High Fertility Bulls Research Articles

Male fertility status is associated with DNA methylation signatures in sperm and transcriptomic profiles of bovine preimplantation embryos

BackgroundInfertility in dairy cattle is a concern where reduced fertilization rates and high embryonic loss are contributing factors. Studies of the paternal contribution to reproductive performance are limited. However, recent discoveries have shown that, in addition to DNA, sperm delivers transcription factors and epigenetic components that are required for fertilization and proper embryonic development. Hence, characterization of the paternal contribution at the time of fertilization is warranted. We hypothesized that sire fertility is associated with differences in DNA methylation patterns in sperm and that the embryonic transcriptomic profiles are influenced by the fertility status of the bull. Embryos were generated in vitro by fertilization with either a high or low fertility Holstein bull. Blastocysts derived from each high and low fertility bulls were evaluated for morphology, development, and transcriptomic analysis using RNA-Sequencing. Additionally, DNA methylation signatures of sperm from high and low fertility sires were characterized by performing whole-genome DNA methylation binding domain sequencing.ResultsEmbryo morphology and developmental capacity did not differ between embryos generated from either a high or low fertility bull. However, RNA-Sequencing revealed 98 genes to be differentially expressed at a false discovery rate < 1%. A total of 65 genes were upregulated in high fertility bull derived embryos, and 33 genes were upregulated in low fertility derived embryos. Expression of the genes CYCS, EEA1, SLC16A7, MEPCE, and TFB2M was validated in three new pairs of biological replicates of embryos. The role of the differentially expressed gene TFB2M in embryonic development was further assessed through expression knockdown at the zygotic stage, which resulted in decreased development to the blastocyst stage. Assessment of the epigenetic signature of spermatozoa between high and low fertility bulls revealed 76 differentially methylated regions.ConclusionsDespite similar morphology and development to the blastocyst stage, preimplantation embryos derived from high and low fertility bulls displayed significant transcriptomic differences. The relationship between the paternal contribution and the embryonic transcriptome is unclear, although differences in methylated regions were identified which could influence the reprogramming of the early embryo. Further characterization of paternal factors delivered to the oocyte could lead to the identification of biomarkers for better selection of sires to improve reproductive efficiency.

Oligo-dT selected spermatozoal transcript profiles differ among higher and lower fertility dairy sires

Spermatozoal messenger RNA (mRNA) has the potential as a molecular marker for sire fertility because this population can reflect gene expression that occurred during spermatogenesis and may have a functional role in early embryonic development. The goal of this study was to compare the oligo-dT selected spermatozoal transcript profiles of higher fertility (Conception Rate (CR) 1.8–3.5) and lower fertility (CR −2.9 to −0.4) sires using Ribonucleic Acid Sequencing (RNA-Seq). A total of 3227 transcripts and 5366 transcripts were identified in the higher and lower fertility populations, respectively. While common transcripts between the two populations were identified (2422 transcripts), several transcripts were also unique to the fertility populations including 805 transcripts that were unique to the higher fertility population and 2944 transcripts that were unique to the lower fertility population. From gene ontological analysis, the transcripts unique to each fertility population differed in Biological Processes (BP), including enrichment of regulatory transcripts for growth and protein kinase activity in the higher fertility bulls. Biological variation in transcript presence among individual sires was also found. Of the candidate fertility spermatozoal transcripts chosen from the RNA-Seq population analysis reported here and previous publications, COX7C was negatively correlated with sire fertility. Using high-throughput sequencing, candidate spermatozoal transcripts were identified for further study as potential markers for sire fertility.

Effect of seminal plasma from high- and low-fertility bulls on cauda epididymal sperm function.

The aim of this study was to characterise the effect of seminal plasma (SP) from bulls of high or low fertility on sperm function. First, the effect of SP on the motility of fresh cauda epididymal spermatozoa (CES) and frozen-thawed ejaculated spermatozoa was assessed (Experiment 1a). Seminal plasma was then collected from bulls of known high and low fertility. Pooled CES were incubated in the SP from each bull, diluted and assessed for motility and viability on Days 1, 2, 3 and 5 after packaging as fresh semen (Experiment 1b). Also assessed were motility, kinematics, viability and mitochondrial membrane potential after thawing (Experiment 1c) as well as hypotonic resistance (Experiment 2) and fertilisation potential using in vitro fertilisation (Experiment 3). Seminal plasma increased the motility of CES (P<0.05); however, there was no effect of SP on the motility and viability of fresh CES or on CES post-thaw motility, viability and mitochondrial membrane potential (P>0.05). The hypotonic resistance of CES was reduced by SP (P<0.05), irrespective of whether the SP was from high- or low-fertility bulls. Seminal plasma from high- or low-fertility bulls had no effect on cleavage or blastocyst rates (P>0.05). In conclusion, SP affects the physiological function of CES but there is no difference between SP from high- or low-fertility bulls.

Involvement of calcium channels and intracellular calcium in bull sperm thermotaxis.

Thermotaxis that sperm migrate to higher temperature area has been confirmed in rabbit and human. In this study, we examined the migration ability of bull sperm in a temperature gradient to confirm thermotaxis and elucidate the involvement of calcium in such thermotaxis, as well as the relation between sperm capacitation and bull fertility. Thermotaxis was evaluated in a temperature gradient of 34–42ºC using a cross-type column 22-mm long, 40-mm wide, and 100-μm deep. Significantly more sperm migrated to the high-temperature area of 39ºC in a 2ºC temperature gradient, and to 40ºC in a 1ºC temperature gradient. In calcium-free, BAPTA containing medium, and EGTA containing medium, the migrated sperm ratio in the two temperature areas was almost the same. In media containing lanthanum, ruthenium red, and 2APB, we could not confirm thermotaxis. Pre- and post-capacitated sperm migrated to the high-temperature area, expressing thermotaxis. The sperm from high-fertility bulls showed clear thermotaxis. Based on these results, thermotaxis of bull sperm was confirmed and the involvement of both calcium channels and intracellular stored calcium in thermotaxis was suggested. Although the sample size of bulls was quite small, the difference in thermotaxis may have been associated with bull fertility. Sperm thermotaxis evaluation has potential as a predictor of bull fertility.

Estimation of endogenous levels of osteopontin, total antioxidant capacity and malondialdehyde in seminal plasma: Application for fertility assessment in buffalo (Bubalus bubalis) bulls.

This study was attempted to identify subfertile bulls by quantifying the endogenous levels of osteopontin (OPN), total antioxidant capacity (TAC) and malondialdehyde (MDA) in seminal plasma of buffalo bulls. On the basis of conception rate, buffalo bulls were classified into two groups: high-fertile (conception rate >50%) and subfertile bulls (conception rate <40%). A total of 100 ejaculates (10 ejaculates from each bull) were collected through artificial vagina method. The concentration of OPN, TAC and catalase (CAT) of high-fertile bulls was found to be higher (p<.05) than that of subfertile bulls. Further, MDA level in seminal plasma was found to be lower (p<.05) in high-fertile bulls compared with subfertile bulls. The fertility status had no effect on the superoxide dismutase (SOD) concentration in seminal plasma of both the groups. The levels of OPN (r=.678, p=0.013) and TAC (r=.648, p=.042) were found to be positively correlated with bull fertility and the level of MDA (r=-.718, p=.019) was found to be negatively correlated with bull fertility. However, the fertility of bulls was not found to be significantly correlated with SOD, CAT and sperm motility. In conclusion, seminal OPN, TAC and MDA tended to be more realistic in identification of subfertile bulls from breeding herds.

Identification of Beef Heifers with Superior Uterine Capacity for Pregnancy.

Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In study one, predominantly Angus heifers were classified based on fertility using serial embryo transfer to select animals with intrinsic differences in pregnancy loss. In each of the four rounds, a single in vitro-produced, high-quality embryo was transferred into heifers on Day 7 postestrus and pregnancy was determined on Days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In study two, fertility-classified heifers were resynchronized and bred with semen from a single high-fertility bull. Blood samples were collected every other day from Days 0 to 36 postmating. Pregnancy rate was determined on Day 28 by ultrasound and was higher in HF (70.4%) than in heifers with low fertility (36.8%; SF and IF). Progesterone concentrations in serum during the first 20 days postestrus were not different in nonpregnant heifers and also not different in pregnant heifers among fertility groups. In study three, a single in vivo-produced embryo was transferred into fertility-classified heifers on Day 7 postestrus. The uteri were flushed on Day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the Day 14 endometrial biopsies of pregnant HF, SF, and IF heifers (n = 5 per group) and analyzed by edgeR-robust analysis. There were 26 differentially expressed genes (DEGs) in the HF compared to SF endometrium, 12 DEGs for SF compared to IF endometrium, and three DEGs between the HF and IF endometrium. Several of the DEG-encoded proteins are involved in immune responses and are expressed in B cells. Results indicate that preimplantation conceptus survival and growth to Day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between Days 14 and 28 when pregnancy recognition signaling and conceptus elongation and implantation must occur for the establishment of pregnancy.

Computer assisted sperm analysis: Relationship between the movement characteristics of buffalo spermatozoa and sire fertility

The present study was undertaken to identify the differences in sperm kinematics between buffalo bulls with different fertility ratings. Murrah buffalo bulls (n=9) that were routinely used for breeding purpose under progeny testing programme were utilized for the study. Bull fertility was determined based on in vivo fertility trials and the conception rates (CR) were adjusted for different non-genetic parameters. Based on the adjusted CR, bulls were classified into high, medium and low fertile group. Frozen semen samples of these bulls were obtained and sperm kinematic parameters were assessed using a computer assisted sperm analyzer. The kinematic parameters analyzed included the curvilinear velocity (VCL), the linear velocity (VSL), the average path velocity (VAP), the amplitude of lateral head displacement (ALH), the linearity (LIN), the straightness coefficient (STR) and the beat cross frequency (BCF). In high fertile bulls, the proportion of motile spermatozoa was higher (p&lt;0.001) than the medium and low fertile bulls. The VAP and VCL of sperm motion were significantly higher (P&lt;0.05) in high fertile bulls compared to either medium or low fertile bulls. The VSL was significantly lower in low fertile bulls (P&lt;0.005) compared to either high or medium fertile bulls. Spermatozoa from high fertile bulls had significantly higher (P&lt;0.05) BCF, STR, ALH and LIN compared to either medium or low fertile bulls. Buffalo bull fertility was significantly and positively correlated with sperm motility, VAP, VSL, VCL and ALH.

Profiling of sperm proteins and association of sperm PDC-109 with bull fertility

The composition of sperm proteins influences the fertilizing ability of sperm and hence the present study was conducted (i) to profile sperm proteins expression patterns in bulls of differing fertility index and (ii) to identify and relate the abundant sperm proteins with bull fertility. The semen samples were collected from Holstein-Friesian bulls (n = 12) varying in conception rate (CR) (high/low). The frozen semen straws (three ejaculates, from each bull) were used to study (a) sperm kinetic parameters, (b) plasmalemma integrity, (c) mitochondrial membrane potential, and (d) chromatin distribution. Three bulls were randomly selected from each group (n = 3) and the neat sperm pellets were subjected to percoll purification, followed by protein isolation using 0.1% Triton X100. The sperm kinetic parameters, plasmalemma integrity, mitochondrial membrane potential, and the chromatin distribution did not differ significantly between groups. The number of acidic (pI; 3.1–5.6, 37%) and basic (pI; 7.9–10.0, 27%) proteins and their pattern of expression varied significantly (p < 0.05) between high and low fertile bulls. The abundant sperm protein spots in 2D-gel electrophoresis (2DE) were identified as seminal plasma protein PDC-109 (i.e., protein with N-terminus aspartic acid, D and carboxy terminus cystine, having 109 amino acids) and its isoform and spermadhesin-1 (SPADH1). The western blot analysis confirmed the presence of PDC-109 isoform proteins at 15.4 kDa (pI 5.3 and 5.5). The seminal plasma protein PDC-109 was abundant in the low fertile when compared to the high fertile group (p < 0.05). This study suggests that the imbalance in acidic and basic sperm proteins may influence sperm fertility and sperm PDC-109 levels above a certain threshold affects bull fertility.

Sperm superoxide dismutase is associated with bull fertility.

Decreasing mammalian fertility and sperm quality have created an urgent need to find effective methods to distinguish non-viable from viable fertilising spermatozoa. The aims of the present study were to evaluate expression levels of ?-tubulin 2C (TUBB2C), heat shock protein 10 (HSP10), hexokinase 1 (HXK1) and superoxide dismutase 1 (SOD1) in spermatozoa from Holstein bulls with varying fertility using western blotting and to analyse the biological networks of these key sperm proteins using a bioinformatics software (Metacore; Thomson-Reuters, Philadelphia, PA, USA). The rationales behind this study were that the sperm proteins play crucial roles in fertilisation and early embryonic development in mammals and ascertaining the biological networks of the proteins helps us better understand sperm physiology and early mammalian development. The results showed that expression of SOD1 was higher in spermatozoa from high fertility bulls (PPin vivo bull fertility. The findings are important because they illuminate molecular and cellular determinants of sperm viability and the identified protein markers can be used to determine bull fertility.

Sperm protamine-status correlates to the fertility of breeding bulls.

During fertilization, spermatozoa make essential contributions to embryo development by providing oocyte activating factors, centrosomal components, and paternal chromosomes. Protamines are essential for proper packaging of sperm DNA; however, in contrast to the studies of oocyte-related female infertility, the influence of sperm chromatin structure on male infertility has not been evaluated extensively. The objective of this study was to determine the sperm chromatin content of bull spermatozoa by evaluating DNA fragmentation, chromatin maturity/protamination, PRM1 protein status, and nuclear shape in spermatozoa from bulls with different fertility. Relationships between protamine 1 (PRM1) and the chromatin integrity were ascertained in spermatozoa from Holstein bulls with varied (high vs. low) but acceptable fertility. Sperm DNA fragmentation and chromatin maturity (protamination) were tested using Halomax assay and toluidine blue staining, respectively. The PRM1 content was assayed using Western blotting and in-gel densitometry, flow cytometry, and immunocytochemistry. Fragmentation of DNA was increased and chromatin maturity significantly reduced in spermatozoa from low-fertility bulls compared to those from high-fertility bulls. Field fertility scores of the bulls were negatively correlated with the percentage of spermatozoa displaying reduced protamination and fragmented DNA using toluidine blue and Halomax, respectively. Bull fertility was also positively correlated with PRM1 content by Western blotting and flow cytometry. However, detection of PRM1 content by Western blotting alone was not predictive of bull fertility. In immunocytochemistry, abnormal spermatozoa showed either a lack of PRM1 or scattered localization in the apical/acrosomal region of the nuclei. The nuclear shape was distorted in spermatozoa from low-fertility bulls. In conclusion, we showed that inadequate amount and localization of PRM1 were associated with defects in sperm chromatin structure, coinciding with reduced fertility in bulls. These findings are highly significant because they reveal molecular and morphological phenotypes of mammalian spermatozoa that influence fertility.

Developmental Potential of Embryos Produced In Vitro by Sperm from Bulls of Contrasting In Vivo Fertility and Ovocytes Retrieved from a Same Donor

Laboratory assessments of sperm traits are poor indicators of fertility. Because the variability in the quality of oocytes collected from different donors drastically influence in vitro embryo production, the aim of this work was to implement an in vitro model to compare the developmental potential of embryos produced by bulls of contrasting in vivo fertility and ovocytes retrieved from the same donor in order to minimize the female related variability. For each trial (n=54), one pair of good quality ovaries of a same donor were split longitudinally and oocytes were recovered by slicing method. Thereafter, matured oocytes were fertilized with sperm of two bulls of low estimated relative conception rates (ERCR +2) contrasting field fertility (6 vs. 6 bulls). Cleavage and blastocyst formation rates were compared. Sperm of high fertility bulls (ERCR+) gave also higher fertilization rates (cleavage) in vitro when compared to low fertility (ERCR-) bulls (odd ratio=1.23). Conversely, the embryonic development to the blastocyst stage was reduced (odd ratio=0.84) in the ERCR+ with respect to the ERCR- bulls. This paradoxical result demonstrates that in vivo bull fertility hardly correlates with in vitro blastocyst yield, but confirms that early events occurring at fertilization are better indicators of the fertility potential. Furthermore, this experimental approach indicates that differences in embryo production rates between bulls of contrasting field fertility may not be outlined in vitro even when bulls are compared using oocytes with variability limited to the same donor.

201 USING THE TRANSCRIPTOME SIGNATURE TO PREDICT PREGNANCY SUCCESS IN BEEF COWS AT 6 DAYS AFTER ARTIFICIAL INSEMINATION

Pregnancy success is critical to the profitability of cattle operations. Attempts to reduce high rates of early embryonic loss mainly focus on the critical phase of embryo recognition by maternal tissue. However, the molecular events driving the uterine tissue toward a favourable stage, facilitating the maternal receptivity, are poorly understood. This study aimed to characterise the endometrial transcriptome profiles of pregnant versus nonpregnant beef cows during early pregnancy and attempted to define a potential set of marker genes that can be valuable for predicting pregnancy outcome. Therefore, pluriparous, cyclic Nellore (Bos indicus) cows were synchronized (n = 51) and artificially inseminated (n = 36) at detected oestrus using semen from a single high-fertility bull. Six days after AI (Day 6), jugular blood samples and an endometrial biopsy from the uterine horn contralateral to the ovary containing the CL were collected. Pregnancy diagnosis was performed by ultrasonography on Days 22 and 30. Based on pregnancy outcome, samples were retrospectively allocated to the following groups: pregnant (P; n = 6) and nonpregnant (NP; n = 5). Both groups had similar plasma progesterone concentrations on Day 6 (less than 1 ng mL–1 between lowest and greatest concentrations). Endometrial biopsies were submitted to RNA-Seq analysis in an Illumina single flow cell line (Illumina Inc., San Diego, CA). The 272 685 768 million filtered reads were mapped to the Bos taurus UMD3.1 reference genome and 14 654 genes were effectively analysed for differential expression between groups. Transcriptome data showed that 216 genes are differently expressed when comparing P v. NP endometrial tissue (adjusted P &lt; 0.1). More specifically, 36 genes showed a significantly up-regulated expression for pregnant cows and 180 are up-regulated for non-pregant cows. Functional enrichment and pathway analyses revealed enriched expression of genes associated with extracellular matrix remodelling in NP cows and nucleotide binding, microsome, and vesicular fraction in P cows. From the 40 top-ranked genes, six that were down- and three that were up-regulated in pregnant cows were further analysed by qRT–PCR in an additional 26 cows. Subsequent quantitative expression data were evaluated using multivariate statistics. Both principal component analysis (PCA; R2 = 0.82 and Q2 = 0.40) and orthogonal projections to latent structures analysis (OPLS), using pregnancy as the dependent variable (R2Y = 0.95 and Q2 = 0.86), efficiently separated P from NP animals. In conclusion, this study characterizes a unique set of genes, expressed in the endometrium as early as 6 days after AI, that indicate a receptive state leading to pregnancy success. Furthermore, expression of such genes can be used as potential markers to efficiently predict pregnancy success.

Identification of putative fertility markers in seminal plasma of crossbred bulls through differential proteomics

Sub-fertility is a major problem in crossbred bulls leading to disintegration of breeding systems and huge economic loss. Identification of some potential biomarkers to determine the latent fertility of bulls accurately has long been the interest of researchers. In this study, we analyzed the proteome of seminal plasma (SP) from bulls with varying fertility to identify the fertility-associated proteins. The proteomic profile of high- and low-fertile bulls was compared by two-dimensional difference gel electrophoresis and differentially expressed proteins were identified through matrix-assisted laser desorption/ionization–time of flight/mass spectrometry. Out of the 18 differentially expressed proteins (P < 0.05), 9 were overexpressed in SP of high-fertile bulls and 9 were overexpressed in SP of low-fertile bulls. The differential expressions ranged from 1.5- to 5.5-fold between the two groups, where protection of telomeres-1 protein (POT1) was highly overexpressed (2.9-fold) in high-fertile group and prostaglandin E2 receptor EP3 (PTGER3) was highly abundant (5.5-fold) in low-fertile group. The protein interaction network was elucidated using STRING software tool, and the functional bioinformatics analysis was done using Blast2Go software. Most of the differentially expressed proteins were found to be involved in cellular processes and biological regulation with binding and catalytic function. It is inferred that the expression of certain proteins in the SP varied with bull fertility, and concurrent appraisal of their expression along with other fertility assays may help in determining bull fertility.

Molecular morphology and function of bull spermatozoa linked to histones and associated with fertility

Sub-par fertility in bulls is influenced by alterations in sperm chromatin, and it might not be solved with increased sperm concentration in artificial insemination. Appropriate histone retention during sperm chromatin condensation plays critical roles in male fertility. The objective of this study was to determine failures of sperm chromatin condensation associated with abnormal persistence or accessibility of histones by aniline blue (ANBL) test, expression levels, and cellular localizations of one variant and two core histones (H3.3, H2B, and H4 respectively) in the spermatozoa of low-fertility (LF) vs high-fertility (HF) bulls. The expression levels and cellular localizations of histones in spermatozoa were studied using immunoblotting, immunocytochemistry, and staining methods. The bioinformatics focused on the sequence identity and evolutionary distance of these proteins among three mammalian species: bovine, mouse, and human. We demonstrated that ANBL staining was different within the LF (1.73 (0.55, 0.19)) and HF (0.67 (0.17, 0.06)) groups (P<0.0001), which was also negatively correlated with in vivo bull fertility (r=-0.90, P<0.0001). Although these histones were consistently detectable and specifically localized in bull sperm cells, they were not different between the two groups. Except H2B variants, H3.3 and H4 showed 100% identity and were evolutionarily conserved in bulls, mice and humans. The H2B variants were more conserved between bulls and humans, than in mice. In conclusion, we showed that H2B, H3.3, and H4 were detectable in bull spermatozoa and that sperm chromatin condensation status, changed by histone retention, is related to bull fertility.

Associations of adiponectin and fertility estimates in Holstein bulls

Adiponectin is a pleiotropic regulator of numerous biological functions, including gonadal steroidogenesis and might play a role in sperm structures and functions. The objectives were to: (1) determine associations among serum concentrations of adiponectin, testosterone, and prolactin, and the sperm DNA fragmentation index; (2) associate sperm adiponectin mRNA abundance with estimates of fertility (sire conception rate); and (3) determine sperm protein expression of adiponectin and its receptor in pre- and postcapacitated sperm from Holstein bulls. In experiment 1, biweekly serum concentrations of adiponectin, prolactin, and testosterone were greater (P < 0.05) for high fertility bulls compared with average and low fertility bulls. Furthermore, sperm DNA fragmentation index was greater (P < 0.05) for low fertility compared with both average and high fertility bulls. In experiment 2, samples of sperm from a single collection from commercial Holstein bulls (N = 34) were used to evaluate relative sperm mRNA expression of adiponectin and its receptors, AdipoR1 and AdipoR2, and protein levels of adiponectin and its receptors, AdipoR1 and AdipoR2, in pre- and postcapacitation sperm. The mRNA abundance of adiponectin and its receptors, AdipoR1 and AdipoR2, were greater for high fertility bulls (>2 to ≤4 sire conception rate) compared with average (≥2 to ≤2) and low (>−2 to ≤−4) fertility bulls. Based on the sperm capacitation assay, average fertility bulls had a greater percentage of acrosome-reacted sperm at 5 hours than high and low fertility bulls, whereas high fertility bulls had a greater percentage of acrosome-reacted sperm than low fertility bulls. After capacitation, levels of adiponectin protein were lower in average fertility bulls, AdipoR1 was lower in all fertility groups, and AdipoR2 was lower in average and high fertility bulls. In conclusion, adiponectin and its receptors had vital roles in sperm structural and functional traits and consequently they were associated with fertility. In addition to its role in steroidogenesis and sperm capacitation, adiponectin might be involved in sperm-egg fusion and fertilization.

66 BUFFALO-BULL SEMEN-FERTILITY EVALUATION IN RELATION TO MOTILITY AND INTEGRITY OF ACROSOME, PLASMA MEMBRANE, AND SPERM DNA

The breeding-soundness examination is conducted to identify and select bulls with an acceptable reproductive efficiency. In buffalo, there is meagre information regarding fertility index in relation to sperm attributes so that a future breeding bull could be selected at the age of maturity. So, to predict the fertility of buffalo breeding bulls, the present study was conducted to assess the motility parameters and integrity of acrosome, plasma membrane, and DNA of cryopreserved semen of high- and low-fertile bulls. The fertility of bulls was classified on the basis of conception rates, where buffalo bulls with conception rate &lt;35% were considered as low-fertility bulls and those with conception rate &gt;55% were considered as high-fertility bulls. A computer-assisted semen analyser was used for motility and viability studies, whereas integrity of acrosome, plasma membrane, and DNA were assessed by Pisum sativum agglutinin–fluorescein isothiocyanate, Annexin-V/PI, and TUNEL assay kit, respectively, under the florescence microscope. At least 200 spermatozoa were evaluated from each group, and results were analysed by ANOVA. The level of significance was observed at P &lt; 0.05. The mean (±SE) values for various motility parameters of sperm for high-fertile bulls were total motility (56.8 ± 3.2%), average path velocity (87.22 ± 1.6 µm s–1), straight linear velocity (68.93 ± 1.9 µm s–1), and curvilinear velocity (156.52 ± 4.3 µm s–1). These values were significantly higher than those of low-fertile bulls (43.8 ± 1.7%, 79.02 ± 2.4 µm s–1, 63.42 ± 1.2 µm s–1, and 142.37 ± 2.8 µm s–1, respectively). The amplitude of lateral head displacement (6.8 ± 0.07 v. 6.5 ± 0.1 µm), beat cross frequency (33.9 ± 0.4 v. 33.43 ± 0.5 Hz), straightness (79.2 ± 0.7 v. 78.7 ± 0.6%), linearity (45.5 ± 0.4 v. 45.5 ± 0.7%), and viability (71.2 ± 0.8 v. 68.9 ± 0.8%) did not differ in both groups. The average percentage of intactness of sperm acrosome of high-fertile bulls was significantly higher (81.82 ± 0.87%) than that of low-fertility bulls (76.86 ± 0.87%). Furthermore, to assess the functionality of plasma membrane of sperm, we analyzed different stages of apoptotic-like events. The percentage of apoptotic sperm differed significantly between high-fertility (15.59 ± 0.75%) and low-fertility (25.94 ± 0.5%) bulls. The percentages of early necrotic, necrotic, and viable sperm did not differ in 2 groups. The DNA integrity in high-fertility (90.24 ± 0.94%) and low-fertility (88.37 ± 0.91%) bulls was not significantly different. In conclusion, the various parameters such as average path velocity, curvilinear velocity, straight linear velocity, and total motility; acrosomal integrity; and percentage of apoptotic sperm are useful for evaluating the semen quality of a bull to reduce the risk of using poor-fertility bulls in an AI program.

Association between mRNA abundance of functional sperm function proteins and fertility of Holstein bulls

Although the existence of a complex population of mRNA in sperm is well documented, its role has not been completely elucidated. The objective of this study was to determine the relationship of mRNA abundance of sperm specific proteins and sire conception rates (SCR; a fertility index) in Holstein bulls. Samples of sperm from a single collection from commercial Holstein bulls (N = 34) were used to evaluate relative mRNA expression of adenylate kinase (AK) 1, integrin beta (IB) 5, Doppel, nerve growth factor, tissue inhibitors of metalloproteinases (TIMP) 2, lactate dehydrogenase C 1, small nuclear ribonucleoprotein polypeptide N, outer dense fiber 2, and phospholipase C zeta (PLCz) 1 in sperm. With the exception of lactate dehydrogenase C 1 and outer dense fiber 2, the mRNA abundances of these proteins were greater (P < 0.05) for high fertility (> +2 to ≤ 4 SCR) bulls compared with average (≥ 2 to ≤ +2) and low fertility (> −2 to ≤ −4) bulls. Of all the multivariate regression models tested, a combination of AK1, IB5, TIMP2, small nuclear ribonucleoprotein polypeptide N, and PLCz1 accounted for 97.4% of the variance in SCR scores. In the absence of PLCz1, the combination of AK1, IB5, Doppel, nerve growth factor, TIMP, and small nuclear ribonucleoprotein polypeptide N accounted for 96.6% of the variance in SCR scores. In addition, immunocytochemistry confirmed that the sperm-specific protein markers evaluated in this study were present in sperm. In conclusion, frozen-thawed semen from bulls with higher AK1, IB5, TIMP, small nuclear ribonucleoprotein polypeptide N 2 and PLCz1 mRNA abundances in the sperm had greater correlations with sire fertility index and may possess greater probabilities of siring calves.

Interrelationships Between Apoptosis and Fertility in Bull Sperm

Male fertility, the ability of sperm to fertilize and activate the egg and support early embryogenesis, is vital for mammalian reproduction. Despite producing adequate numbers of sperm with normal motility and morphology, some males suffer from low fertility whose molecular mechanisms are not known. The objective was to determine apoptosis in sperm from high and low fertility bulls and its relationship with male fertility. DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) in the sperm were determined using TUNEL, Annexin V, and immunoblotting approaches, respectively. Amounts of apoptotic spermatozoa were 2.86 (± 1.31) and 3.00 (± 0.96) in high and low fertility bulls, respectively (P=0.548), and were not correlated with fertility. There was a negative correlation between early necrotic spermatozoa and viable spermatozoa (r = –0.99, P<0.0001). Fertility scores were correlated with live spermatozoa detected by eosin-nigrosin test and necrotic spermatozoa determined via flow cytometry (r = –0.49, P<0.006 and r = –0.266, P<0.0113, respectively). BAX level was higher in low fertile group than high fertile group; however, this difference was not statistically significant due to the variations of bull samples (Bull 1–3 vs. Bull 4–5) in low fertile group (P<0.283). BCL-2 was not detectable in any of the sperm samples. The results shed light onto molecular and cellular underpinnings of male fertility.

Dynamics of microRNAs in bull spermatozoa

BackgroundMicroRNAs are small non-coding RNAs that regulate gene expression and thus play important roles in mammalian development. However, the comprehensive lists of microRNAs, as well as, molecular mechanisms by which microRNAs regulate gene expression during gamete and embryo development are poorly defined. The objectives of this study were to determine microRNAs in bull sperm and predict their functions.MethodsTo accomplish our objectives we isolated miRNAs from sperm of high and low fertility bulls, conducted microRNA microarray experiments and validated expression of a panel of microRNAs using real time RT-PCR. Bioinformatic approaches were carried out to identify regulated targets.ResultsWe demonstrated that an abundance of microRNAs were present in bovine spermatozoa, however, only seven were differentially expressed; hsa-aga-3155, -8197, -6727, -11796, -14189, -6125, -13659. The abundance of miRNAs in the spermatozoa and the differential expression in sperm from high vs. low fertility bulls suggests that the miRNAs possibly play important functions in the regulating mechanisms of bovine spermatozoa.ConclusionIdentification of specific microRNAs expressed in spermatozoa of bulls with different fertility phenotypes will help better understand mammalian gametogenesis and early development.

Novel Protein Markers as an Indicator of Male Fertility.

In both humans and animals, approximately half of all cases of infertility derived from male factor or sub-fertility. However, the molecular mechanisms of sperm fertility defects are not well known. Coupled with the fact that the mature spermatozoa have lost the transcription, translation, and protein synthesis, proteomic analysis is acceptable to understand comprehensive process of sperm molecular function related with fertility. The recent developments in proteomic study have amplified the identification of biomarkers associated with fertility. Despite these recent efforts, no studies have clearly defined the relationship between the proteome and male fertility after proteomic study. Therefore, we used proteomic approaches to study fertility-related marker expression in the spermatozoa of high and low fertility bulls, and we used western blot analysis to evaluate whether the proteins identified by 2-DE were correlated with the fertility of 20 individual bovine spermatozoa. We identified eight proteins that were differentially expressed in the spermatozoa of high and low fertility bulls. Five proteins, ENO1, ATP5B, ASPP2, AHSG, and GPX4, were more highly represented in the spermatozoa of high fertility bulls; whereas three proteins, VDAC2, ropporin-1, and UQCRC2, were more highly represented in the spermatozoa of low fertility bulls. The expression levels of ENO1 were greater in the spermatozoa of high fertility bulls than low fertility bulls (Figure 2 A), and ENO1 expression was strongly positively correlated with NRR (r = 0.517, p < 0.05, Figure 3 A). There were no differences in protein expression between high and low fertility bulls for ATP5B and PHGPx4 (Figure 2 B and Figure 2 C), and the expression levels of these proteins were not significantly correlated with NRR (ATP5B: r = 0.1, PHGPx4: r = 0.053, both p < 0.05; Figure 3 B and Figure 3 C). The expression levels of UQCRC2 and VDAC2 were significantly negatively correlated with historic NRR (UQCRC2: r = −0.503, p < 0.05, Figure 2 D; VDAC2: r = 0.475, p < 0.05, Figure 2 E). To my best knowledge, we suggested that concurrent comparisons between protein expression and male fertility by western blot analysis may represent a good in vitro model system to diagnosis the male fertility. Moreover, these novel proteins will used to not only major male fertility markers and further explore the mechanism of male fertility, but will also provide new targets for male contraceptives. This research was supported by the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ008415), Rural Development Administration, Republic of Korea.