Hostname: page-component-7d684dbfc8-d9hj2 Total loading time: 0 Render date: 2023-09-30T23:39:48.879Z Has data issue: false Feature Flags: { "corePageComponentGetUserInfoFromSharedSession": true, "coreDisableEcommerce": false, "coreDisableSocialShare": false, "coreDisableEcommerceForArticlePurchase": false, "coreDisableEcommerceForBookPurchase": false, "coreDisableEcommerceForElementPurchase": false, "coreUseNewShare": true, "useRatesEcommerce": true } hasContentIssue false

Sperm chromatin protamination influences embryo development in unsexed and sexed bull semen

Published online by Cambridge University Press:  15 January 2021

Thiago Velasco Guimarães Silva*
Laboratory of In Vitro Fertilization, Institute of Biological Science, Federal University of Pará, Belém, Pará, Brazil
Priscila Di Paula Bessa Santana
Federal Rural University of Amazon, Belém, Pará, Brazil
Eduardo Baia de Souza
Laboratory of In Vitro Fertilization, Institute of Biological Science, Federal University of Pará, Belém, Pará, Brazil
Ana Júlia Mota de Lima
Laboratory of In Vitro Fertilization, Institute of Biological Science, Federal University of Pará, Belém, Pará, Brazil
Caroline de Araújo Santos
Laboratory of In Vitro Fertilization, Institute of Biological Science, Federal University of Pará, Belém, Pará, Brazil
Nathália Nogueira da Costa Almeida
Laboratory of In Vitro Fertilization, Institute of Biological Science, Federal University of Pará, Belém, Pará, Brazil
Vanessa Cunha de Brito
Laboratory of In Vitro Fertilization, Institute of Biological Science, Federal University of Pará, Belém, Pará, Brazil
Arnaldo Algaranhar Gonçalves
Laboratory of In Vitro Fertilization, Institute of Biological Science, Federal University of Pará, Belém, Pará, Brazil
Sebastião Tavares Rolim Filho
Federal Rural University of Amazon, Belém, Pará, Brazil
Marcela da Silva Cordeiro
Federal Institute of Pará, Ananindeua, Pará, Brazil
Simone do Socorro Damasceno Santos
Laboratory of In Vitro Fertilization, Institute of Biological Science, Federal University of Pará, Belém, Pará, Brazil
Moysés dos Santos Miranda
Laboratory of In Vitro Fertilization, Institute of Biological Science, Federal University of Pará, Belém, Pará, Brazil
Otávio Mitio Ohashi
Laboratory of In Vitro Fertilization, Institute of Biological Science, Federal University of Pará, Belém, Pará, Brazil
Author for correspondence: Thiago Velasco Guimarães Silva. Federal University of Pará, Belém, Pará, Brazil. Tel: +55 91988823358. E-mail:


Sex selection through sperm sorting offers advantages in regards selection pressure in high-producing livestock. However, the sex-sorting process results in sperm membrane and DNA damage that ultimately decrease fertility. We hypothesized that given the role of protamines in DNA packaging, protamine deficiency could account, at least partially, for the DNA damage observed following sperm sex sorting. To test this, we compared protamine status between unsexed and sexed spermatozoa from two bulls using the fluorochrome chromomycin A3 (CMA3) and flow cytometry. Then, we assessed embryo development following in vitro fertilization (IVF) using the same sperm treatments. Overall, sperm protamination was not different between sexed and unsexed semen. However, one of the two bulls displayed higher rates of protamine deficiency for both unsexed and sexed semen (P < 0.05). Moreover, unsexed semen from this bull yielded lower blastocyst (P < 0.05) and blastocyst hatching rates than unsexed sperm from the other bull. CMA3-positive staining was negatively correlated with cleavage (R2 85.1, P = 0.003) and blastocyst hatching (R2 87.6, P = 0.006) rates in unsexed semen. In conclusion, while the sex-sorting process had no effect on sperm protamine content, we observed a bull effect for sperm protamination, which correlated to embryo development rates following IVF.

Research Article
© The Author(s), 2021. Published by Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)


Aoki, V and Carrell, D (2004). Human protamines and the developing spermatid: their structure, function, expression and relationship with male infertility. Asian J Androl 5, 315–24.Google Scholar
Aoki, VW, Liu, L and Carrell, DT (2005). Identification and evaluation of a novel sperm protamine abnormality in a population of infertile males. Hum Reprod 20, 1298–306.CrossRefGoogle Scholar
Barceló-Fimbres, M, Campos-Chillón, L F and Seidel, GE Jr (2011). In vitro fertilization using non-sexed and sexed bovine sperm: sperm concentration, sorter pressure, and bull effects. Reprod Domest Anim 46, 495502.CrossRefGoogle ScholarPubMed
Beyhan, Z, Johnson, LA and First, NL (1999). Sexual dimorphism in IVM-IVF bovine embryos produced from X and Y chromosome-bearing spermatozoa sorted by high speed flow cytometry. Theriogenology 52, 3548.CrossRefGoogle Scholar
Bianchi, PG, Manicardi, GC, Bizzaro, D, Bianchi, U and Sakkas, D (1993). Effect of Deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human mature spermatozoa. Biol Reprod 49, 1083–8.CrossRefGoogle ScholarPubMed
Bodmer, M, Janett, F, Hässig, M, Daas, N den, Reichert, P and Thun, R (2005). Fertility in heifers and cows after low dose insemination with sex-sorted and non-sorted sperm under field conditions. Theriogenology 64, 1647–55.CrossRefGoogle ScholarPubMed
Boe-Hansen, GB, Morris, ID, Ersbøll, AK, Greve, T and Christensen, P (2005). DNA integrity in sexed bull sperm assessed by neutral Comet assay and sperm chromatin structure assay. Theriogenology 63, 1789–802.CrossRefGoogle ScholarPubMed
Botigelli, RC, Schwarz, KL, Zaffalon, FG, Del Collado, M Castro, FC, Fernandes, H and Leal, CLV (2017). Influence of nitric oxide and phosphodiesterases during in vitro maturation of bovine oocytes on meiotic resumption and embryo production. Zygote 25, 321–30.CrossRefGoogle ScholarPubMed
Botigelli, RC, Razzaa, EM, Pioltinea, EM, Fontesa, PK, Schwarzb, KRL, Leal, CLV and Nogueira, MFG (2018). Supplementing in vitro embryo production media by NPPC and sildenafil affect the cytoplasmic lipid content and gene expression of bovine cumulus–oocyte complexes and embryos. Reprod Biol 1, 6675.CrossRefGoogle Scholar
Butler, ST, Hutchinson, IA, Cromie, AR and Shalloo, L (2014). Applications and cost benefits of sexed semen in pasture-based dairy production systems. Animal, 8, 165172.CrossRefGoogle ScholarPubMed
Carrell, DT (2012). Epigenetics of the male gamete. Fertil Steril 97, 267–74.CrossRefGoogle ScholarPubMed
Carrell, DT, Emery, BR and Hammoud, S (2007). Altered protamine expression and diminished spermatogenesis: what is the link? Hum Reprod Update 13, 313–27.CrossRefGoogle ScholarPubMed
Carrell, DT, Emery, BR and Hammoud, S (2008). The aetiology of sperm protamine abnormalities and their potential impact on the sperm epigenome. Int J Androl 31, 537–45.CrossRefGoogle ScholarPubMed
Carvalho, JO, Sartori, R, Machado, GM, Mourão, GB and Dode, MAN (2010). Quality assessment of bovine cryopreserved sperm after sexing by flow cytometry and their use in in vitro embryo production. Theriogenology 74, 1521–30.CrossRefGoogle ScholarPubMed
Carvalho, JO, Silva, LP, Sartori, R and Dode, MAN (2013). Nanoscale differences in the shape and size of X and Y chromosome-bearing bovine sperm heads assessed by atomic force microscopy. PLoS One 8, e59387.CrossRefGoogle Scholar
Castro, LS, Siqueira, AFP, Hamilton, TRS, Mendes, CM, Visintin, JA and Assumpção, MEOA (2018). Effect of bovine sperm chromatin integrity evaluated using three different methods on in vitro fertility. Theriogenology, 107, 142–8.CrossRefGoogle ScholarPubMed
Catteeuw, M, Wydooghe, E, Mullaart, E, Knijn, HM and Van Soom, A (2017). In vitro production of bovine embryos derived from individual donors in the Corral® dish. Acta Vet Scand 59, 41.CrossRefGoogle ScholarPubMed
Cree, LH, Balhorn, R and Brewer, LR (2011). Single molecule studies of DNA-protamine interactions. Protein Pept Lett 18, 802–10.CrossRefGoogle ScholarPubMed
Diógenes, MN, Guimarães, ALS, Leme, LO and Dode, MAN (2017). Bovine in vitro embryo production: the effects of fibroblast growth factor 10 (FGF10). J Assist Reprod Genet 34, 383–90.CrossRefGoogle Scholar
Dogan, S, Vargovic, P, Oliveira, R, Belser, LE, Kaya, A, Moura, A, Sutovsky, P, Parrish, J, Topper, E and Memili, E (2015). Sperm protamine-status correlates to the fertility of breeding bulls. Biol Reprod 92, 92.CrossRefGoogle ScholarPubMed
Fathi, Z, Tavalaee, M, Kiani, A, Deemeh, MR, Modaresi, M and Nasr-Esfahani, MH (2011). Flow cytometry: a novel approach for indirect assessment of protamine deficiency by CMA3 staining, taking into account the presence of M540 or apoptotic bodies. Int J Fertil Steril 5, 128–33.Google ScholarPubMed
Fernández-Díez, C, González-Rojo, S, Lombó, M and Herráez, MP (2016). Impact of sperm DNA damage and oocyte-repairing capacity on trout development. Reproduction 152, 5767.CrossRefGoogle ScholarPubMed
Fortes, MRS, Satake, N, Corbet, DH, Corbet, NJ, Burns, BM, Moore, SS and Boe-Hansen, GB (2014). Sperm protamine deficiency correlates with sperm DNA damage in Bos indicus bulls. Andrology 2, 370–8.CrossRefGoogle ScholarPubMed
Francis, S, Yelumalai, S, Jones, C and Coward, K (2014). Aberrant protamine content in sperm and consequential implications for infertility treatment. Hum Fertil 17, 80–9.CrossRefGoogle ScholarPubMed
Frijters, ACJ, Mullaart, E, Roelofs, RMG, van Hoorne, RP, Moreno, JF, Moreno, O and Merton, JS (2009). What affects fertility of sexed bull semen more, low sperm dosage or the sorting process? Theriogenology 71, 64–7.CrossRefGoogle ScholarPubMed
Garcia-Herreros, M, Carter, TF, Villagómez, DAF, MacAulay, AD, Rath, D, King, WA and Lonergan, P (2010). Incidence of chromosomal abnormalities in bovine blastocysts derived from unsorted and sex-sorted spermatozoa. Reprod Fertil Dev 22, 1272–8.CrossRefGoogle ScholarPubMed
Giacone, F, Cannarella, R, Mongioì, LM, Alamo, A, Condorelli, RA, Calogero, AE and La Vignera, S (2019). Epigenetics of male fertility: effects on assisted reproductive techniques. World J Men Health 37, 148–56.CrossRefGoogle ScholarPubMed
Holm, P, Booth, PJ, Schmidt, MH, Greve, T and Callesen, H (1999). High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. Theriogenology 52, 683700.CrossRefGoogle ScholarPubMed
Inaba, Y, Abe, R, Geshi, M, Matoba, S, Nagai, T and Somfai, T (2016). Sex sorting of spermatozoa affects developmental competence of in vitro fertilized oocytes in a bull-dependent manner. J Reprod Dev 62, 451–6.CrossRefGoogle Scholar
Iranpour, FG, Nasr-Esfahani, MH, Valojerdi, MR and al-Taraihi, TM (2000). Chromomycin A3 staining as a useful tool for evaluation of male fertility. J Assist Reprod Genet 17, 60–6.CrossRefGoogle ScholarPubMed
Leibfried, L and First, NL (1979). Characterization of bovine follicular oocytes and their ability to mature in vitro . J Anim Sci 48, 7686.CrossRefGoogle ScholarPubMed
Lu, KH, Cran, DG and Seidel, GE Jr (2000). In vitro fertilization with flow-cytometrically-sorted bovine sperm. Theriogenology 52, 1393–405.CrossRefGoogle Scholar
Ménézo, Y, Dale, B and Cohen, M (2010). DNA damage and repair in human oocytes and embryos: a review. Zygote 18, 357–65.CrossRefGoogle ScholarPubMed
Miller, D, Brinkworth, M and Iles, D (2010). Paternal DNA packaging in spermatozoa: more than the sum of its parts? DNA, histones, protamines and epigenetics. Reproduction 139, 287301.CrossRefGoogle Scholar
Morton, KM, Herrmann, D, Sieg, B, Struckmann, C, Maxwell, WM, Rath, D, Evans, G, Lucas-Hahn, A, Niemann, H and Wrenzycki, C (2007). Altered mRNA expression patterns in bovine blastocysts after fertilisation in vitro using flow-cytometrically sex-sorted sperm. Mol Reprod Dev 74, 931–40.CrossRefGoogle ScholarPubMed
Nanassy, L, Liu, L, Griffin, J and Carrell, DT (2011). The clinical utility of the protamine 1/protamine 2 ratio in sperm. Protein Pept Lett 18, 772–7.CrossRefGoogle ScholarPubMed
Ni, K, Spiess, A-N, Schuppe, H-C and Steger, K (2016). The impact of sperm protamine deficiency and sperm DNA damage on human male fertility: a systematic review and meta-analysis. Andrology 4, 789–99.CrossRefGoogle ScholarPubMed
Palma, GA, Olivier, NS, Neumüller, C and Sinowatz, F (2008). Effects of sex-sorted spermatozoa on the efficiency of in vitro fertilization and ultrastructure of in vitro produced bovine blastocysts. Anat Histol Embryol 37, 6773.Google ScholarPubMed
Parrish, JJ, Susko-Parrish, J, Winer, M and First, NL (1988). Capacitation of bovine sperm by heparin. Biol Reprod 38, 1171–80.CrossRefGoogle ScholarPubMed
Perrini, C, Esposti, P, Cremonesi, F and Consiglio, AL (2018). Secretome derived from different cell lines in bovine embryo production in vitro . Reprod Fertil Dev 30, 658–71.CrossRefGoogle ScholarPubMed
Puglisi, R, Vanni, R, Galli, A, Balduzzi, D, Parati, K, Bongioni, G, Crotti, G, Duchi, R, Galli, C, Lazzari, G and Aleandri, R (2006). In vitro fertilisation with frozen–thawed bovine sperm sexed by flow cytometry and validated for accuracy by real-time PCR. Reproduction 132, 519–26.CrossRefGoogle ScholarPubMed
Rahman, MB, Vandaele, L, Rijsselaere, T, Maes, D, Hoogewijs, M, Frijters, A, Noordman, J, Granados, A, Dernelle, E, Shamsuddin, M, Parrish, JJ and Van Soom, A (2011). Scrotal insulation and its relationship to abnormal morphology, chromatin protamination and nuclear shape of spermatozoa in Holstein-Friesian and Belgian Blue bulls. Theriogenology 76, 1246–57.CrossRefGoogle ScholarPubMed
Rajabi, H, Mohseni-Kouchesfehani, H, Mohammadi-Sangcheshmeh, A, Farifteh-Nobijari, F and Salehi, M (2016). Pronuclear epigenetic modification of protamine deficient human sperm following injection into mouse oocytes. Syst Biol Reprod Med 62, 125–32.CrossRefGoogle ScholarPubMed
Sartori, R, Souza, AH, Guenther, JN, Caraviello, DZ, Geiger, LN, Schenk, JL and Wiltbank, MC (2004). Fertilization rate and embryo quality in superovulated Holstein heifers artificially inseminated with X-sorted or unsorted sperm. Anim Reprod 1, 8690.Google Scholar
Seidel, GE (2007). Overview of sexing sperm. Theriogenology 68, 443–6.CrossRefGoogle ScholarPubMed
Simões, R, Feitosa, WB, Mendes, CM, Marques, MG, Nicacio, AC, de Barros, FR, Visintin, JA and Assumpção, ME (2009). Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency. Biotech Histochem 84, 7983.CrossRefGoogle ScholarPubMed
Stringfellow, DA and Seidel, SM (1998). Manual of the International Embryo Transfer Society (3rd edn). International Embryo Transfer Society, IETS.Google Scholar
Tavalaee, M, Razavi, S and Nasr-Esfahani, MH (2009). Influence of sperm chromatin anomalies on assisted reproductive technology outcome. Fertil Steril 91, 1119–26.CrossRefGoogle ScholarPubMed
Tavalaee, M, Kiani, A, Arbabian, M, Deemeh, MR and Esfahani, MHN (2010). Flow cytometry: a new approach for indirect assessment of sperm protamine deficiency. Int J Fertil Steril 3, 184.Google Scholar
Wilson, RD, Fricke, PM, Leibfried-Rutledge, ML, Rutledge, JJ, Penfield, CMS and Weigel, KA (2006). In vitro production of bovine embryos using sex-sorted sperm. Theriogenology 65, 1007–15.CrossRefGoogle ScholarPubMed
Xu, J, Chaubal, SA and Du, F (2009). Optimizing IVF with sexed sperm in cattle. Theriogenology 71, 3947.CrossRefGoogle ScholarPubMed
Zhao, XM, Ren, JJ, Zhao, SJ, Cui, LS, Hao, HS, Wang, HY, Du, WH, Qin, T, Liu, Y, Wang, D and Zhu, HB (2014). Apoptosis-Like events and in vitro fertilization capacity of sex-sorted bovine sperm. Reprod Domest Anim 49, 543–9.CrossRefGoogle ScholarPubMed