Freezability genetics in rabbit semen

[EN] The aim of this study was to estimate the heritability of semen freezability and to estimate the genetic correlation between frozen-thawed sperm traits and the growth rate in a paternal rabbit line. Estimated heritabilities showed that frozen-thawed semen traits are heritable (ranged between 0.08 and 0.15). In the case of Live-FT (percentage of viable sperm after freezing), the estimated heritability is the highest one, and suggests the possibility of effective selection. After the study of genetic correlations it seems that daily weight gain (DG) was negatively correlated with sperm freezability, but no further conclusions could be drawn due to the high HPD95%. More data should be included in order to obtain better accuracy for the estimates of these genetic correlations. If the results obtained at present study were confirmed, it would imply that selection for DG could alter sperm cell membranes or seminal plasma composition, both components related to sperm cryoresistance. (C) 2017 Elsevier Inc. All rights reserved.This work was supported by the Generalitat Valenciana research program (Prometeo II 2014/036). Lavara R. acknowledges the partial support received from Generalitat Valenciana under VALid program (APOST/2014/034) and from Ministry of Economy and Competitiveness under subprogramme "Formation posdoctoral" (FPDI-2013-16707).Lavara García, R.; Moce Cervera, ET.; Baselga Izquierdo, M.; Vicente Antón, JS. (2017). Freezability genetics in rabbit semen. Theriogenology. 102:54-58. https://doi.org/10.1016/j.theriogenology.2017.07.013S545810

Semen evaluation of two selected lines of rabbit bucks

[EN] Twenty rabbit bucks of 9 months of age were used to evaluate semen quality of two lines of New Zealand rabbit bucks selected for litter size at weaning (A line) and growth rate from weaning to slaughter (R line). The morphological semen characteristics indicated that the A line spermatozoa had greater acrosome integrity (+3.6 percentage units; P<0.01) and smaller sperm head size (for example, ¿1.46 ¿m2 for sperm head area) than in the R line. Seminal functional traits were also significantly higher for the A line (+13.4 percentage units for viability, +10.6 percentage units for hypo-osmotic swelling test (HOST) and +3.3 g/L for seminal plasma protein. However, no differences were detected between lines for motility parameters and seminal plasma protein electrophoretic profiles. Both lines had the same twelve bands with the following molecular weights to the nearest 1 kD: 124, 117, 99, 86, 75, 62, 40, 32, 21, 19, 10 and 6 kD. A relationship (r=0.308 for A line and 0.359 for R line; P<0.01) was found between the integrity of the plasmatic membrane (viability rate) and tail membrane (HOST) of the spermatozoa in the A line, but not in the R line, which had greater sperm head size. There was also a significant positive correlation coefficient between sperm concentration and either viability or some kinetic traits (r=0.567 and 0.575 for VCL, r=0.584 and 0.561 for VSL and r=0.588 and 0.588 for VAP, for A and R lines, respectively; P<0.001). We concluded that the A line seems to have better semen characteristics than the R line. We also found an interesting correlation among the seminal morphological, functional and kinetic traits, which could possibly be used to facilitate semen evaluation.Hosam M. Safaa was supported by a grant from ICSC-World Laboratory, Switzerland. This work has been supported by the Spanish Research Project (CICYT AGL2004-02710/GAN). The authors are indebted to F. Marco-Jiménez, Universidad Politécnica de Valencia, Valencia, Spain, and G.M.K. Mehaisen, Department of Animal Production, Faculty of Agricultural, Cairo University, Giza, Egypt, for their excellent cooperation throughout this study and to D.A.A. El-Sayed, Faculty of Agricultural, Cairo University, Giza, Egypt, for her help in preparing the manuscript.Safaa, H.; Vicente, J.; Lavara, R.; Viudes De Castro, M. (2008). Semen evaluation of two selected lines of rabbit bucks. World Rabbit Science. 16(3). doi:10.4995/wrs.2008.62216

Embryo vitrification in rabbits: Consequences for progeny growth

[EN] The objective of this research is to examine if there are any effects of the rederivation procedures on rabbit growth pattern and on weight of different organ in adults. For this purpose, three experiments were conducted on two different groups of animals (control group and vitrified transferred group) to evaluate the possible effect of embryo manipulation (vitrification and transfer procedures) on future growth traits. The first experiment studies body weight from 1 to 9 weeks of age from the two groups. The second experiment describes the growth curve of progeny from experimental groups and analyzes their Gompertz curve parameters, including the estimation of adult body weight. The third experiment has been developed to study if there are any differences in different organ weight in adult males from the two experimental groups. In general, the results indicate that rederivation procedures had effect on the phenotypic expression of growth traits. The results showed that rabbit produced by vitrification and embryo transfer had higher body weight in the first four weeks of age than control progeny. Results from body weight (a parameter) and b parameter estimated by fitting the Gompertz growth curve did not show any difference between experimental groups. However, differences related with growth velocity (k parameter of the Gompertz curve) were observed among them, showing that the control group had higher growth velocity than the vitrified transferred group. In addition, we found that liver weight at 40th week of age exhibits significant differences between the experimental groups. The liver weight was higher in the control males than in the VF males. Although the present results indicate that vitrification and transfer procedures might affect some traits related with growth in rabbits, further research is needed to assess the mechanisms involved in the appearance of these phenotypes and if these phenotypes could be transferred to the future progeny.This study was supported by the Generalitat Valenciana research program (Prometeo II 2014/036) and Spanish Research Projects (CICYT AGL2011-29831-C03-01; AGL2014-53405-C2-1-P). Lavara R. acknowledges the partial support received from Generalitat Valenciana under VALid+ program (APOST/2014/034) and from Ministry of Economy and Competitiveness under subprogramme "Formacion posdoctoral" (FPDI-2013-16707).Lavara García, R.; Baselga Izquierdo, M.; Marco Jiménez, F.; Vicente Antón, JS. (2015). Embryo vitrification in rabbits: Consequences for progeny growth. Theriogenology. 84(5):674-680. https://doi.org/10.1016/j.theriogenology.2015.04.025S67468084

Long-term and transgenerational effects of cryopreservation on rabbit embryos

The short-term effects of cryopreservation and embryo transfer are well documented (reduced embryo viability, changes in pattern expression), but little is known about their long-term effects. We examined the possibility that embryo vitrification and transfer in rabbit could have an impact on the long-term reproductive physiology of the offspring and whether these phenotypes could be transferred to the progeny. Vitrified rabbit embryos were warmed and transferred to recipient females (F0). The offspring of the F0 generation were the F1 generation (cryopreserved animals). Females from F1 generation offspring were bred to F1 males to generate an F2 generation. In addition, two counterpart groups of noncryopreserved animals were bred and housed simultaneously to F1 and F2 generations (CF1 and CF2, respectively). The reproductive traits studied in all studied groups were litter size (LS), number born alive at birth (BA), and postnatal survival at Day 28 (number of weaned/number born alive expressed as percentage). The reproductive traits were analyzed using Bayesian methodology. Features of the estimated marginal posterior distributions of the differences between F1 and their counterparts (F1 - CF1) and between F2 and their counterparts (F2 - CF2) in reproductive characters found that vitrification and transfer procedures cause a consistent increase in LS and BA between F1 and CF1 females (more than 1.4 kits in LS and more than 1.3 BA) and also between F2 and CF2 females (0.96 kits in LS and 0.94 BA). We concluded that embryo cryopreservation and transfer procedures have long-term effects on derived female reproduction (F1 females) and transgenerational effects on female F1 offspring (F2 females).Lavara García, R.; Baselga Izquierdo, M.; Marco Jiménez, F.; Vicente Antón, JS. (2014). Long-term and transgenerational effects of cryopreservation on rabbit embryos. Theriogenology. 81(7):988-992. doi:10.1016/j.theriogenology.2014.01.030S98899281

Evaluation by re-derivation of a paternal line after 18 generations on seminal traits, proteome and fertility

[EN] Males from a paternal line selected for growth traits were used to produce semen doses at insemination centres and farms in a breeding scheme for rabbit meat production. The aim of this study was to assess whether a program of selection by daily gain in fattening period changed the seminal traits, plasma and sperm proteome and the fertility of semen when used in artificial insemination. Thirty-nine males from a paternal line were obtained by re-derivation from vitrified embryos with a difference of 18 generations (G21V and G39V). Sperm production parameters, morphological traits, sperm motility parameters and viability were evaluated from ejaculates. Seminal plasma and sperm proteome of three pool ejaculates from 10 mature males of each group were analysed and semen doses were used to inseminate 311 females. Only the percentage of abnormal sperm showed significant differences, with G21V presenting fewer abnormal sperm than G39V (10.5 +/- 2.63 vs 23.8 +/- 1.98). The discriminant analysis (DA-PLS) showed a clear effect of the generation for plasma and sperm proteome. In seminal plasma, 643 proteins were reported and 64 proteins were differentially expressed, of which 56 were overexpressed in G39V (87.5%). Sperm proteome reported 1360 proteins with 132 differentially abundant proteins. Of the total, 89 proteins were overexpressed in G39V (67.4%). From the 64 and 132 differentially abundant proteins of plasma and sperm, 19 and 26 had a FC >1.5, 12 and 13 of them belonging to the Oryctolagus cuniculus taxonomy, respectively. Despite observing differences in important proteins related to capacitation, sperm motility or immunoprotection and consequently to the fertilization process (TMPRSS2, Serpin family, Farn71f1, ATPase H+ transporting accessory protein 2, carbonic anhydrase 2, UDP-glucose glycoprotein glucosyltransferase 2), no differences in fertility and prolificacy were detected when commercial seminal doses were used for insemination from both male groups. However, overabundance of KIAA1324 protein can be related to the increase in abnormal sperm after selection by growth rate.This research was supported by AGL2017-85162-C2-1-R research project funded by Ministerio de Economia, Industria y Competitividad (MICINN, Spain). X Garcia-Dominguez was supported by a research grant from MICINN (BES-2015-072429). English text version was revised by N. Macowan English Language Service.Juárez, JD.; Marco-Jiménez, F.; Talaván, AM.; García-Domínguez, X.; Viudes-De-Castro, MP.; Lavara, R.; Vicente Antón, JS. (2020). Evaluation by re-derivation of a paternal line after 18 generations on seminal traits, proteome and fertility. Livestock Science. 232:1-13. https://doi.org/10.1016/j.livsci.2019.103894S113232Antalis, T. M., Bugge, T. H., & Wu, Q. (2011). Membrane-Anchored Serine Proteases in Health and Disease. Proteases in Health and Disease, 1-50. doi:10.1016/b978-0-12-385504-6.00001-4Bezerra, M. J. B., Arruda-Alencar, J. M., Martins, J. A. M., Viana, A. G. A., Viana Neto, A. M., Rêgo, J. P. A., … Moura, A. A. (2019). Major seminal plasma proteome of rabbits and associations with sperm quality. Theriogenology, 128, 156-166. doi:10.1016/j.theriogenology.2019.01.013Brun, J.-M., Theau-Clément, M., & Bolet, G. (2002). The relationship between rabbit semen characteristics and reproductive performance after artificial insemination. Animal Reproduction Science, 70(1-2), 139-149. doi:10.1016/s0378-4320(01)00197-xBrun, J.-M., Theau-Clément, M., Esparbié, J., Falières, J., Saleil, G., & Larzul, C. (2006). Semen production in two rabbit lines divergently selected for 63-d body weight. Theriogenology, 66(9), 2165-2172. doi:10.1016/j.theriogenology.2006.07.004Brun, J. M., Sanchez, A., Ailloud, E., Saleil, G., & Theau-Clément, M. (2016). Genetic parameters of rabbit semen traits and male fertilising ability. Animal Reproduction Science, 166, 15-21. doi:10.1016/j.anireprosci.2015.12.008Bünger, L., Lewis, R. M., Rothschild, M. F., Blasco, A., Renne, U., & Simm, G. (2005). Relationships between quantitative and reproductive fitness traits in animals. Philosophical Transactions of the Royal Society B: Biological Sciences, 360(1459), 1489-1502. doi:10.1098/rstb.2005.1679Casares-Crespo, L., Fernández-Serrano, P., Vicente, J. S., Marco-Jiménez, F., & Viudes-de-Castro, M. P. (2018). Rabbit seminal plasma proteome: The importance of the genetic origin. Animal Reproduction Science, 189, 30-42. doi:10.1016/j.anireprosci.2017.12.004Casares-Crespo, L., Fernández-Serrano, P., & Viudes-de-Castro, M. P. (2019). Proteomic characterization of rabbit (Oryctolagus cuniculus) sperm from two different genotypes. Theriogenology, 128, 140-148. doi:10.1016/j.theriogenology.2019.01.026Castellini, C., Lattaioli, P., Moroni, M., & Minelli, A. (2000). Effect of seminal plasma on the characteristics and fertility of rabbit spermatozoa. Animal Reproduction Science, 63(3-4), 275-282. doi:10.1016/s0378-4320(00)00181-0Castellini, C., Cardinali, R., Dal Bosco, A., Minelli, A., & Camici, O. (2006). Lipid composition of the main fractions of rabbit semen. Theriogenology, 65(4), 703-712. doi:10.1016/j.theriogenology.2005.05.053Castellini, C., Mourvaki, E., Cardinali, R., Collodel, G., Lasagna, E., Del Vecchio, M. T., & Dal Bosco, A. (2012). Secretion patterns and effect of prostate-derived granules on the sperm acrosome reaction of rabbit buck. Theriogenology, 78(4), 715-723. doi:10.1016/j.theriogenology.2012.02.012Courtens, J., Bolet, G., & Theau-Clément, M. (1994). Effect of acrosome defects and sperm chromatin decondensation on fertility and litter size in the rabbit. Preliminary electron-microscopic study. Reproduction Nutrition Development, 34(5), 427-437. doi:10.1051/rnd:19940504Choucair, F., 2018. Unraveling the sperm transcriptome by nextgeneration sequencing and the global epigenetic and landscape in infertile men. Molecular Biology.Université Côted’ Azur; Université libanaise, NNT:2018AZUR4058. https://tel.archives-ouvertes.fr/tel-01958881.Davis, B. K., & Davis, N. V. (1983). Binding by glycoproteins of seminal plasma membrane vesicles accelerates decapacitation in rabbit spermatozoa. Biochimica et Biophysica Acta (BBA) - Biomembranes, 727(1), 70-76. doi:10.1016/0005-2736(83)90370-xEllerman, D. A., Myles, D. G., & Primakoff, P. (2006). A Role for Sperm Surface Protein Disulfide Isomerase Activity in Gamete Fusion: Evidence for the Participation of ERp57. Developmental Cell, 10(6), 831-837. doi:10.1016/j.devcel.2006.03.011Estany, J., Camacho, J., Baselga, M., & Blasco, A. (1992). Selection response of growth rate in rabbits for meat production. Genetics Selection Evolution, 24(6), 527. doi:10.1186/1297-9686-24-6-527García-Tomás, M., Sánchez, J., Rafel, O., Ramon, J., & Piles, M. (2006). Variability, repeatability and phenotypic relationships of several characteristics of production and semen quality in rabbit. Animal Reproduction Science, 93(1-2), 88-100. doi:10.1016/j.anireprosci.2005.06.011García-Tomás, M., Sánchez, J., Rafel, O., Ramon, J., & Piles, M. (2006). Heterosis, direct and maternal genetic effects on semen quality traits of rabbits. Livestock Science, 100(2-3), 111-120. doi:10.1016/j.livprodsci.2005.08.004Garénaux, E., Kanagawa, M., Tsuchiyama, T., Hori, K., Kanazawa, T., Goshima, A., … Kitajima, K. (2015). Discovery, Primary, and Crystal Structures and Capacitation-related Properties of a Prostate-derived Heparin-binding Protein WGA16 from Boar Sperm. Journal of Biological Chemistry, 290(9), 5484-5501. doi:10.1074/jbc.m114.635268Gerena, R. L., Irikura, D., Urade, Y., Eguchi, N., Chapman, D. A., & Killian, G. J. (1998). Identification of a Fertility-Associated Protein in Bull Seminal Plasma As Lipocalin-Type Prostaglandin D Synthase1. Biology of Reproduction, 58(3), 826-833. doi:10.1095/biolreprod58.3.826Gervasi, M. G., & Visconti, P. E. (2017). Molecular changes and signaling events occurring in spermatozoa during epididymal maturation. Andrology, 5(2), 204-218. doi:10.1111/andr.12320Jeyendran, R. S., Van der Ven, H. H., Perez-Pelaez, M., Crabo, B. G., & Zaneveld, L. J. D. (1984). Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. Reproduction, 70(1), 219-228. doi:10.1530/jrf.0.0700219Kim, T. S., Heinlein, C., Hackman, R. C., & Nelson, P. S. (2006). Phenotypic Analysis of Mice Lacking the Tmprss2 -Encoded Protease. Molecular and Cellular Biology, 26(3), 965-975. doi:10.1128/mcb.26.3.965-975.2006Kwon, J. T., Ham, S., Jeon, S., Kim, Y., Oh, S., & Cho, C. (2017). Expression of uncharacterized male germ cell-specific genes and discovery of novel sperm-tail proteins in mice. PLOS ONE, 12(7), e0182038. doi:10.1371/journal.pone.0182038Larzul, C., Gondret, F., Combes, S., & de Rochambeau, H. (2005). Divergent selection on 63-day body weight in the rabbit: response on growth, carcass and muscle traits. Genetics Selection Evolution, 37(1), 105. doi:10.1186/1297-9686-37-1-105Lavara, R., Mocé, E., Lavara, F., Viudes de Castro, M. P., & Vicente, J. S. (2005). Do parameters of seminal quality correlate with the results of on-farm inseminations in rabbits? Theriogenology, 64(5), 1130-1141. doi:10.1016/j.theriogenology.2005.01.009Lavara, R., Vicente, J. S., & Baselga, M. (2010). Genetic parameter estimates for semen production traits and growth rate of a paternal rabbit line. Journal of Animal Breeding and Genetics, 128(1), 44-51. doi:10.1111/j.1439-0388.2010.00889.xLavara, R., Vicente, J. S., & Baselga, M. (2012). Estimation of genetic parameters for semen quality traits and growth rate in a paternal rabbit line. Theriogenology, 78(3), 567-575. doi:10.1016/j.theriogenology.2012.03.002Lavara, R., Vicente, J. S., & Baselga, M. (2013). Genetic variation in head morphometry of rabbit sperm. Theriogenology, 80(4), 313-318. doi:10.1016/j.theriogenology.2013.04.015Law, R. H., Zhang, Q., McGowan, S., Buckle, A. M., Silverman, G. A., Wong, W., … Whisstock, J. C. (2006). Genome Biology, 7(5), 216. doi:10.1186/gb-2006-7-5-216Leone, M. G., Haq, H. A., & Saso, L. (2002). Lipocalin type prostaglandin D-synthase: which role in male fertility? Contraception, 65(4), 293-295. doi:10.1016/s0010-7824(02)00280-9Lestari, S. W., Miati, D. N., Seoharso, P., Sugiyanto, R., & Pujianto, D. A. (2017). Sperm Na+, K+-ATPase α4 and plasma membrane Ca2+-ATPase (PMCA) 4 regulation in asthenozoospermia. Systems Biology in Reproductive Medicine, 63(5), 294-302. doi:10.1080/19396368.2017.1348565Liao, T.-T., Xiang, Z., Zhu, W.-B., & Fan, L.-Q. (2009). Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry. Asian Journal of Andrology, 11(6), 683-693. doi:10.1038/aja.2009.59Llobat, L., Marco-Jiménez, F., Peñaranda, D., Thieme, R., Navarrete, A., & Vicente, J. (2011). mRNA Expression in Rabbit Blastocyst and Endometrial Tissue of Candidate Gene Involved in Gestational Losses. Reproduction in Domestic Animals, 47(2), 281-287. doi:10.1111/j.1439-0531.2011.01855.xLoveland, K., Major, A., Butler, R., Jans, D., Miyamoto, Y., & Young, J. (2015). Putting things in place for fertilization: discovering roles for importin proteins in cell fate and spermatogenesis. Asian Journal of Andrology, 17(4), 537. doi:10.4103/1008-682x.154310Lukefahr, S. D., Odi, H. B., & Atakora, J. K. (1996). Mass selection for 70-day body weight in rabbits. Journal of Animal Science, 74(7), 1481. doi:10.2527/1996.7471481xMa, Q., Li, Y., Luo, M., Guo, H., Lin, S., Chen, J., … Gui, Y. (2017). The expression characteristics of FAM71D and its association with sperm motility. Human Reproduction, 32(11), 2178-2187. doi:10.1093/humrep/dex290Marai, I. F. ., Habeeb, A. A. ., & Gad, A. . (2002). Rabbits’ productive, reproductive and physiological performance traits as affected by heat stress: a review. Livestock Production Science, 78(2), 71-90. doi:10.1016/s0301-6226(02)00091-xMocé, E., Vicente, J. S., & Lavara, R. (2003). Effect of freezing–thawing protocols on the performance of semen from three rabbit lines after artificial insemination. Theriogenology, 60(1), 115-123. doi:10.1016/s0093-691x(02)01329-8Naturil-Alfonso, C., Lavara, R., Millán, P., Rebollar, P. G., Vicente, J. S., & Marco-Jiménez, F. (2016). Study of failures in a rabbit line selected for growth rate. World Rabbit Science, 24(1), 47. doi:10.4995/wrs.2016.4016Nizza, A., Di Meo, C., & Taranto, S. (2003). Effect of Collection Rhythms and Season on Rabbit Semen Production. Reproduction in Domestic Animals, 38(6), 436-439. doi:10.1046/j.1439-0531.2003.00458.xOsada, T., Watanabe, G., Kondo, S., Toyoda, M., Sakaki, Y., & Takeuchi, T. (2001). Male Reproductive Defects Caused by Puromycin-Sensitive Aminopeptidase Deficiency in Mice. Molecular Endocrinology, 15(6), 960-971. doi:10.1210/mend.15.6.0643Pascual, J. J., García, C., Martínez, E., Mocé, E., & Vicente, J. S. (2004). Rearing management of rabbit males selected by high growth rate: the effect of diet and season on semen characteristics. Reproduction Nutrition Development, 44(1), 49-63. doi:10.1051/rnd:2004016Pascual, J. J., Marco-Jiménez, F., Martínez-Paredes, E., Ródenas, L., Fabre, C., Juvero, M. A., & Cano, J. L. (2016). Feeding programs promoting daily feed intake stability in rabbit males reduce sperm abnormalities and improve fertility. Theriogenology, 86(3), 730-737. doi:10.1016/j.theriogenology.2016.02.026Pérez-Patiño, C., Parrilla, I., Li, J., Barranco, I., Martínez, E. A., Rodriguez-Martínez, H., & Roca, J. (2019). The Proteome of Pig Spermatozoa Is Remodeled During Ejaculation. Molecular & Cellular Proteomics, 18(1), 41-50. doi:10.1074/mcp.ra118.000840Peralta-Arias, R. D., Vívenes, C. Y., Camejo, M. I., Piñero, S., Proverbio, T., Martínez, E., … Proverbio, F. (2015). ATPases, ion exchangers and human sperm motility. REPRODUCTION, 149(5), 475-484. doi:10.1530/rep-14-0471Piles, M., & Tusell, L. (2011). Genetic correlation between growth and female and male contributions to fertility in rabbit. Journal of Animal Breeding and Genetics, 129(4), 298-305. doi:10.1111/j.1439-0388.2011.00975.xPiles, M., Mocé, M. L., Laborda, P., & Santacreu, M. A. (2013). Feasibility of selection for male contribution to embryo survival as a way of improving male reproductive performance and semen quality in rabbits1. Journal of Animal Science, 91(10), 4654-4658. doi:10.2527/jas.2013-6446Rahman, M. S., Kwon, W.-S., & Pang, M.-G. (2017). Prediction of male fertility using capacitation-associated proteins in spermatozoa. Molecular Reproduction and Development, 84(9), 749-759. doi:10.1002/mrd.22810Roca, J., Martínez, S., Orengo, J., Parrilla, I., Vázquez, J. M., & Martínez, E. A. (2005). Influence of constant long days on ejaculate parameters of rabbits reared under natural environment conditions of Mediterranean area. Livestock Production Science, 94(3), 169-177. doi:10.1016/j.livprodsci.2004.10.011De Rochambeau, H., de la Fuente, L., Rouvier, R., & Ouhayoun, J. (1989). Sélection sur la vitesse de croissance post-sevrage chez le lapin. Genetics Selection Evolution, 21(4), 527. doi:10.1186/1297-9686-21-4-527Saeed, A. I., Sharov, V., White, J., Li, J., Liang, W., Bhagabati, N., … Quackenbush, J. (2003). TM4: A Free, Open-Source System for Microarray Data Management and Analysis. BioTechniques, 34(2), 374-378. doi:10.2144/03342mt01Samanta, L., Parida, R., Dias, T. R., & Agarwal, A. (2018). The enigmatic seminal plasma: a proteomics insight from ejaculation to fertilization. Reproductive Biology and Endocrinology, 16(1). doi:10.1186/s12958-018-0358-6Sabés-Alsina, M., Planell, N., Torres-Mejia, E., Taberner, E., Maya-Soriano, M. J., Tusell, L., … Lopez-Bejar, M. (2015). Daily exposure to summer circadian cycles affects spermatogenesis, but not fertility in an in vivo rabbit model. Theriogenology, 83(2), 246-252. doi:10.1016/j.theriogenology.2014.09.013Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V., Sagliocco, F., Wilm, M., Vorm, O., … Mann, M. (1996). Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels. Proceedings of the National Academy of Sciences, 93(25), 14440-14445. doi:10.1073/pnas.93.25.14440Shilov, I. V., Seymour, S. L., Patel, A. A., Loboda, A., Tang, W. H., Keating, S. P., … Schaeffer, D. A. (2007). The Paragon Algorithm, a Next Generation Search Engine That Uses Sequence Temperature Values and Feature Probabilities to Identify Peptides from Tandem Mass Spectra. Molecular & Cellular Proteomics, 6(9), 1638-1655. doi:10.1074/mcp.t600050-mcp200Theau-Clément, M., Bolet, G., Sanchez, A., Saleil, G., & Brun, J. M. (2015). Some factors that influence semen characteristics in rabbits. Animal Reproduction Science, 157, 33-38. doi:10.1016/j.anireprosci.2015.03.011Thundathil, J. C., Rajamanickam, G. D., & Kastelic, J. P. (2018). Na/K-ATPase and Regulation of Sperm Function. Animal Reproduction, 15(Suppl. 1), 711-720. doi:10.21451/1984-3143-ar2018-0024Tusell, L., Legarra, A., García-Tomás, M., Rafel, O., Ramon, J., & Piles, M. (2012). Genetic basis of semen traits and their relationship with growth rate in rabbits1. Journal of Animal Science, 90(5), 1385-1397. doi:10.2527/jas.2011-4165Vicente, J. (2004). Study of fertilising capacity of spermatozoa after heterospermic insemination in rabbit using DNA markers. Theriogenology, 61(7-8), 1357-1365. doi:10.1016/j.theriogenology.2003.08.009Vicente, J. S., Llobat, L., Viudes-de-Castro, M. P., Lavara, R., Baselga, M., & Marco-Jiménez, F. (2012). Gestational losses in a rabbit line selected for growth rate. Theriogenology, 77(1), 81-88. doi:10.1016/j.theriogenology.2011.07.019Viudes-de-Castro, M. P., & Vicente, J. S. (1997). Effect of sperm count on the fertility and prolificity rates of meat rabbits. Animal Reproduction Science, 46(3-4), 313-319. doi:10.1016/s0378-4320(96)01628-4Viudes-de-Castro, M. P., Mocé, E., Lavara, R., Marco-Jiménez, F., & Vicente, J. S. (2014). Aminopeptidase activity in seminal plasma and effect of dilution rate on rabbit reproductive performance after insemination with an extender supplemented with buserelin acetate. Theriogenology, 81(9), 1223-1228. doi:10.1016/j.theriogenology.2014.02.003Viudes de Castro, M. P., Casares-Crespo, L., Monserrat-Martínez, A., & Vicente, J. S. (2015). Determination of enzyme activity in rabbit seminal plasma and its relationship with quality semen parameters. World Rabbit Science, 23(4), 247. doi:10.4995/wrs.2015.4064Vizcaíno, J. A., Deutsch, E. W., Wang, R., Csordas, A., Reisinger, F., Ríos, D., … Hermjakob, H. (2014). ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nature Biotechnology, 32(3), 223-226. doi:10.1038/nbt.2839Wandernoth, P. M., Mannowetz, N., Szczyrba, J., Grannemann, L., Wolf, A., Becker, H. M., … Wennemuth, G. (2015). Normal Fertility Requires the Expression of Carbonic Anhydrases II and IV in Sperm. Journal of Biological Chemistry, 290(49), 29202-29216. doi:10.1074/jbc.m115.698597Weininger, R. B., Fisher, S., Rifkin, J., & Bedford, J. M. (1982). Experimental studies on the passage of specific IgG to the lumen of the rabbit epididymis. Reproduction, 66(1), 251-258. doi:10.1530/jrf.0.0660251Yan, M., Zhang, X., Pu, Q., Huang, T., Xie, Q., Wang, Y., … Gu, J. (2016). Immunoglobulin G Expression in Human Sperm and Possible Functional Significance. Scientific Reports, 6(1). doi:10.1038/srep2016

Breeding programmes to improve male reproductive performance and efficiency of insemination dose production in paternal lines: feasibility and limitations

[EN] This paper aims at reviewing the current genetic knowledge of the issues related to the efficient use of bucks in artificial insemination (AI). Differences between lines have been found relevant in semen production and quality traits not necessarily related to their specialization as maternal or paternal lines. Accurate heritability estimates indicate that genetic selection for increasing semen production by improving male libido and reducing the number of rejected ejaculates may not be effective. However, total sperm produced per ejaculate appears to be as an interesting trait to select for, despite that genetic correlation between ejaculate volume and sperm concentration has not been yet accurately estimated. Semen pH has shown low to medium heritability estimates and a low coefficient of variation, therefore it is not advisable to attempt improvement by direct selection. In general, sperm motility traits have shown low heritabilities but, the rate of motile sperms per ejaculate has been considered as convenient to select for. Morphological characteristics of the spermatozoa have revealed as medium to highly heritable. There are evidences of high genetic correlations between sperm traits before and after freezing-thawing. There are few studies regarding the estimation of heterosis of seminal traits but results indicate important and favorable direct and maternal heterosis in crosses between maternal lines. However, this has not been confirmed in a cross between two paternal lines. Until now, attempts to find parametric or non-parametric functions to predict ejaculate fertility through seminal characteristics recorded in routinely evaluations have been very unsatisfactory. Hence, it may be necessary to find other semen quality markers, or to evaluate some of the currently used ones in a more precise manner or closer to the AI time in order to improve the ability to predict ejaculate fertility. Several seminal characteristics phenotypically correlated to male fertility, could be considered as potential traits to select for in order to genetically improving this trait. However, only the semen pH has been checked for this purpose, and a negative result has been obtained. Other traits can be studied in the future but bearing in mind that the required experiments will need large number of bucks for an accurate estimation of the genetic correlation of the trait with male fertility. This means that these experiments will be expensive and difficult to set up. The most common criterion to select paternal lines, average daily gain, seems not to be genetically correlated to male fertility and seminal traits. Thus, selection for average daily gain has no detrimental consequences on these traits, and a multi-trait selection, including growth rate and seminal traits directly related to an efficient AI semen dose production, is feasible in paternal lines. The male contribution to fertility after natural mating and after AI with semen doses with high concentration is negligible, but it has been found that, under more restrictive conditions of AI, male contributions to fertility and litter size are low but higher in magnitude than the ones obtained after natural mating. The genetic correlation between the female and male contributions to fertility has been found to be moderate to high and positive.This study was supported by the Generalitat Valenciana Research Programme (Prometeo 2009/125) and Spanish Research Projects (INIA RTA2005-00088-CO2; INIA RTA2008-00070-CO2; CICYT AGL2008-03274). Raquel Lavara was supported by a research grant from the Spanish Ministry of Education (MEC, FPU AP2007-03755) and Llibertat Tusell by a research grant from INIA.Piles, M.; Tusell, L.; Lavara García, R.; Baselga Izquierdo, M. (2013). Breeding programmes to improve male reproductive performance and efficiency of insemination dose production in paternal lines: feasibility and limitations. World Rabbit Science. 21(2):61-75. doi:10.4995/wrs.2013.1240SWORD617521

Development of Cheaper Embryo Vitrification Device Using the Minimum Volume Method

[EN] This study was designed to compare the efficiency of the Cryotop and Calibrated plastic inoculation loop (CPIL) devices for vitrification of rabbit embryos on in vitro development and implantation rate, offspring rate at birth and embryonic and fetal losses. CPIL is a simple tool used mainly by microbiologists to retrieve an inoculum from a culture of microorganisms. In experiment 1, embryos were vitrified using a Cryotop device and a CPIL device. There were no significant differences in hatched/hatching blastocyst stage rates after 48 h of culture among the vitrified groups (62±4.7% and 62±4.9%, respectively); however, the rates were significantly lower (P<0.05) than those of the fresh group (95±3.4%). In experiment 2, vitrified embryos were transferred using laparoscopic technique. The number of implanted embryos was estimated by laparoscopy as number of implantation sites at day 14 of gestation. At birth, total offspring were recorded. Embryonic and fetal losses were calculated as the difference between implanted embryos and embryos transferred and total born at birth and implanted embryos, respectively. The rate of implantation and development to term was similar between both vitrification devices (56±7.2% and 50±6.8% for implantation rate and 40±7.1% and 35±6.5% for offspring rate at birth); but significantly lower than in the fresh group (78±6.6% for implantation rate and 70±7.2% for offspring rate at birth, P<0.05). Likewise, embryonic losses were similar between both vitrification devices (44±7.2% and 50±6.8%), but significantly higher than in the fresh group (23±6.6%, P < 0.05). However, fetal losses were similar between groups (10±4.4%, 15±4.8% and 8±4.2%, for vitrified, Cryotop or CPIL and fresh, respectively). These results indicate that the CPIL device is as effective as the Cryotop device for vitrification of rabbit embryos, but at a cost of 0.05 per device.This research was supported by the projects Spanish Research project AGL2014-53405-C2-1-P Comision Interministerial de Ciencia y Tecnologia (FMJ, JSV) and Generalitat Valenciana research program (Prometeo II 2014/036, JSV, FMJ).Marco Jiménez, F.; Jiménez Trigos, ME.; Almela-Miralles, V.; Vicente Antón, JS. (2016). Development of Cheaper Embryo Vitrification Device Using the Minimum Volume Method. PLoS ONE. 11(2):1-9. https://doi.org/10.1371/journal.pone.0148661S1911

Effect of Embryo Vitrification on Rabbit Foetal Placenta Proteome during Pregnancy

Very limited information on the post-implantatory effects of vitrification has been published till now. We observed in a previous study that the vitrification procedure for the cryopreservation of embryos introduced transcriptomic and proteomic modifications in the rabbit foetal placenta at the middle of gestation. Now, we have conducted a proteomic study to determine whether protein alterations in the foetal placenta induced by the vitrification procedure remain during pregnancy. In this study, we used 2D-DIGE and mass spectrometry (MALDI-TOF-TOF and LC-MS/MS analysis) to identify the protein changes during middle and late stages of gestation (Day 14 and Day 24, respectively) in rabbit foetal placenta. We identified 11 differentially expressed proteins at Day 14 and 13 proteins at Day 24. Data are available via ProteomeXchange with identifiers PXD001840 and PXD001836. In addition, we demonstrate the presence of three proteins, serum albumin, isocitrate dehydrogenase 1 [NADP+], and phosphoglycerate mutase 1, which were altered during pregnancy. We demonstrate the existence of changes in foetal placental protein during pregnancy induced by the vitrification procedure, which brings into question whether vitrification effects observed during foetal development could lead to physiological and metabolic disorders in adulthood. This effect, taken together with other effects reported in the literature, suggests that embryo cryopreservation is not neutral.This work was supported by the Generalitat Valenciana research program (Prometeo 2014/036) and the Spanish Research Projects (CICYT AGL2011-29831-C03-01). M. D. Saenz-de-Juano was supported by a research grant from Generalitat Valenciana (Programa VALI+d, ACIF/2011/254). Nofima AS provided support in the form of salaries for author KH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the Author Contributions section.Saenz De Juano Ribes, MDLD.; Vicente Antón, JS.; Hollung, K.; Marco Jiménez, F. (2015). Effect of Embryo Vitrification on Rabbit Foetal Placenta Proteome during Pregnancy. PLoS ONE. 10(4):e0125157-e0125157. https://doi.org/10.1371/journal.pone.0125157Se0125157e012515710

EFFECT OF DONOR STRAIN AND MATURATION STAGE OF RABBIT OOCYTES ON RESULTS OF PENETRATION TEST OF RABBIT SEMEN

[EN] The objective of this work was to develop a homologous in vitre penetration test to evaluate !he behaviour of rabbit sperm. Three treatments were applied to sperm (fresh, treated with heparin and frozen with a DMSO-sucrose extender), and two types of oocytes (immature or in vitre matured) belonging to tour different rabbit lines were used. The test was performed in TCM-199, under the following conditions: 37°C, 5% C02 and saturated humidi!y for 6 hours. After incubation, oocytes were denuded and they were observad under rnicroscope to record the nurnber of sperrnatozoa/oocyte. Results obtained showed significan! differences between ali !he treatments of sperm in the percentage of oocytes with adhered sperrnatozoa (80%, 57%, 28% oocytes with adhered spermatozoa for fresh, treated with heparin and frozen semen, respectively, P<0.05). At !he same time, larger number of sperm/penetrated oocyte were observad for fresh semen !han for !he other treatments (21.3± 3. 7, 3.9 ± 0.6 and 2.3 ± 0.2 sperm/oocyte for fresh, treated with heparin and frozen semen, respectively, P<0.01 ). The resistance of fresh semen to !he incubation conditions was higher !han that of frozen or treated with heparin semen, which could determine the differences observed between thern in the results of !he test. From the results obtained, it could be concluded that immature oocytes could be used to evaluate !he behaviour of rabbit sperm, since even fresh semen were capable to ad he re to !he surface of these oocytes; on !he other hand, further studies are necessary to improve the resistance a! the incubation conditions of frozen semen, which will probably perrnit to predict its fertility in vivo.[FR] L'objectif de ce travail était de développer un test in vitro homologue de pénétration des oocytes, pour évaluer le comportement des spermatoza·ides de lapins. Trois traitements ont été appliqués sur la semence (fraiche, traitée avec de l'héparine, ou congelée avec un dilueur contenant du DMSO et du sucrose) et 2 types d'oocytes (immatures ou maturés in vitro) appartenaient a 4 lignées différentes. Le test a été réalisé dans du TCM-199 dans les · conditions suivantes : 37°C, 5% de C02, atmosphére saturée en humidité pendan! 6 heures). Aprés incubation, les oocytes ont été dénudés et observés sous un microscope pour enregistrer le nombre de spermatozordes par oocyte. Les résultats obtenus mettent en évidence des différences significatives entre tous les traitements de la semence sur le pourcentage d'oocytes présentant des spermatozordes accolés (80%, 57% et 28 %, respectivement pour la semence fraiche, traitée avec l'héparine ou congelée, p<0.05). En méme temps, un plus grand nombre de spermatozo·ides/ oocyte pénétré est observé pour la semence fraiche comparé aux autres traitements (21.3± 3. 7 vs 3.9 ± 0.6 et 2.3 ± 0.2 spermatozo"ides/oocytes respectivement pour la semen ce fraTche, traitée avec l'héparine ou congelé p<0.01 ). Une meilleure résistance de la semence fraiche aux conditions d'incubation pourrait expliquer les différences obtenues. On peut conclure que les oocytes immatures peuvent étre utilisés pour évaluer le comportement des spermatozordes de lapins, puisque méme les spermatozo"ides éjaculés sont capables d'adhérer a la surface des oocytes. Cependant, de nouvelles études seront nécessaires pour améliorer la résistance des spermatozo'ides aux conditions d'incubation de la semence préalablement congelée, afin de prédire la fertilité in vivo.This work was supported by CICYT project nº AGF98-0470-C02-01. The authors thank Mr. Neil Macowan for revising the English version.Mocé, E.; Vicente, J.; Lavara, R. (2002). EFFECT OF DONOR STRAIN AND MATURATION STAGE OF RABBIT OOCYTES ON RESULTS OF PENETRATION TEST OF RABBIT SEMEN. World Rabbit Science. doi:10.4995/wrs.2002.476SWORD10

Effect of Cooling Rate to 5 degrees C, Straw Size and Farm on Fertilizing Ability of Cryopreserved Rabbit Sperm

High fertility and prolificacy in rabbits are currently only achieved using fresh sperm. This study was conducted to determine if the cooling rate to 5 degrees C, the straw size and the farm where artificial inseminations are performed have an impact on the fertilizing ability of rabbit sperm cryopreserved with an extender containing dimethyl sulphoxide (DMSO; 1.75 M) and sucrose (0.05 M). Slow cooling to 5 degrees C improved neither fertility rate (58 vs 56% kindling rate for fast and slow cooling, respectively) nor prolificacy (6.5 vs 8.7 total born for slow and fast cooling, respectively; p &lt; 0.05) compared to fast cooling rate to 5 degrees C. The straw size did not have an effect on either fertility or prolificacy (47 vs 57% kindling rate and 6.3 vs 6.8 total born for sperm loaded into 0.25 and 0.5 ml straws, respectively). In addition, similar results were obtained between farms (46-57% kindling rate and 4.9-6.7 total born), although this effect should be studied further. In conclusion, with this extender, slow cooling does not present a beneficial effect on sperm fertilizing ability and either 0.25 or 0.5 ml straws can be used to freeze the sperm, obtaining similar results after artificial insemination. In addition, similar results were obtained between farms when using cryopreserved sperm, and these results were lower than those obtained after artificial insemination with fresh semen. Therefore, new approaches are needed to improve the results obtained when cryopreserved sperms are used before this type of sperm can be used for commercial purposes