Quality and in vitro fertility of Uruguayan Criollo bulls’ semen cryopreserved in two commercial diluents

  • Yael Filipiak https://orcid.org/0000-0003-4916-6282
  • Eileen Armstrong Universidad de la República
  • Rafael Aragunde Universidad de la República
  • Danilo Fila Universidad de la República
  • Jorge Alvaro Gil Laureiro Universidad de la República
  • Valentina Alvarez Universidad de la República
  • Marcos Pereira Universidad de la República
  • Juan C. Boggio Universidad de la República
  • Clara Larocca Universidad de la República
  • Fernando Vila Universidad de la República
  • María Silvia Llambi Dellacasa Universidad de la República
Keywords: Creole bovine, in vitro fertilization, sperm, semen quality, CASA


We evaluated the quality and in vitro fertility (IVF) of semen from the Uruguayan Criollo cattle bank of the Veterinary Faculty. We thawed semen from 8 bulls (0850, 0853, 1026, 1035, 3733, 5834, 9701, 9714), diluted in Triladyl® (T) and Andromed® (A) (with/without egg yolk) and evaluated by CASA: concentration, motility, kinetics, morphology and performed a hyposmotic test, after 2h we repeated motility and kinetics. T-test (5% significance) for time and diluents was applied and percentiles to categorize the 16 samples. The best samples were: T-9714, T-1026, A-9701, A-9714, T-0853, T-9701; except bull 0850 (A and T), the others were acceptable. For IVF we aspirated 404 A-quality oocyte-cumulus complexes (OCC) from bovine ovaries from a slaughterhouse, washed and matured them in TCM with 5% BCS and hormones, in 100μl drops, covered with mineral oil, in incubator (38.5ºC, 5% CO2 and 95% humidity), 22h. 8 groups of OCC were inseminated with selected semen: 1026, 3733, 5834 and 9701 (A and T), which was capacitated by centrifugation in Percoll® gradients (90/45%), in Talp-Sperm and adjusted with Talp-Fert to 2x106sperm/ml, drops were formed, covered with mineral oil and placed the OCC inside, 18h. The zygotes were denuded and cultured in CR1aa with BCS (5%). We evaluated cleavage (48h) and embryo development (ED) on day 7 (IETS standards). Results were analyzed with Chi square (5% significance). There were no significant differences in cleavage between diluents (A:143/207; T:128/197), nor ED (69 and 68 respectively) (p>0.05). There were no significant differences in cleavage between the 1026 (73/109) bull's semen and 9701 (68/101) (p>0.05), nor between them and 5834 (77/102) and 3733 (53/92); although, 5834 had higher cleavage rate than 3733 (p=0.008). There were no significant differences in ED between bulls (p>0.05). This work was important to assess the quality and fertility of the semen bank.


Download data is not yet available.


Aguilar, G.F., K.K. Amaro, G. Hernández, F. Fernández Reyes. 2013. Evaluación de dos diluyentes para la conservación de semen ovino: yema de huevo, lecitina de soya. División: Ciencias Biológicas y de la Salud, Departamento de Producción Agrícola y Animal. Universidad Autónoma Metropolitana. Unidad Xochimilco. México.

Aires, V. A., K-D. Hinsch, F. Mueller-Schloesser, K. Bogner, S. Mueller-Schloesser and E. Hinsch. 2003. In vitro and in vivo comparison of egg yolk-based and soybean lecithinbased extenders for cryopreservation of bovine semen. Theriogenology 60(2): 269-79. https://doi.org/10.1016/S0093-691X(02)01369-9

Amann, R. P., and D. Walberski. 2014. Computer-assited sperm analysis (CASA): Capabilities and potential developments. Theriogenology 81: 5-17. https://doi.org/10.1016/j.theriogenology.2013.09.004

Amann, R. P., and J. M. DeJarnette. 2012. Impact of genomic selection of AI dairy sires on their likely utilization and methods to estimate fertility: A paradigm shift. Review. Theriogenology 77:795– 817. https://doi.org/10.1016/j.theriogenology.2011.09.002

Amann, R. P., and S. P. S. Gill. 2000. Correlation or diagnosis and prediction? Proc. 14th ICAR, Stockholm 1, 69 (2: 3).

Andrabi, S. 2009. Factors affecting the quality of cryopreserved buffalo (Bubalus bubalis) bull spermatozoa. Reprod. Domest. Anim. 44(3):552-569. https://doi.org/10.1111/j.1439-0531.2008.01240.x

Aragunde, R., D. Fila, J. C. Boggio E. Devincenzi, E. Armstrong, S. Llambí. 2019. Caracterización de la criopreservación de semen de Bovinos Criollo Uruguayo. X Encuentro Lationoamericano y del Caribe de Biotecnología Agropecuaria y XII Simposio REDBIO Argentina. REDBIO. 12-15 de noviembre de 2019, Uruguay.

Blom, E. 1950. Interpretation of spermatic cytology in Bulls. Fertil. Steril. 1(3): 223-238. https://doi.org/10.1016/s0015-0282(16)30183-2

Carballo, D., R. Canseco, R. García, F. Montiel. 2009. Comparación de dos diluyentes comerciales para criopreservar semen de bovino bajo condiciones de campo en el trópico húmedo. Avances en la Investigación Agrícola, Pecuaria, Forestal y Acuícola en el Trópico Mexicano.

Christensen, P., R. Labouriau, A. Birck, G.B. Boe-Hansen, J. Pedersen, and S. Borchersen. 2011. Relationship among seminal quality measures and field fertility of young dairy bulls using low-dose inseminations. J. Dairy. Sci. 94: 1744-54. https://doi.org/10.3168/jds.2010-3087

Cormier, N., M. A. Sirard, and J. L. Bailey. 1997. Premature capacitation of bovine spermatozoa is initiated by cryopreservation. J. Androl. 18: 461–468. https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.1997.tb01953.x

Eid L. N., S. P. Lorton, and J. .J. Parrish, 1994. Paternal influence on S-phase in the first cell cycle of the bovine embryo. Biol. Reprod. 51: 1232–1237. https://doi.org/10.1095/biolreprod51.6.1232

FAO. 2012. Cryoconservation of Animal Genetic Resources. FAO Animal Production and Health Guidelines, no12, Roma.

Filipiak, Y., y C. Larocca. 2012. Utilización del azul tripán para diferenciar ovocitos bovinos vivos y muertos en fertilización in vitro. Arch. Zootec. 61 (234): 309-312. https://dx.doi.org/10.4321/S0004-05922012000200017

Fiser P. S., and R. W. Fairfull. 1986. The effect of rapid cooling (cold shock) of ram semen photoperiod and egg yolk in diluents on the survival of spermatozoa before and after frezzing. Cryobiol. 23(6): 518-524. https://doi.org/10.1016/0011-2240(86)90061-1

Freneau, G. E., P. J. Chenoweth, R. Ellis, G. Rupp. 2010. Sperm morphology of beef bulls evaluated by two different methods. Anim. Reprod. Sci. 118: 176-181. https://doi.org/10.1016/j.anireprosci.2009.08.015

Fukui Y, H Kohno, T. Togari, M. Hiwasa, K. Okabe. 2008. Fertility after artificial insemination using a soybean based semen extender in sheep. J Reprod Dev. 4(4): 286-289. https://doi.org/10.1262/jrd.20004

Galarza, A. 2013. Eficacia de dos diluyentes: tris + lecitina de soya (Andromed®) y tris + yema de huevo (Triladyl ®), en la crioconservación de semen de toro de la raza Jersey en Cuenca-Ecuador. Tesis de Maestría. Facultad de Ciencias Agropecuarias. Universidad de Cuenca, Ecuador.

Hummersted, R. H., J. K. Graham, and J. P. Nolan. 1990. Cryopreservation of mammaliam sperm: what we ask them to survive. J. Androl. 11(1): 73-88. PMID: 2179184.

IETS. 2013. Manual of the International Embryo Transfer Society. A procedural guide and general information for the use of embryo transfer technology emphasizing sanitary procedures. Fourth edition.

Kastelic, J. P., and J. C. Thundathil. 2008. Breeding Soundness Evaluation and Semen Analysis for Predicting Bull Fertility. Reprod. Dom. Anim. 43(2): 368–373. https://doi.org/10.1111/j.1439-0531.2008.01186.x

Kastelic, J. P. 2013. Male involvement in fertility and factors affecting semen quality in bulls. Anim. Front. 3(4): 20-25. https://doi.org/10.2527/af.2013-0029

Koziol, J. H., and C. L. Armstrong. 2018. Society for Theriogenology Manual for Breeding Soundness Examination of Bulls. Second Edition. https://www.therio.org/store/ViewProduct.aspx?id=12061863

Kruger, T. F., R. Menkveld, F. Stander, C. J. Lombard, J. P. Van der Merwe, J. Van Zyl, and K. Smith. 1986. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil. Steril. 46(6): 1118-1123. https://doi.org/10.1016/s0015-0282(16)49891-2

Larocca, C., y Y. Filipiak. 2017. Semen bovino sexado congelado-descongelado en producción de embriones in vitro. Int. J. Morphol., 35(1): 371-375. http://dx.doi.org/10.4067/S0717-95022017000100059

Larsen, L., T. Scheike, T.K. Jensen, J.P. Bonde, E. Ernst, N.H. Hjollund, Y. Zhou, N.E. Skakkebaek, and A. Giwercman. 2000. Computer-assisted semen analisys parameters as predictors for fertility of men from the general population. Human Reprod. 15(7): 1562-1567. https://doi.org/10.1093/humrep/15.7.1562

Larson, J. L., and D. J. Miller. 2000. Can relative spermatozoa galactosyltransferase activity be predictive of dairy bull fertility? J. Dairy Sci. 83: 2473–2479. https://doi.org/10.3168/jds.s0022-0302(00)75139-3

Liebfried, L., and N. L. First. 1979. Characterization of bovine follicular oocytes and their ability to mature in vitro. J. Anim. Sci. 48:76-86. https://doi.org/10.2527/jas1979.48176x

Llambí, S., y E. Armstrong. 2016. Evaluación reproductiva, creación de un banco de germoplasma y relevamiento poblacional de bovinos y cerdos Criollos del Uruguay. Proyecto de Investigación y Desarrollo CSIC, llamado 2016. Facultad de Veterinaria, Udelar. Montevideo, Uruguay.

Lozano, H. 2009. Factores que afectan la calidad seminal en toros. Rev. Med. Vet. Zoot. 56: 258-272. https://www.redalyc.org/pdf/4076/407639221010.pdf

Marquant-Le Guienne, B., P. Humblot, M. Thibier, and C. Thibault. 1990. Evaluation of bull semen fertility by homologous in vitro fertilization tests. Reprod. Nutr. Dev 30: 259–266. https://doi.org/10.1051/rnd:19900212

Minitube. 2018. Andromed®. Egg yolk free medium for bull semen. Disponible en: https://www.minitube.com/pdf/index/13503-0200_Leaflet-AndroMed_en_181002.pdf [consulta: 27/07/2020].

Morrel, J. M., A. S. Valeanu, N. Lundeheim, and A. Johannisson. 2018. Sperm quality in frozen beef and dairy bull semen. Acta Vet. Scand. 60: 41. https://doi.org/10.1186/s13028-018-0396-2

Muiño, R., C. Tamargo, C. O. Hidalgo, and A .I. Peña. 2007. Identification of sperm subpopulations with defined motility characteristics in ejaculates from Holstein bulls: effects of cryopreservation and between-bull variation. Anim. Reprod. Sci. 109(1-4): 27-39. https://doi.org/10.1016/j.anireprosci.2007.10.007

Nuñez, A. L., y A. A. Rubio. 2015. Comparación de la calidad biológica del semen bovino poscongelado utilizando como crioprotector leche al 2% de grasa, Andromed y Continental one step. Tesis de grado. Escuela Agrícola Panamericana, Zamorano. Honduras.

Oehninger, S., D Franken, N. Alexander, and G. D. Hodgen. 1992. Hemizona assay and its impact on the identification and treatment of human sperm dysfunctions. Andrología 24: 307-321. https://doi.org/10.1111/j.1439-0272.1992.tb02660.x

Oliveira Moura, L. C., M. Corona Da Silva, P. Pereira das Neves Snoeck. 2010. Diferentes soluções de teste hiposmótico para sêmen ovino. Rev. Bras. Med. Vet., 32(3): 146-150. https://1library.org/document/download/z3l58vmz

Phillips, N. J., M. R. Mcgowan, S. D. Johnston, and D. G. Mayer. 2004. Relationship between thirty post-thaw spermatozoal characteristics and the field fertility of 11 high-use Australian dairy AI sires. Anim. Reprod. Sci. 81: 47–61. https://doi.org/10.1016/j.anireprosci.2003.10.003

Rehman, F. U. 2012. Substitution of animal protein source with plant protein in semen extenders of various cattle breeds. Tesis de Master of Science (Hons) in Livestock Management. Livestock Management Department, Khyber Pakhtunkhwa Agricultural University, Peshawar-Pakistan.

Rodríguez-Martínez, H. 2003. Laboratory Semen Assessment and Prediction of Fertility: still Utopia? Reprod. Dom. Anim. 38: 312–318. https://doi.org/10.1046/j.1439-0531.2003.00436.x

Saacke, R. G., J. C. Dalton, S. Nadir, R. L. Nebel, J. H. Bame. 2000. Relationship of seminal traits and insemination time to fertilization rate and embryo quality. Anim. Reprod. Sci. 60-61: 663-667. https://doi.org/10.1016/s0378-4320(00)00137-8

Spitzer, J. C., F. M. Hopkins, and P. J. Chenoweth. 2011. New guidelines for breeding soundness evaluation (BSE) of bulls. Beef Cattle Information, Clemson University Cooperative Extension. http://media.clemson.edu/public/extension/beef_cattle/bc_2011.pdf

Salomon, S., and W. Maxwell. 1995. Frozen storage of ram semen I. Processing, frezzing, thawing and fertility after cervical insemination. Anim. Reprod. Sci. 37(3–4): 185-249. https://doi.org/10.1016/0378-4320(94)01327-I

Silveira, E. C., L. A. Bortolloti, F. Morotti, K. C. Silva-Santos, G. M. G. Santos, E. R. Andrade, and M. M. Seneda. 2013. Insemination of four cows per dose of frozen semen with a fixed-time artificial insemination protocol. Anim. Reprod. 10(2): 124-126. https://www.animal-reproduction.org/article/5b5a604ff7783717068b46b4/pdf/animreprod-10-2-124.pdf

Shi, K. S., K. H. Lu, and I. Gordon. 1990. Effect of bulls on fertilization of bovine oocytes and their subsequent development in vitro. Theriog. 33: 324. https://doi.org/10.1016/s0093-691x(02)01124-x

Thun, R., M. Hurtado, and F. Janett. 2002. Comparison of Biociphos-Plus and TRIS-egg yolk extender for cryopreservation of bull semen. Theriog. 57: 1087-1094. https://doi.org/10.1016/s0093-691x(01)00704-x

Trimeche, A., J. Yvon, M. Vidament, E. Palmer, and M. Magistrini. 1999. Effects of glutamine, proline, histidine, and betaine on post.thaw motility of stallion spermatozoa. Theriog. 52(1): 181-91. https://doi.org/10.1016/s0093-691x(99)00120-x

Truelson, S. L., J. K. Graham, R. G. Mortimer, and T. G. Field. 1996. In vitro penetration into bovine oocytes and zona-free hamster oocytes by bull spermatozoa treated with liposomes. J. Dairy Sci. 79:991–999. https://doi.org/10.3168/jds.s0022-0302(96)76450-0

Üstüner, B., S. Alcay, Z. Nur, H. Sagirkaya, and M. K. Soylu. 2013. Effect of egg yolk and soybean lecithin on tris-based extender in post-thaw ram semen quality and in vitro fertility. Kafkas Univ. Vet. Fak. Derg. 20(3): 393-398. https://www.cabdirect.org/cabdirect/abstract/20143102997

Valverde, A., y M. Madrigal-Valverde. 2018. Sistemas de análisis computadorizado de semen en la reproducción animal1. Agronomía Mesoamericana 29, 2:469-484 http://dx.doi.org/10.15517/ma.v29i2.30613

Van Wagtendonk-de Leeuw, A., R. Haring, L. Kaal-Lansbergen, and J. H. Den Daas. 2000. Fertility results using bovine semen cryopreserved with extenders based on egg yolk and soybean extract. Theriog. 54(1): 57-67. https://doi.org/10.1016/S0093-691X(00)00324-1

Watson, P. F. 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fertil. Dev. 7: 871-891. https://doi.org/10.1071/rd9950871

Zhang, B. R., B. Larsson, N. Lundeheim, M.G. Haard, and H. Rodriguez-Martinez. 1999. Prediction of bull fertility by combined in vitro assessments of frozen-thawed semen from young dairy bulls entering an AI-programme. Int. J. Androl. 22: 253–260. https://doi.org/10.1046/j.1365-2605.1999.00178.x

Zhang, B.R., B. Larsson, N. Lundeheim, H. Rodriguez-Martinez. 1997. Relationship between embryo development in vitro and 56-day non return rates of cows inseminated with frozen-thawed semen from dairy bulls. Theriog. 48: 221–231.

How to Cite
Filipiak, Yael, Eileen Armstrong, Rafael Aragunde, Danilo Fila, Jorge Alvaro Gil Laureiro, Valentina Alvarez, Marcos Pereira, Juan C. Boggio, Clara Larocca, Fernando Vila, and María Silvia Llambi Dellacasa. 2020. “Quality and in Vitro Fertility of Uruguayan Criollo bulls’ Semen Cryopreserved in Two Commercial Diluents”. Archivos Latinoamericanos De Producción Animal 28 (3-4), 133-43. https://ojs.alpa.uy/index.php/ojs_files/article/view/2807.