Subfertile Animals Research Articles

ADR-2 regulates fertility and oocyte fate in Caenorhabditis elegans.

RNA-binding proteins (RBPs) play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the adenosine deaminase acting on RNA family of RBPs and the sole adenosine-to-inosine RNA-editing enzyme in Caenorhabditis elegans, can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RBPs that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogeneous nuclear ribonucleoprotein family of RBPs. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing-dependent and RNA editing-independent mechanisms in regulating embryogenesis.

ADR-2 regulates fertility and oocyte fate in C. elegans.

RNA binding proteins play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the Adenosine DeAminase acting on RNA (ADAR) family of RNA binding proteins and the sole adenosine-to-inosine RNA editing enzyme in C. elegans, can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RNA binding proteins that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogenous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals, and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing-dependent and independent mechanisms in regulating embryogenesis.

Effect of platelet-rich plasma (PRP) on post-thaw quality, kinematics and in vivo fertility of fertile and subfertile buffalo (Bubalus bubalis) spermatozoa.

This study investigated the effect of adding platelet-rich plasma (PRP) in semen extender prior cryopreservation on post-thaw quality, kinematics, and in vivo fertility of fertile and subfertile buffalo spermatozoa. Eleven buffalo bulls were classified based on their conception rate (CR) into fertile (n = 8, CR > 55%) and subfertile (n = 3, CR < 35%) groups. Ejaculates were collected with artificial vagina, pooled, and dispensed into 6 aliquots, diluted with Tris-egg yolk-glycerol extender supplemented with different proportions of PRP [0% (control), 5%, 10%, 15%, 20%, and 25%] followed by cryopreservation using standard procedures. Post-thaw sperm quality, kinematics, antioxidant activity, cryosurvival rate, and in vivo fertility were compared between fertile and subfertile groups and among proportions of PRP within each group. The results showed that 15% PRP greatly (P < 0.001) improved sperm characteristics, average path velocity, and curvilinear velocity of the subfertile group. Interestingly, 5%, 10%, and 15% PRP greatly (P < 0.001) reduced malondialdehyde content and improved enzymatic (glutathione peroxidase and superoxide dismutase) and total antioxidant capacity in fertile and subfertile groups. However, these three proportions of PRP significantly (P < 0.001) improved the cryosurvival rate of the subfertile group; only 15% PRP greatly improved CR of subfertile (60.83% vs. 34.17%) animals to be comparable with that of fertile ones treated with 5 (59.17%) and 10% (60.83%) PRP. In conclusion, adding 15% PRP to semen extender before cryopreservation is recommended to improve post-thaw quality, antioxidant activity, and in vivo fertility of buffalo semen particularly of the subfertile animals.

Classification of the sperm chromatin compaction alterations in bulls (Bos taurus) and its correlation with the efficiency of the in vitro production of embryos

This paper proposes to classify the sperm chromatin compaction alterations in bulls, according to the affected area location and its objective is evaluating the correlation of the intensity, the heterogeneity and these kinds of chromatin decompaction with the rates of cleavage and the formation of blastocysts of in vitro production of embryos (IVPE). It was used several subfertile animals sperm samples, which were evaluated using the toluidine blue staining and computer image analysis, making possible the categorization of the chromatin decompaction according to their location. The percentages of chromatin decompaction and heterogeneity were also evaluated. IVPEs were done and the rates of cleavage and of blastocysts were correlated with the chromatin characteristics. It made possible the classification of the chromatin decompaction according to the head affected part in at least four types: base decompaction, basal half decompaction, central axis decompaction, total decompaction. Based on the correlation, it can be implicated that each type of classification has different influences on the bull fertility. It made possible understanding that sperms amount with 5% or more of chromatin decompaction intensity interferes in the bull fertility and this condition can be featured as an uncompensable defect, while the heterogeneity of chromatin is not an important factor in the IVPE results.

Sperm-Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus-Oocyte Complex.

We have recently described a new model to study gamete interaction in mammalian species. The model recreates the spherical surface of the oocyte by using magnetic Sepharose beads coated with a layer of a recombinant protein involved in gamete interaction (such as ZP2, or the IZUMO1 receptor JUNO) and an external layer of cumulus oophorus cells, thus mimicking, to some extent, a native cumulus-oocyte complex. Once generated, this 3D model can be used in a sperm-binding assay to obtain valuable information about the molecular basis of gamete interaction, since different recombinant proteins can be used to coat the bead surface, thus generating a variety of models to be used for several species. Furthermore, thanks to the ability of the model to decoy sperm, the physiological status of the bound sperm can be studied, making this a powerful tool to select sperm with high fertilizing capacity, to unmask subfertile animals in livestock breeding centers, or for toxicological studies. Here, we describe how to generate and use this model for sperm-binding assays, using porcine sperm as an example, and ZP2, a protein from zona pellucida, as the recombinant protein of interest. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of the in vitro 3D model Alternate Protocol 1: Binding cumulus oophorus cells to the model Basic Protocol 2: Quality control of the model by SDS-PAGE electrophoresis and western blot Support Protocol 1: Immunochemistry to confirm proper protein distribution on surface of beads Support Protocol 2: Elution of recombinant conjugated proteins Basic Protocol 3: Sperm-binding assay Alternate Protocol 2: Sperm preparation by the swim-up method.

Weighted Gene Correlation Network Meta-Analysis Reveals Functional Candidate Genes Associated with High- and Sub-Fertile Reproductive Performance in Beef Cattle.

Improved reproductive efficiency could lead to economic benefits for the beef industry, once the intensive selection pressure has led to a decreased fertility. However, several factors limit our understanding of fertility traits, including genetic differences between populations and statistical limitations. In the present study, the RNA-sequencing data from uterine samples of high-fertile (HF) and sub-fertile (SF) animals was integrated using co-expression network meta-analysis, weighted gene correlation network analysis, identification of upstream regulators, variant calling, and network topology approaches. Using this pipeline, top hub-genes harboring fixed variants (HF × SF) were identified in differentially co-expressed gene modules (DcoExp). The functional prioritization analysis identified the genes with highest potential to be key-regulators of the DcoExp modules between HF and SF animals. Consequently, 32 functional candidate genes (10 upstream regulators and 22 top hub-genes of DcoExp modules) were identified. These genes were associated with the regulation of relevant biological processes for fertility, such as embryonic development, germ cell proliferation, and ovarian hormone regulation. Additionally, 100 candidate variants (single nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs)) were identified within those genes. In the long-term, the results obtained here may help to reduce the frequency of subfertility in beef herds, reducing the associated economic losses caused by this condition.

Copy-number variation introduced by long transgenes compromises mouse male fertility independently of pachytene checkpoints.

Long transgenes are often used in mammalian genetics, e.g., to rescue mutations in large genes. In the course of experiments addressing the genetic basis of hybrid sterility caused by meiotic defects in mice bearing different alleles of Prdm9, we discovered that introduction of copy-number variation (CNV) via two independent insertions of long transgenes containing incomplete Prdm9 decreased testicular weight and epididymal sperm count. Transgenic animals displayed increased occurrence of seminiferous tubules with apoptotic cells at 18days postpartum (dpp) corresponding to late meiotic prophase I, but not at 21 dpp. We hypothesized that long transgene insertions could cause asynapsis, but the immunocytochemical data revealed that the adult transgenic testes carried a similar percentage of asynaptic pachytene spermatocytes as the controls. These transgenic spermatocytes displayed less crossovers but similar numbers of unrepaired meiotic breaks. Despite slightly increased frequency of metaphase I spermatocytes with univalent chromosome(s) and reduced numbers of metaphase II spermatocytes, cytological studies did not reveal increased apoptosis in tubules containing the metaphase spermatocytes, but found an increased percentage of tubules carrying apoptotic spermatids. Sperm counts of subfertile animals inversely correlated with the transcription levels of the Psmb1 gene encoded within these two transgenes. The effect of the transgenes was dependent on sex and genetic background. Our results imply that the fertility of transgenic hybrid animals is not compromised by the impaired meiotic synapsis of homologous chromosomes, but can be negatively influenced by the increased expression of the introduced genes.

Analysis of the uterine lumen in fertility-classified heifers: II. Proteins and metabolites†.

Survival and growth of the bovine conceptus is dependent on endometrial secretions or histotroph. Previously, serial blastocyst transfer was used to classify heifers as high fertile (HF), subfertile (SF), or infertile (IF). Here, we investigated specific histotroph components (proteins and metabolites) in the uterine lumen of day 17 fertility-classified heifers. Interferon tau (IFNT) was more abundant in uterine lumenal fluid (ULF) of pregnant HF than SF animals as the conceptus was longer in HF heifers. However, no differences in endometrial expression of selected classical and nonclassical interferon-stimulated genes (ISGs) were observed, suggesting that IFNT signaling in the endometrium of pregnant HF and SF heifers was similar. Pregnancy significantly increased the abundance of several proteins in ULF. Based on functional annotation, the abundance of a number of proteins involved in energy metabolism, oxidative stress, amino acid metabolism, and cell proliferation and differentiation were greater in the ULF of pregnant HF than SF heifers. Metabolomics analysis found that pregnancy only changed the metabolome composition of ULF from HF heifers. The majority of the metabolites that increased in the ULF of pregnant HF as compared to SF heifers were associated with energy and amino acid metabolism. The observed differences in ULF proteome and metabolome are hypothesized to influence uterine receptivity with consequences on conceptus development and survival in fertility-classified heifers.

BEEF SPECIES-RUMINANT NUTRITION CACTUS BEEF SYMPOSIUM: Maternal immune modulation prior to embryo arrival in the uterus is important for establishment of pregnancy in cattle1.

In 1953, Sir Peter Medawar first recognized the allogeneic properties of a developing conceptus and rationalized that an "immune-tolerant" physiological state must exist during pregnancy. Early theories speculated that the conceptus evaded the maternal immune system completely, but 40 yr after Medawar's observations, Wegmann proposed that the maternal immune system shifts the cytokine profile away from inflammatory cytokine production when an embryo is present. The economic consequences and production losses of subfertile animals have been well documented in studies evaluating calving distribution. Despite advances in understanding infertility or subfertility, few technologies exist to identify subfertile animals or improve fertility beyond hormonal intervention associated with synchronization protocols. Work in rodents and some livestock species indicates that the uterine immune cell population shifts dramatically after copulation and these early immune-modulated events establish a receptive uterine environment. Clearly, as evident in embryo transfer, the presence of a conceptus is sufficient to establish communication for pregnancy establishment but does not rule out the importance of other physiological events to prime the maternal immune system prior to blastocyst arrival in the uterus. In support of this concept, work in our laboratory and by others has demonstrated that autologous intrauterine transfer of peripheral immune cells prior to embryo transfer can increase pregnancy rates and accelerate conceptus development in women and cattle. Understanding aberrant immune regulation in subfertile animals may provide markers for subfertility or targets for clinical intervention to enhance fertility, particularly when using reproductive technologies.

Chronic inflammatory and degenerative endometrial lesions in subfertile Criollo Limonero cattle; a B. taurus Latin-American breed threatened with extinction; A case-control study

Criollo Limonero is a tropical Bos taurus breed for sustainable dual purpose (milk and beef) production in the South-American tropics, which is currently threatened with extinction. The objective was to perform a clinical evaluation and histopathological assessment of uterine biopsy samples of repeat breeder (RB) Criollo Limonero cattle to determine the occurrence of pathological conditions as potential causes of subfertility. Twenty-four Criollo Limonero cattle [18 cows (5–13 years old) and 6 heifers (6–7.5 years old)] that had failed to conceive after four or more services were considered for this study. Additionally, five cows with history of adequate reproductive performance were used as a control group. Animals were submitted to physical exam, vaginoscopy, and ultrasonographical evaluation of the reproductive tract. Uterine biopsy samples were collected for histopathological evaluation. Vaginoscopy revealed that 41.7% of the RB cattle had abnormal vaginal secretions, while abnormal secretions were not observed in any control cow. Ultrasonographical examination of the uterus revealed the presence of free uterine fluid in 20.8% of the RB animals, while none of the control cows had fluid in the uterine lumen. In addition, ovarian cysts were observed in 25.0% of the RB animals. Histopathological evaluation of the endometrial biopsies revealed that mononuclear leukocyte infiltration, dilated uterine glands, and periglandular fibrosis were the most prevalent lesions in the sub-fertile animals. Chronic endometritis characterized by inflammatory (mononuclear leukocyte infiltration) and degenerative (dilated glands and periglandular fibrosis) endometrial lesions, and ovarian cysts were the most frequent reproductive pathologies observed in the studied subfertile Criollo Limonero cattle, suggesting a strong association with their reduced fertility.

Uterine influences on conceptus development in fertility-classified animals

A major unresolved issue is how the uterus influences infertility and subfertility in cattle. Serial embryo transfer was previously used to classify heifers as high-fertile (HF), subfertile (SF), or infertile (IF). To assess pregnancy loss, two in vivo-produced embryos were transferred into HF, SF, and IF heifers on day 7, and pregnancy outcome was assessed on day 17. Pregnancy rate was substantially higher in HF (71%) and SF (90%) than IF (20%) heifers. Elongating conceptuses were about twofold longer in HF than SF heifers. Transcriptional profiling detected relatively few differences in the endometrium of nonpregnant HF, SF, and IF heifers. In contrast, there was a substantial difference in the transcriptome response of the endometrium to pregnancy between HF and SF heifers. Considerable deficiencies in pregnancy-dependent biological pathways associated with extracellular matrix structure and organization as well as cell adhesion were found in the endometrium of SF animals. Distinct gene expression differences were also observed in conceptuses from HF and SF animals, with many of the genes decreased in SF conceptuses known to be embryonic lethal in mice due to defects in embryo and/or placental development. Analyses of biological pathways, key players, and ligand-receptor interactions based on transcriptome data divulged substantial evidence for dysregulation of conceptus-endometrial interactions in SF animals. These results support the ideas that the uterus impacts conceptus survival and programs conceptus development, and ripple effects of dysregulated conceptus-endometrial interactions elicit loss of the postelongation conceptus in SF cattle during the implantation period of pregnancy.

Isolation and identification of proteins from swine sperm chromatin and nuclear matrix

The aim of this study was to perform a proteomic analysis to isolate and identify proteins from the swine sperm nuclear matrix to contribute to a database of swine sperm nuclear proteins. We used prechilled diluted semen from seven boars (19 to 24 weekold) from the commercial line Landrace x Large White x Pietran. The semen was processed to separate the sperm heads and extract the chromatin and nuclear matrix for protein quantification and analysis by mass spectrometry, by LTQ Orbitrap ELITE mass spectrometer (Thermo-Finnigan) coupled to a nanoflow chromatography system (LC-MS/MS). We identified 222 different proteins in the sample; a total of 159 (71.6%) were previously described as present in the somatic or sperm nuclei of other species, 41 (18.5%) did not have a previously reported nuclear presence and 22 (9.9%) had not been characterized. The most abundant family of proteins corresponded to ribosomal (13.1%), followed by cytoskeleton (12.2%), uncharacterized (9.9%), histones (5.4%), proteasome subunits (3.6%) and heat shock (1.8%). The other proteins clustered in other families accounted for 54% of the total proteins. The protein isolation of the nuclear matrix of the swine spermatozoa was satisfactory, thus demonstrating that the protocol used was efficient. Several proteins were identified and described. However, it was not possible to identify some protein structures. Therefore, this study helps to establish a starting point for future proteomic studies comparing fertile and sub-fertile animals.

Evaluación de la aptitud reproductiva del toro

&lt;p&gt;La evaluación de la aptitud reproductiva del toro es un examen sencillo, práctico y económico que permite identificar animales subfértiles e infértiles que pueden llegar a afectar el éxito de un programa reproductivo; en consecuencia, debería realizarse de manera periódica, sin embargo, su utilización en nuestro país es baja. Esta evaluación involucra tres pasos fundamentales: el examen físico (conformación general, órganos sexuales internos y externos), la evaluación de la libido y la colecta y evaluación de semen. Se realiza una revisión de los aspectos que contemplan la evaluación reproductiva del toro, con el fin de determinar la aptitud reproductiva de los machos destinados para tal fin.&lt;/p&gt;

A Nonsense Mutation in TMEM95 Encoding a Nondescript Transmembrane Protein Causes Idiopathic Male Subfertility in Cattle

Genetic variants underlying reduced male reproductive performance have been identified in humans and model organisms, most of them compromising semen quality. Occasionally, male fertility is severely compromised although semen analysis remains without any apparent pathological findings (i.e., idiopathic subfertility). Artificial insemination (AI) in most cattle populations requires close examination of all ejaculates before insemination. Although anomalous ejaculates are rejected, insemination success varies considerably among AI bulls. In an attempt to identify genetic causes of such variation, we undertook a genome-wide association study (GWAS). Imputed genotypes of 652,856 SNPs were available for 7962 AI bulls of the Fleckvieh (FV) population. Male reproductive ability (MRA) was assessed based on 15.3 million artificial inseminations. The GWAS uncovered a strong association signal on bovine chromosome 19 (P = 4.08×10−59). Subsequent autozygosity mapping revealed a common 1386 kb segment of extended homozygosity in 40 bulls with exceptionally poor reproductive performance. Only 1.7% of 35,671 inseminations with semen samples of those bulls were successful. None of the bulls with normal reproductive performance was homozygous, indicating recessive inheritance. Exploiting whole-genome re-sequencing data of 43 animals revealed a candidate causal nonsense mutation (rs378652941, c.483C>A, p.Cys161X) in the transmembrane protein 95 encoding gene TMEM95 which was subsequently validated in 1990 AI bulls. Immunohistochemical investigations evidenced that TMEM95 is located at the surface of spermatozoa of fertile animals whereas it is absent in spermatozoa of subfertile animals. These findings imply that integrity of TMEM95 is required for an undisturbed fertilisation. Our results demonstrate that deficiency of TMEM95 severely compromises male reproductive performance in cattle and reveal for the first time a phenotypic effect associated with genomic variation in TMEM95.

DNA methylation is correlated with gene expression during early pregnancy in Bos taurus

Coordinated regulation of endometrial gene expression is essential for successful pregnancy establishment. A nonreceptive uterine environment may be a key contributor to pregnancy loss, as the majority of pregnancy losses occur prior to embryo implantation. DNA methylation has been highlighted as a potential contributor in regulating early pregnancy events in the uterus. It was hypothesized that DNA methylation regulates expression of key genes in the uterus during pregnancy. The correlation between DNA methylation and gene expression was tested. Endometrial samples from fertile and subfertile dairy cow strains were obtained at day 17 of pregnancy or the reproductive cycle. Microarrays were used to characterize genome-wide DNA methylation profiles and data compared with previously published transcription profiles. 39% of DNA methylation probes assayed mapped to RefSeq genes with transcription measurements. Correlations among gene expression and DNA methylation were assessed, and the 1,000 most significant correlations used for subsequent analysis. Of these, 52% percent were negatively correlated with gene expression. When this gene list was compared with previously reported gene expression studies on the same tissues, 42% were differentially expressed when pregnant and cycling animals were compared, and 11% were differentially expressed when pregnant fertile and subfertile animals were compared. DNA methylation status was correlated with gene expression in several pathways implicated in early pregnancy events. Although these data do not provide direct evidence of a causative association between DNA methylation and gene expression, this study provides critical support for an effect of DNA methylation in early pregnancy events and highlights candidate genes for future studies.

Medidas corporales y concentración sérica y folicular de lípidos y glucosa en vacas Brahman fértiles y subfértiles

El objetivo de este estudio fue evaluar las diferencias en las medidas corporales en vacas Brahman fértiles y subfértiles, establecer las diferencias en la concentración de colesterol, lipoproteínas y glucosa en suero y líquido folicular, y desarrollar un modelo para predecir subfertilidad en vacas Brahman a partir de las medidas corporales. Se seleccionaron vacas Brahman registradas, agrupadas en fértiles (15) y subfértiles (15) según historial reproductivo. Se tomaron muestras de suero y líquido folicular (diámetro &gt;8 mm) para determinar colesterol, lipoproteínas, triacilgliceroles y glucosa. Las vacas subfértiles mostraron un fenotipo masculino, con medidas corporales mayores, y concentraciones de colesterol y HDL séricos y foliculares más bajas que las vacas fértiles. El colesterol y el HDL se correlacionaron positivamente entre los compartimientos en ambos grupos. La subfertilidad es más probable en vacas pesadas con hombros amplios y mayor perímetro torácico. El tamaño corporal en vacas Brahman subfértiles se relacionó con su apariencia, con cambios en los metabolitos séricos y foliculares y con las concentraciones de colesterol y HDL.

IDENTIFICATION OF FERTILITY MARKERS IN EQUINE SEMEN USING PROTEOMICS TECHNIQUES

Current semen evaluation techniques are useful in identifying subfertile animals, however they are not reliable indicators of fertility. As sperm and seminal plasma proteins have known effects on both in vivo and in vitro fertilization, their potential as markers of semen quality and stallion fertility are important areas of research. The objectives of this study were to determine if specific proteins in the stallion semen could be related to semen quality characteristics and in vivo fertility using proteomics techniques. Seven fertile stallions were regularly collected and bred to a total of 142 mares for the 2006 breeding season. Fresh semen for the study was collected on three occasions in the middle of the breeding season (Sandy Ridge Stallion Station, Bassano, Alberta), each two weeks apart, and the sperm and seminal plasma were immediately separated by centrifugation and frozen until subjected to 2D gel electrophoresis. The sperm and seminal plasma proteins were separated on 24 cm 12 % acrylamide gels, and pH 3–10 in the first dimension (GE Healthcare, Baie d'Urfé, QC). All protein species were analyzed and matched across stallions using Progenesis software (Non-Linear Dynamics, Durham, NC). The quantified proteins were compared to visual semen quality characteristics and in vivo fertility using the mixed models procedure with repeated measures as well as correlation analysis in SAS (SAS Institute, Cary, NC). The stallions ranged from 50 % to 100 % for first cycle pregnancy rate, and overall pregnancy rate ranged from 75 to 100 %. When semen quality was assessed, there were differences (P<0.05) in sperm concentration, total number of sperm and semen volume. There were no differences reported in progressively motile sperm (PMS), however there were replicates where stallions showed large variation in their motility, down to 35 % PMS. Only preliminary data has been obtained for the proteomics analysis at this time. Over 3000 proteins for each gel were visualized and quantified using the Progenesis software, and 32 proteins in the spermatozoa and 8 proteins in seminal plasma differed across stallions (P<0.05). For the seminal plasma, 3 proteins were positively correlated with PMS, and 5 were negatively correlated with PMS (P<0.05). Interestingly, none of these proteins were correlated with fertility, however 14 different proteins were either positively or negatively correlated to fertility. For sperm proteins, 3 proteins were positively correlated with PMS and 9 were negatively correlated with PMS (P<0.05). All proteins of interest will be identified and verified using mass spectrometry techniques. Candidate protein markers reported in previous studies, such as osteopontin, will also be characterized in these samples. Subjecting equine sperm and seminal plasma to proteomics techniques has revealed many potential candidates of fertility in both sperm and seminal plasma in preliminary results. Determination of a reliable marker that is associated with stallion fertility would be very useful for screening stallions for breeding potential and also for the improvement of reproductive technologies. (poster)

The quantification of lipid and protein oxidation in stallion spermatozoa and seminal plasma: Seasonal distinctions and correlations with DNA strand breaks, classical seminal parameters and stallion fertility

The goal of this work was to correlate oxidative stress caused by reactive oxygen species (ROS) and DNA damage with classic semen parameters in spermatozoa and seminal plasma of fertile and subfertile stallions. Oxidation was measured in both lipids and proteins, using the thiobarbituric acid reactive species (TBARS) assay and the DNPH carbonyl groups assay, respectively. Sperm DNA damage was monitored using the TUNEL assay. These parameters were monitored in samples obtained during the breeding and the non-breeding seasons. In general, fertile stallions showed better classical semen parameters, and those parameters improved from the non-breeding to the breeding season, although an increase in sperm production was accompanied by a decrease in the semen quality from subfertile stallions in the breeding season. In terms of oxidation levels we found that there were clear differences whether lipids or proteins were considered. In the breeding season there seemed to be a tendency towards normalizing lipid oxidation in spermatozoa and seminal plasma, and protein oxidation in the seminal plasma, of both fertile and subfertile animals. Thus, differences monitored in the non-breeding season were no longer visible. Interestingly, a higher level of protein oxidation was found in the sperm of fertile animals in the breeding season. Considering that there were positive correlations between sperm protein oxidation and sperm motility and vitality, these results suggests that the oxidation of semen proteins may be important for sperm function. On the other hand, lipid oxidation in the seminal plasma seemed to be a general indicator for sperm damage. In the non-breeding season positive correlations between lipid and protein oxidation levels in both sperm and seminal plasma and several defects in sperm function were found, but only for subfertile animals, thus suggesting that lipid and protein oxidation may aid in the identification of subfertile stallions during the non-breeding season. Levels of ROS production never seemed to result in compromised sperm DNA integrity, indicating that measurements were within physiological levels and/or that there is an efficient antioxidant activity in stallion sperm cells.

Capacitation, acrosome function and chromatin structure in stallion sperm

In general, fertility in breeding stallions is lower and more variable than in the other farm animal species, primarily because selection is based on pedigree, looks and/or athletic performance, with little consideration of fertility or fertility potential. Moreover, because the average stallion breeds only a limited number of mares per year and in-field fertility is influenced significantly by non-stallion factors such as management and mare fertility, meaningful fertility data are hard to come-by. Unfortunately, generating usable figures would involve impractically high costs, time and numbers of mares. Instead, a breeding soundness examination (BSE), based on assessments of sperm number, motility and morphological normality and of mating ability, is often carried out with the ostensible aim of identifying animals with the “potential for good fertility”. In fact, the BSE generally succeeds only in ruling out those stallions with a very clear reason for sub-fertility, and still fails to identify some seriously sub-fertile animals. Thus, the routine BSE has very limited use as a predictor of subsequent fertility. This paper reviews assays developed for identifying capacitated, acrosome-reacted and DNA-damaged sperm, and assesses their utility for improving our ability to predict a stallion's fertility prior to the onset of his breeding career.

Differential susceptibility to chromatid breaks induced by bleomycin in sub-fertile and fertile bovines

The rate of chromatid breaks was studied in cows with a history of sub-fertility by means of a test based on measurement of the average of breaks induced in lymphocytes of peripheral blood cultures. Fourteen female specimens were divided into two groups: fertile and sub-fertile. Peripheral blood lymphocytes were cultured and prepared for cytogenetic analysis. Two types of culture were established for each animal to evaluate the response of peripheral blood lymphocyte cultures to the genotoxic effects of bleomycin. The first culture did not receive bleomycin treatment (spontaneous chromosome aberrations). Our results showed that median breaks per cell (b/c) (± semirange) for spontaneous culture of the fertile and sub-fertile animals and bleomycin sensitivity assay for fertile and sub-fertile animals were 0.00 ± 0.06, 0.02 ± 0.03, 0.08 ± 0.05 and 0.22 ± 0.09, respectively. There was no significant difference ( P > 0.05) in the chromosomal breakage in lymphocytes not exposed to bleomycin; however, in comparing the number of chromatid breaks per cell in cultures treated with bleomycin, the sub-fertile group showed a significantly higher ( P < 0.05) level than the fertile group. These findings have implications both for identifying cattle with less than optimum fertility as well as for providing potential avenues to study the origins of sub-fertility.