (P = 0.000), and both forms of corneal ectasia (FFKC and frank KC eyes), with an AUC of 0.966 (P = 0.000). The sensitivity and specificity of the TBI was 97.5 and 77.8 % in detecting FFKC and 97.5 and 89.7 % in detecting any KC, respectively, with a cut-off value of 0.375. The morphological index BAD_D and the biomechanical index CBI were also very useful in distinguishing eyes with any KC from normal eyes, with AUCs of 0.965 and 0.934, respectively. The SSI was significantly different between KC, FFKC and normal eyes (P = 0.000), indicating an independent decrease in corneal stiffness in KC eyes. Conclusions The combination of a Scheimpflug-based biomechanical analyser and tomography could increase the accuracy in detecting subclinical keratoconus in Chinese patients. The TBI was the most valuable index for detecting subclinical keratoconus, with a high sensitivity and specificity. Evaluation of corneal biomechanical properties in refractive surgery candidates could be helpful for recognizing potential keratoconic eyes and increasing surgical safety.

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21 THE INJECTION OF CORTISOL TO UTERUS INCREASES THE IMPLANTATION RATE AND LITTER SIZE IN PIG ARTIFICIAL INSEMINATION USING CYROPRESERVED SPERMATOZOA

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab21 ◽

2011 ◽

Vol 23 (1) ◽

pp. 117

Author(s):

M. Shimada ◽

T. Okazaki

Keyword(s):

Litter Size ◽

Seminal Plasma ◽

Artificial Insemination ◽

Implantation Rate ◽

Fertilization Rate ◽

Dependent Manner ◽

Vitro Fertilization ◽

Novel Method

Cryopreserved boar spermatozoa are not routinely available to swine artificial insemination (AI) because conception and farrowing rates, along with litter size, have remained low. We have reported the positive roles of seminal plasma in frozen–thawed sperm functions (Okazaki et al. 2009 Theriogenology 71, 491–498). Moreover, the injection of seminal plasma to uterus with frozen–thawed spermatozoa significantly increased the implantation rate. Thus, the factors in seminal plasma act not only on sperm but also on uterus to induce successful fertilization and implantation in pig AI using cryopreserved spermatozoa. To test this hypothesis, we identified the factors in seminal plasma and then developed novel pig AI method using cryopreserved spermatozoa. The sperm-rich fraction was collected weekly from each boar using the gloved-hand technique. The seminal plasma was removed just after collection by centrifuge and then was frozen as described in our previous study (Okazaki et al. 2009 Theriogenology 71, 491–498). When the frozen–thawed sperm was incubated with Fluo-3/AM to determine the level of intercellular Ca2+, the level of Ca2+ was increased in a time-dependent manner, and spontaneous capacitation that was judged by tyrosine phosphorylation of sperm protein by Western blotting (Shimada et al. 2008 Development 135, 2001–2011), was also induced in post-thawed sperm. The addition of EGTA to thawing solution significantly suppressed the Ca2+-induced capacitation. Moreover, the treatment increased fertilization rate in in vitro fertilization and in vivo in artificial insemination as similar as those in sperm with seminal plasma. The same number of blastocyst was collected from uterus by AI using post-thawed sperm with EGTA. However, the pregnancy rate remained low, and the number of leukocytes in the uterus was increased. In the next experiment, we examined in seminal plasma, the level of cortisol that has been known to play an important role in controlling immune function. The results showed that cortisol (1.0 ng mL–1) was detected in seminal plasma. When the sows of natural oestrus were twice artificial inseminated with or without cortisol, the injection of cortisol (5 μg/50 mL) to uterus with sperm significantly decreased the number of leukocytes in the uterus or endometrium at 24 to 36 h after AI. The low number of leukocytes in the uterus was similar to that in uterus injected fresh semen. The cortisol injection significantly increased the implantation rate and litter size of sows as compared to AI without cortisol (implantation rate; 83% v. 51%, litter size; 10.6 v. 7.3). From these results, we concluded that the injection of cortisol with frozen–thawed spermatozoa by EGTA-containing solution was a novel method of pig AI using cryopreserved spermatozoa. This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, and JST-Grant (No. 12-068 and No. 12-104).

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Exploring Seminal Plasma GSTM3 as a Quality and In Vivo Fertility Biomarker in Pigs—Relationship with Sperm Morphology

Antioxidants ◽

10.3390/antiox9080741 ◽

2020 ◽

Vol 9 (8) ◽

pp. 741

Author(s):

Marc Llavanera ◽

Ariadna Delgado-Bermúdez ◽

Yentel Mateo-Otero ◽

Lorena Padilla ◽

Xavier Romeu ◽

...

Keyword(s):

Antioxidant Enzyme ◽

Seminal Plasma ◽

Cell Biology ◽

Sperm Morphology ◽

Sperm Quality ◽

Mammalian Species ◽

Enzyme Linked Immunosorbent Assay ◽

Epididymal Maturation

Glutathione S-transferases Mu 3 (GSTM3) is an essential antioxidant enzyme whose presence in sperm has recently been related to sperm cryotolerance, quality and fertility. However, its role in seminal plasma (SP) as a predictor of the same sperm parameters has never been investigated. Herein, cell biology and proteomic approaches were performed to explore the presence, origin and role of SP-GSTM3 as a sperm quality and in vivo fertility biomarker. GSTM3 in SP was quantified using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit specific for Sus scrofa, whereas the presence of GSTM3 in testis, epididymis and accessory sex glands was assessed through immunoblotting analysis. Sperm quality and functionality parameters were evaluated in semen samples at 0 and 72 h of liquid-storage, whereas fertility parameters were recorded over a 12-months as farrowing rate and litter size. The presence and concentration of GSTM3 in SP was established for the first time in mammalian species, predominantly synthesized in the epididymis. The present study also evidenced a relationship between SP-GSTM3 and sperm morphology and suggested it is involved in epididymal maturation rather than in ejaculated sperm physiology. Finally, the data reported herein ruled out the role of this antioxidant enzyme as a quality and in vivo fertility biomarker of pig sperm.

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Influence of transcervical infusion of seminal plasma on the farrowing rate and litter size in artificially inseminated sows

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/1678-4162-11227 ◽

2020 ◽

Vol 72 (5) ◽

pp. 1691-1697

Author(s):

I. Stančić ◽

I. Radović ◽

S. Dragin ◽

M. Mirkov ◽

I. Pihler ◽

...

Keyword(s):

Litter Size ◽

Seminal Plasma ◽

Artificial Insemination ◽

Physiological Solution ◽

Fertility Rate ◽

Pig Production ◽

Plasma Infusion ◽

Breeding Herd ◽

Pig Farm ◽

Farrowing Rate

ABSTRACT Recent studies have focused on the use of seminal plasma to increase sow fertility after classical intracervical artificial insemination (AI). The aim of the present study was to investigate the influence of seminal plasma infusion, prior to the application of conventional AI dose, on the fertility rate in sows. A total of 114 sows were treated with intrauterine infusion of 30ml seminal plasma (SP), while 114 control sows were infused by physiological solution (PS), immediately before the application of conventional AI dose. The experiment was conducted at one commercial pig farm in Serbia, which is comprised of 1,500 sows in the breeding herd. Intrauterine infusion of seminal plasma produced significantly (P<0.05) higher farrowing rate (93.8%) and significantly (P<0.01) more live-born piglets per litter (12.27), compared with the control sows (83.33% farrowing rate and 10.48 piglets). The present results show that intrauterine infusion of seminal plasma can be a useful tool for increasing the fertility rate in artificially inseminated sows, under the conditions of practical intensive pig production.

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Seminal Plasma Anti-Müllerian Hormone: A Potential AI-Boar Fertility Biomarker?

Biology ◽

10.3390/biology9040078 ◽

2020 ◽

Vol 9 (4) ◽

pp. 78 ◽

Cited By ~ 1

Author(s):

Isabel Barranco ◽

Beatriz Fernandez-Fuertes ◽

Lorena Padilla ◽

Ariadna Delgado-Bermúdez ◽

Asta Tvarijonaviciute ◽

...

Keyword(s):

Seminal Plasma ◽

Sperm Quality ◽

Sperm Concentration ◽

Predictive Biomarker ◽

Membrane Lipid ◽

Enzyme Linked Immunosorbent Assay ◽

Lipid Disorder ◽

Size Number ◽

Farrowing Rate

The anti-Müllerian hormone (AMH), a Sertoli cell-secreted glycoprotein that is present in seminal plasma (SP), is considered as a marker of spermatogenesis in humans. This study aimed to evaluate the presence of this hormone in boar SP, together with its putative relationship with sperm quality, function, and in vivo fertility parameters in liquid-stored semen samples. The concentration of SP-AMH was assessed in 126 ejaculates from artificial insemination (AI)-boars (n = 92) while using a commercial Enzyme-Linked ImmunoSorbent Assay (ELISA) kit with monoclonal antibodies specific for Sus scrofa AMH (CEA228Po, Cloud-clone). Sperm quality (concentration, motility, viability, and acrosome damage) and functionality (membrane lipid disorder and intracellular H2O2 generation) were assessed in semen samples at 0 and 72 h of liquid-storage. In addition, fertility parameters from 3113 sows inseminated with the AI-boars were recorded in terms of farrowing rate, litter size, number of stillbirths per litter, and the duration of pregnancy over a 12-month period. The results revealed that the SP-AMH concentration varied widely among boar ejaculates, with no differences among breeds. Moreover, the SP-AMH concentration proved to be a good predictive biomarker for sperm concentration (p ˂ 0.05), but poor for other sperm quality, functionality, and in vivo fertility parameters of liquid-stored semen samples from AI-boars.

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