Impact of proximal cytoplasmic droplets on quality traits and in-vitro embryo production efficiency of cryopreserved bull spermatozoa

Background: Proximal cytoplasmic droplets (PCDs), a remnant of germ cell cytoplasm, are common non-specific morphological defects in bovine semen. This study evaluated the effect of higher percentages of PCDs on the quality of frozen-thawed bovine semen, embryo production and early embryo development.Methods: Three ejaculates from each of five (group 1: PCD <= 1%, control) and eight adult Bos indicus bulls (group 2: PCD >= 24%) were analysed. Semen samples were examined for: post-thaw motility, vigour of movement, concentration, sperm morphology, slow thermoresistance test (STT), membrane integrity, acrosome status, mitochondrial function using fluorescent probes association (FITC-PSA, PI and JC-1) and sperm chromatin integrity using acridine orange assay. Two bulls from group 2, with 28.5% and 48.5% PCD, respectively, and three bulls from the control group, each with 0% PCD, were selected for IVF (in vitro fertilisation).Results: Semen analyses revealed a significant correlation (P < 0.01) between increased rates of PCD and sperm quality traits. Nevertheless, no differences were observed in sperm motility and vigour either before or after the STT or in the percentage of intact acrosomes (analysed by differential interference contrast microscopy (DIC) after STT), but membrane integrity, acrosome status (evaluated with FITC-PSA staining method after thawing) and mitochondrial function were reduced, when compared with group 1 (P < 0.05). The higher incidence of PCD was positively correlated to chromatin damage, especially after three hours of incubation at 37 degrees C. IVF showed similar results for bull C2 (group 1, control) and bull P2 (group 2, group with higher PCDs).Conclusion: Higher PCD levels influenced spermatozoa quality traits. IVF and embryo development data showed that cleavage, blastocyst formation and blastocyst hatching may have been influenced by the interaction of morphology traits and individual bull effects

Influence of Sex on Basal and Dickkopf-1 Regulated Gene Expression in the Bovine Morula

<div><p>Sex affects function of the developing mammalian embryo as early as the preimplantation period. There were two goals of the current objective. The first was to determine the degree and nature of differences in gene expression between female and male embryos in the cow at the morula stage of development. The second objective was to determine whether DKK1, a molecule known to alter differentiation of the blastocyst, would affect gene expression differently for female and male morulae. In Experiment 1, female and male embryos were treated with DKK1 at Day 5 after insemination. Morulae were harvested 24 h after treatment, pooled in groups of 20 for microarray analysis and RNA subjected to analysis of gene expression by microarray hybridization. There were 662 differentially expressed genes between females and males and 128 of these genes had a fold change ≥ 1.5 between the two sexes. Of the genes upregulated in females, 49.5% were located in the X chromosome. Functional analysis predicted that cell survival was greater in female embryos. Experiment 2 involved a similar design except that transcripts for 12 genes previously reported to be affected by sex, DKK1 or the interaction were quantified by quantitative polymerase chain reaction. Expression of all genes tested that were affected by sex in experiment 1 was affected in a similar manner in Experiment 2. In contrast, effects of DKK1 on gene expression were largely not repeatable in Experiment 2. The exception was for the Hippo signaling gene <i>AMOT</i>, which was inhibited by DKK1. In Experiment 3, embryos produced by fertilization with unsorted sperm were treated with DKK1 at Day 5 and abundance of transcripts for <i>CDX2</i>, <i>GATA6</i>, and <i>NANOG</i> determined at Days 5, 6 and 7 after insemination. There was no effect of DKK1 on expression of any of the three genes. In conclusion, female and male bovine embryos have a different pattern of gene expression as early as the morula stage, and this is due to a large extent to expression of genes in the X chromosomes in females. Differential gene expression between female and male embryos is likely the basis for increased resistance to cell death signals in female embryos and disparity in responses of female and male embryos to changes in the maternal environment.</p></div

Effects of sex, DKK1 and the interaction on expression of genes involved in blastocyst differentiation and pluripotency.^a

<p>^a Units are log2 of fluorescent intensity.</p><p>Effects of sex, DKK1 and the interaction on expression of genes involved in blastocyst differentiation and pluripotency.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133587#t002fn001" target="_blank">^a</a></p

Effect of day after insemination on expression of <i>CDX2</i>, <i>NANOG</i> and <i>GATA6</i> (Experiment 3).

<p>Data are shown as ΔC_t data (left panel) and as fold-change relative to Day 5 controls (right panel). Data are least-squares means ± SEM (left) or were calculated from the least-squares means of ΔC_t (right). Embryos were morulae (Days 5 and 6) or blastocysts (Day 7). Day of insemination is considered Day 0. Blue bars represent control and red bars represent DKK1. Day effects are indicated by superscripts. Days with different superscripts differ (P<0.05). There were no effects of DKK1 or day x DKK1.</p

Differences in gene expression between female and male embryos is predictive that cell survival is enhanced in females.

<p>Genes upregulated in females are in red while genes upregulated in males are in green. Cellular and molecular functions predicted to be activated are in orange while functions predicted to be inhibited in blue. Orange arrows represents predicted activation of the function, blue arrows represent predicted inhibition, yellow arrows indicates that the relationship is inconsistent with the prediction while gray lines represent that the effect is not predicted. Note <i>EXO5</i> is identified as DEM1 in the microarray database.</p

Number of differentially expressed genes due to DKK1, sex and the interaction.

<p>Number of differentially expressed genes due to DKK1, sex and the interaction.</p

Effect of sex and DKK1 on AMOT expression in Day 6 morula (Experiment 2).

<p>Data are shown as ΔC_t data (top panel) and as fold-change relative to female controls (bottom panel). Data are least-squares means ± SEM (top) or were calculated from the least-squares means of ΔC_t (bottom). RNA abundance was affected by sex (P = 0.0527) and DKK1 ((P = 0.0222) but not by the interaction (P = 0.5791).</p

Comparison of the fold-change in gene expression due to sex, DKK1 and DKK1 x sex as determined by microarray and by qPCR. ^a

<p>^a Abbreviation: fem—female.</p><p>Comparison of the fold-change in gene expression due to sex, DKK1 and DKK1 x sex as determined by microarray and by qPCR. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133587#t003fn001" target="_blank">^a</a></p

Membrane lipid profile monitored by mass spectrometry detected differences between fresh and vitrified in vitro-produced bovine embryos

This study aimed to evaluate the impact of vitrification on membrane lipid profile obtained by mass spectrometry (MS) of in vitro-produced bovine embryos. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) has been used to obtain individual embryo membrane lipid profiles. Due to conditions of analysis, mainly membrane lipids, most favorably phosphatidylcholines (PCs) and sphingomyelins (SMs) have been detected. The following ions described by their mass-to-charge ratio (m/z) and respective attribution presented increased relative abundance (1.2-20×) in the vitrified group: 703.5 [SM (16:0) + H]+; 722.5 [PC (40:3) + Na]+; 758.5 [PC (34:2) + H]+; 762.5 [PC (34:0) + H]+; 790.5 [PC (36:0) + H]+ and 810.5 [PC (38:4) + H]+ and/or [PC (36:1) + Na]+. The ion with a m/z 744.5 [PCp (34:1) and/or PCe (34:2)] was 3.4-fold more abundant in the fresh group. Interestingly, ions with m/z 722.5 or 744.5 indicate the presence of lipid species, which are more resistant to enzymatic degradation as they contain fatty acyl residues linked through ether type bonds (alkyl ether or plasmalogens, indicated by the lowercase 'e' and 'p', respectively) to the glycerol structure. The results indicate that cryopreservation impacts the membrane lipid profile, and that these alterations can be properly monitored by MALDI-MS. Membrane lipids can therefore be evaluated by MALDI-MS to monitor the effect of cryopreservation on membrane lipids, and to investigate changes in lipid profile that may reflect the metabolic response to the cryopreservation stress or changes in the environmental conditions.This study aimed to evaluate the impact of vitrification on membrane lipid profile obtained by mass spectrometry (MS) of in vitro-produced bovine embryos. Matrix-assisted laser desorption ionization– mass spectrometry (MALDI–MS) has been used to obtain in235732741CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOsem informaçãoAlmodin, C.G., Minguetti-Camara, V.C., Paixao, C.L., Pereira, P.C., Embryo development and gestation using fresh and vitrified oocytes (2010) Hum. Reprod, 25, pp. 1192-1198Apparicio, M., Ferreira, C.R., Tata, A., Santos, V.G., Alves, A.E., Mostachio, G.Q., Pires-Butler, E.A., Vicente, W.R., Chemical composition of lipids present in cat and dog oocyte by matrix-Assisted desorption ionization mass spectrometry (MALDI- MS (2012) Reprod. Domest. Anim, 47, pp. 113-117Arav, A., Zeron, Y., Leslie, S.B., Behboodi, E., Anderson, G.B., Crowe, J.H., Phase transition temperature and chilling sensitivity of bovine oocytes (1996) Cryobiology, 33, pp. 589-599Bartz, R., Li, W.H., Venables, B., Zehmer, J.K., Roth, M.R., Welti, R., Anderson, R.G., Chapman, K.D., Lipidomics reveals that adiposomes store ether lipids and mediate phospholipid traffic (2007) J. Lipid Res, 48, pp. 837-847Berry, K.A., Hankin, J.A., Barkley, R.M., Spraggins, J.M., Caprioli, R.M., Murphy, R.C., Maldi imaging of lipid biochemistry in tissues by mass spectrometry (2011) Chem. Rev, 111, pp. 6491-6512Byrne, A.T., Southgate, J., Brison, D.R., Leese, H.J., Analysis of apoptosis in the preimplantation bovine embryo using tunel (1999) J. Reprod. Fertil, 117, pp. 97-105Cabral, E.C., Sevart, L., Spindola, H.M., Coelho, M.B., Sousa, I.M., Queiroz, N.C., Foglio, M.A., Riveros, J.M., Pterodon pubescens oil: Characterisation, certification of origin and quality control via mass spectrometry fingerprinting analysis (2013) Phytochem. Anal, 24, pp. 184-192Camargo, L.S., Boite, M.C., Wohlres-Viana, S., Mota, G.B., Serapiao, R.V., As, W.F., Viana, J.H., Nogueira, L.A., Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro (2011) Cryobiology, 63, pp. 256-262Cortes, C., Vapnik, V., (1995) Support-Vector Networks, 20, pp. 273-297. , Saitta L, ed. Machine LearningCortezzi, S.S., Cabral, E.C., Trevisan, M.G., Ferreira, C.R., Setti, A.S., Braga, D.P., Figueira, R.E.C., Borges, E., Prediction of embryo implantation potential by mass spectrometry fingerprinting of the culture medium (2013) Reproduction, 145, pp. 453-462Dinnyes, A., Nedambale, L., Cryopreservation of manipulated embryos: Tackling the double jeopardy (2009) Reprod. Fertil. Dev, 21, pp. 45-59Eberlin, L.S., Abdelnur, P.V., Passero, A., De Sa, G.F., Daroda, R.J., De Souza, V., Eberlin, M.N., Analysis of biodiesel and biodiesel-petrodiesel blends by high performance thin layer chromatography combined with easy ambient sonic-spray ionization mass spectrometry (2009) Analyst, 134, pp. 1652-1657Fahy, E., Sud, M., Cotter, D., Subramaniam, S., Lipid maps online tools for lipid research (2007) Nucleic Acids Res, 35, pp. 606-612Ferreira, C.R., Saraiva, S.A., Catharino, R.R., Garcia, J.S., Gozzo, F.C., Sanvido, G.B., Santos, L.F., Eberlin, M.N., Single embryo and oocyte lipid fingerprinting by mass spectrometry (2010) J. Lipid Res, 51, pp. 1218-1227Ferreira, C.R., Eberlin, L.S., Hallett, J.E., Cooks, R.G., Single oocyte and single embryo lipid analysis by desorption electrospray ionization mass spectrometry (2012) J. Mass Spectrom, 47, pp. 29-33Fuchs, B., Süss, R., Schiller, J., An update of malditof mass spectrometry in lipid research (2011) Prog. Lipid Res, 50, p. 132Gardner, D.K., Dissection of culture media for embryos: The most important and less important components and characteristics (2008) Reprod. Fertil. Dev, 20, pp. 9-18Hankin, J.A., Murphy, R.C., Relationship between 392 MALDI IMS intensity and measured quantity of selected phospholipids in rat brain sections (2010) Anal. Chem, 82, pp. 8476-8484Horvath, G., Seidel, G.E., Jr., Vitrification of bovine oocytes after treatment with cholesterol-loaded methyl-cyclodextrin (2006) Theriogenology, 66, pp. 1026-1033Huwiler, A., Kolter, T., Pfeilschifter, J., Sandhoff, K., Physiology and pathophysiology of sphingolipid metabolism and signaling (2000) Biochim. Biophys. Acta, 1485, pp. 63-99Kaplan, M.R., Simoni, R.D., Intracellular transport of phosphatidylcholine to the plasma membrane (1985) J. Cell Biol, 101, pp. 441-445Kim, J.Y., Kinoshita, M., Ohnishi, M., Fukui, Y., Lipid and fatty acid analysis of fresh and frozen-Thawed immature and in vitro matured bovine oocytes (2001) Reproduction, 122, pp. 131-138Lapa, M., Marques, C.C., Alves, S.P., Vasques, M.I., Baptista, M.C., Carvalhais, I., Silva Pereira, M., Pereira, R.M., Effect of trans-10 cis-12 conjugated linoleic acid on bovine oocyte competence and fatty acid composition (2011) Reprod. Domest. Anim, 46, pp. 904-910Leibo, S.P., Preservation of ova and embryos by freezing (1981) New Technologies in Animal Breeding, pp. 127-139. , eds B.G. Brackett, G.E. Seidel Jr & S.M. Seidel New York. Academic PressMcKeegan, P.J., Sturmey, R.G., The role of fatty acids in oocyte and early embryo development (2011) Reprod. Fertil. Dev, 24, pp. 59-67Merrill, A.H., Schmelz, E.M., Dillehay, D.L., Spiegel, S., Shayman, J.A., Schroeder, J.J., Riley, R.T., Wang, E., Sphingolipids-The enigmatic lipid class: Biochemistry, physiology, and pathophysiology (1997) Toxicol. Appl. Pharmacol, 142, pp. 208-225Paula-Lopes, F.F., Hansen, P.J., Heat shockinduced apoptosis in preimplantation bovine embryos is a developmentally regulated phenomenon (2002) Biol. Reprod, 66, pp. 1169-1177Pereira, R.M., Baptista, M.C., Vasques, M.I., Horta, A.E., Portugal, P.V., Bessa, R.J., Silva, J.C., Marques, C.C., Cryosurvival of bovine blastocysts is enhanced by culture with trans-10 cis-12 conjugated linoleic acid (10t, 12c CLA (2007) Anim. Reprod. Sci, 98, pp. 293-301Rocha-Frigoni, N.A.S., Leão, B.C.S., Nogueira Accorsi É., M.F., Mingoti, G.Z., Reduced levels of intracellular reactive oxygen species and apoptotic status are not correlated with increases in cryotolerance of bovine embryos produced in vitro in the presence of antioxidants (2014) Reprod. Fertil. Dev, 26, pp. 797-805Räty, M., Ketoja, E., Pitkänen, T., Ahola, V., Kananen, K., Peippo, J., In vitro maturation supplements affect developmental competence of bovine cumulus-oocyte complexes and embryo quality after vitrification (2011) Cryobiology, 63, pp. 245-255Saraiva, S.A., Cabral, E.C., Eberlin, M.N., Catharino, R.R., Amazonian vegetable oils and fats: Fast typification and quality control via triacylglycerol (tag) profiles from dry matrix-Assisted laser desorption/ionization time-offlight (maldi-Tof) mass spectrometry fingerprinting (2009) J. Agric. Food Chem, 57, pp. 4030-4034Seidel, G.E., Jr., Modifying oocytes and embryos to improve their cryopreservation (2006) Theriogenology, 65, pp. 228-235Sturmey, R.G., Reis, A., Leese, H.J., McEvoy, T.G., Role of fatty acids in energy provision during oocytematuration and early embryo development (2009) Reprod. Domest. Anim, 44, pp. 50-58Sudano, M.J., Paschoal, D.M., Rascado, T.A.S., Magalhães, L.C., Crocomo, L.F., De Lima-Neto, J.F., Landim-Alvarenga, F.A.C., Lipid content and apoptosis of in vitro produced bovine embryos as determinants of susceptibility to vitrification (2011) Theriogenology, 75, pp. 1211-1220Sudano, M.J., Santos, V.G., Tata, A., Ferreira, C.R., Paschoal, D.M., MacHado, R., Buratini, J., Landim-Alvarenga, F.D., Phosphatidylcholine and sphingomyelin profiles vary in Bos taurus indicus and Bos taurus taurus in vitro and in vivo-produced blastocysts (2012) Biol. Reprod, 87, pp. 1211-1220Tata, A., Sudano, M.J., Santos, V.G., Landim-Alvarenga, F.D., Ferreira, C.R., Eberlin, M.N., Optimal single-embryo mass spectrometry fingerprinting (2013) J. Mass Spectrom, 48, pp. 844-849Vajta, G., Rindom, N., Peura, T.T., Holm, P., Greve, T., Callesen, H., The effect of media, serum and temperature on in vitro survival of bovine blastocysts after open pulled straw (OPS) vitrification (1999) Theriogenology, 52, pp. 939-948Van Meer, G., Voelker, D.R., Feigenson, G.W., Membrane lipids: Where they are and how they behave (2008) Nat. Rev. Mol. Cell. Biol, 9, pp. 112-124Vance, J.E., Tasseva, G., Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells (2013) Biochim. Biophys. Acta, 1831, pp. 543-554Wiegmann, K., Schütze, S., Kampen, E., Himmler, A., MacHleidt, T., Krönke, M., Human 55-kDa receptor for tumor necrosis factor coupled to signal transduction cascades (1992) J. Biol. Chem, 267, pp. 7997-8001Wold, S., Esbensen, K., Geladi, P., Principal component analysis (1987) Chemometrics and Intelligent Laboratory Systems, pp. 37-52. , Amsterdam. Elsevier Science PublishersZeron, Y., Sklan, D., Arav, A., Effect of polyunsaturated fatty acid supplementation on biophysical parameters and chilling sensitivity of ewe oocytes (2002) Mol. Reprod. Dev, 61, pp. 271-27

Membrane lipid profile of in vitro-produced embryos is affected by vitrification but not by long-term dietary supplementation of polyunsaturated fatty acids for oocyte donor beef heifers

CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICODietary rumen-protected polyunsaturated fatty acids (PUFAs) rich in linoleic acid (LA) may affect embryo yield, and LA can modulate the molecular mechanisms of lipid uptake in bovine blastocysts produced in vitro. In embryos, membrane lipids, such as phos29612171230CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO554730/2010-3306746/2012-3BCSL and GZM were awarded scholarships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil (#554730/2010-3 and #306746/2012-3). The authors thank Elanco, Grupo Guabi, Ingámed, Alta Genetics Brazil, Ouro Fino Saúde Animal, Wa