Bacterial contamination of semen has become an important contributor to the reduced shelf life of insemination doses in the poultry industry, which is why antibiotics (ATBs) are an important component of semen extenders. Due to a global rise in antimicrobial resistance, the aim of this study was to assess the efficiency of selected commercially available semen extenders to prevent possible bacterial contamination of rooster ejaculates. Two selected extenders free from or containing 31.2 µg/mL kanamycin (KAN) were used to process semen samples from 63 healthy Lohmann Brown roosters. Phosphate-buffered saline without ATBs was used as a control. The extended samples were stored at 4 °C for 24 h. Sperm motility, viability, mitochondrial activity, DNA integrity and the oxidative profile of each extended sample were assessed following 2 h and 24 h of storage. Furthermore, selective media were used to quantify the bacterial load and specific bacterial species were identified with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The results indicate that semen extenders enriched with KAN ensured a significantly higher preservation of sperm quality in comparison to their KAN-free counterparts. Bacterial load was significantly decreased in diluents supplemented with ATBs (p ≤ 0.001); however, KAN alone was not effective enough to eradicate all bacteria since several Escherichia coli, Enterococcus faecalis, Enterococcus faecium and Micrococcus luteus were retrieved from samples extended in KAN-supplemented commercial extenders. As such, we may suggest that more focus should be devoted to the selection of an optimal combination and dose of antibiotics for poultry extenders, which should be accompanied by a more frequent bacteriological screening of native as well as extended poultry semen.

AgroBioTech Research Centre Slovak University of Agriculture in Nitra Tr A Hlinku 2 94976 Nitra Slovakia

Department of Bioenergetics Food Analysis and Microbiology Institute of Food Technology and Nutrition University of Rzeszow Cwiklinskiej 1 35 601 Rzeszow Poland

Department of Neuroscience 2nd Faculty of Medicine Charles University 5 Úvalu 84 15006 Prague Czech Republic

Institute of Applied Biology Faculty of Biotechnology and Food Sciences Slovak University of Agriculture in Nitra Tr A Hlinku 2 94976 Nitra Slovakia

Institute of Biotechnology Faculty of Biotechnology and Food Sciences Slovak University of Agriculture in Nitra Tr A Hlinku 2 94976 Nitra Slovakia

Institute of Fruit Science Viticulture and Enology Faculty of Horticulture and Landscape Engineering Slovak University of Agriculture Tr A Hlinku 2 94976 Nitra Slovakia

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Scanes C. The Global Importance of Poultry. Poult. Sci. 2007;86:1057–1058. doi: 10.1093/ps/86.6.1057. PubMed DOI

Birhanu M.Y., Osei-Amponsah R., Yeboah Obese F., Dessie T. Smallholder poultry production in the context of increasing global food prices: Roles in poverty reduction and food security. Anim. Front. 2023;13:17–25. doi: 10.1093/af/vfac069. PubMed DOI PMC

Wen C., Mai C., Cai R., Gou Q., Zhang B., Li J., Sun C., Yang N. Inheritance of the duration of fertility in chickens and its correlation with laying performance. Genet. Sel. Evol. 2022;54:41. doi: 10.1186/s12711-022-00733-7. PubMed DOI PMC

Mohan J., Sharma S., Kolluri G., Dhama K. History of artificial insemination in poultry, its components and significance. Worlds Poult. Sci. J. 2018;74:475–488. doi: 10.1017/S0043933918000430. DOI

Bakst M.R., Dymo J.S. Artificial Insemination in Poultry. In: Lemma A., editor. Success in Artificial Insemination—Quality of Semen and Diagnostics Employed. 1st ed. InTech; London, UK: 2013. pp. 175–195.

Donoghue A.M., Wishart G.J. Storage of poultry semen. Anim. Reprod. Sci. 2000;62:213–232. doi: 10.1016/S0378-4320(00)00160-3. PubMed DOI

Blesbois E., Brillard J.P. Specific features of in vivo and in vitro sperm storage in birds. Animal. 2007;1:1472–1481. doi: 10.1017/S175173110700081X. PubMed DOI

Partyka A., Niżański W. Advances in storage of poultry semen. Anim. Reprod. Sci. 2022;246:106921. doi: 10.1016/j.anireprosci.2021.106921. PubMed DOI

Bustani G.S., Baiee F.H. Semen extenders: An evaluative overview of preservative mechanisms of semen and semen extenders. Vet. World. 2021;14:1220–1233. doi: 10.14202/vetworld.2021.1220-1233. PubMed DOI PMC

Morrell J.M., Wallgren M. Alternatives to antibiotics in semen extenders: A review. Pathogens. 2014;3:934–946. doi: 10.3390/pathogens3040934. PubMed DOI PMC

Maasjost J., Mühldorfer K., de Jäckel S.C., Hafez H.M. Antimicrobial Susceptibility Patterns of Enterococcus faecalis and Enterococcus faecium Isolated from Poultry Flocks in Germany. Avian Dis. 2015;59:143–148. doi: 10.1637/10928-090314-RegR. PubMed DOI

Tvrdá E., Petrovičová M., Benko F., Ďuračka M., Kováč J., Slanina T., Galovičová L., Žiarovská J., Kačániová M. Seminal Bacterioflora of Two Rooster Lines: Characterization, Antibiotic Resistance Patterns and Possible Impact on Semen Quality. Antibiotics. 2023;12:336. doi: 10.3390/antibiotics12020336. PubMed DOI PMC

Cox N.A., Stern N.J., Wilson J.L., Musgrove M.T., Buhr R.J., Hiett K.L. Isolation of Campylobacter spp. from Semen Samples of Commercial Broiler Breeder Roosters. Avian Dis. 2002;46:717–720. doi: 10.1637/0005-2086(2002)046[0717:IOCSFS]2.0.CO;2. PubMed DOI

Ahmed K. Bacterial Flora of Poultry Semen and Their Antibiotic Sensitivity Pattern. Int. J. Appl. Pure Sci. Agric. 2015;1:39–41.

Reiber M.A., McInroy J.A., Conner D.E. Enumeration and Identification of Bacteria in Chicken Semen. Poult. Sci. 1995;74:795–799. doi: 10.3382/ps.0740795. PubMed DOI

Sayed M.A.M., Abd-Elhafeez H.H., Afifi O.S., Marzouk M.W., El-Sherry T.M. Sperm tendency to agglutinate in motile bundles in relation to sperm competition and fertility duration in chickens. Sci. Rep. 2022;12:18860. doi: 10.1038/s41598-022-22049-8. PubMed DOI PMC

Omprakash A., Venkatesh G. Effect of vaginal douching and different semen extenders on bacterial load and fertility in turkeys. Br. Poult. Sci. 2006;47:523–526. doi: 10.1080/00071660600829209. PubMed DOI

Haines M.D., Parker H.M., McDaniel C.D., Kiess A.S. Impact of 6 different intestinal bacteria on broiler breeder sperm motility in vitro. Poult. Sci. 2013;92:2174–2181. doi: 10.3382/ps.2013-03109. PubMed DOI

Tvrdá E., Petrovičová M., Benko F., Ďuračka M., Galovičová L., Slanina T., Kačániová M. Curcumin Attenuates Damage to Rooster Spermatozoa Exposed to Selected Uropathogens. Pharmaceutics. 2022;15:65. doi: 10.3390/pharmaceutics15010065. PubMed DOI PMC

Tvrdá E., Ďuračka M., Benko F., Lukáč N. Bacteriospermia—A formidable player in male subfertility. Open Life Sci. 2022;17:1001–1029. doi: 10.1515/biol-2022-0097. PubMed DOI PMC

Dhama K., Singh R.P., Karthik K., Chakraborty S., Tiwari R., Wani M.Y., Mohan J. Artificial Insemination in Poultry and Possible Transmission of Infectious Pathogens: A Review. Asian J. Anim. Vet. Adv. 2014;9:211–228. doi: 10.3923/ajava.2014.211.228. DOI

Al-Bahry S.N., Mahmoud I.Y., Al Musharafi S.K., Paulson J.R. Consumption of Contaminated Eggs: A Public Health Concern. Med. Res. Arch. 2015;2:22–28. doi: 10.18103/mra.v2i4.366. DOI

Mehdi Y., Létourneau-Montminy M.P., Gaucher M.L., Chorfi Y., Suresh G., Rouissi T., Brar S.K., Côté C., Ramirez A.A., Godbout S. Use of antibiotics in broiler production: Global impacts and alternatives. Anim. Nutr. 2018;4:170–178. doi: 10.1016/j.aninu.2018.03.002. PubMed DOI PMC

Roberts J.R., Souillard R., Bertin J. Avian diseases which affect egg production and quality. In: Nys Y., Bain M., Van Immerseel F., editors. Improving the Safety and Quality of Eggs and Egg Products. 1st ed. Woodhead Publishing; Ambridge, UK: 2011. pp. 376–393.

Lenický M., Slanina T., Kačániová M., Galovičová L., Petrovičová M., Ďuračka M., Benko F., Kováč J., Tvrdá E. Identification of Bacterial Profiles and Their Interactions with Selected Quality, Oxidative, and Immunological Parameters of Turkey Semen. Animals. 2021;11:1771. doi: 10.3390/ani11061771. PubMed DOI PMC

Gale C., Brown K.I. The Identification of Bacteria Contaminating Collected Semen and the Use of Antibiotics in Their Control. Poult. Sci. 1961;40:50–55. doi: 10.3382/ps.0400050. DOI

Alkali I.M., Asuku S.O., Umar M.B., Abba A., Mustapha A., Bukar M.M., Waziri M.A. Microbial Contaminants in Fresh and Extended Turkey Semen and their Sensitivity to Antibiotics. Nig. Vet. J. 2020;41:1–6. doi: 10.4314/nvj.v41i1.1. DOI

Sexton T.J., Jacobs L.A., McDaniel G.R. A new poultry semen extender. 4. Effect of antibacterials in control of bacterial contamination in chicken semen. Poult. Sci. 1980;59:274–281. doi: 10.3382/ps.0590274. PubMed DOI

Duracka M., Lukac N., Kacaniova M., Kantor A., Hleba L., Ondruska L., Tvrda E. Antibiotics Versus Natural Biomolecules: The Case of In Vitro Induced Bacteriospermia by Enterococcus faecalis in Rabbit Semen. Molecules. 2019;24:4329. doi: 10.3390/molecules24234329. PubMed DOI PMC

Rakha B.A., Zafar Z., Ansari M.S., Akhter S., Qadeer S., Akhter A., Waseem K., Santiago-Moreno J. Influence of Bacterial Contamination and Antibiotic Sensitivity on Cryopreserved Sperm Quality of Indian Red Jungle Fowl. Biopreserv. Biobank. 2023. advance online publication . PubMed DOI

Gross S., Seinige D., Kehrenberg C., Oliveira M., Siebert U. Occurrence of Antimicrobial-Resistant Escherichia coli in Marine Animals in the North and Baltic Sea: Preliminary Results; Proceedings of the 50th Annual IAAAM Conference; Durban, South Africa. 18–22 May 2019.

Faisal A.J., Salman H.A. Determination of Semen Quality and Antibacterial Susceptibility Pattern of Bacteria Isolated from Semen of Iraqi Subjects. Microbiol. Biotechnol. Lett. 2021;49:587–593. doi: 10.48022/mbl.2108.08006. DOI

Goularte K.L., Voloski F.L.S., Redú J.F.M., Ferreira C.E.R., Vieira A.D., Duval E.H., Mondadori R.G., Lucia T., Jr. Antibiotic resistance in microorganisms isolated in a bull semen stud. Reprod. Domest. Anim. 2020;55:318–324. doi: 10.1111/rda.13621. PubMed DOI

Tvrdá E., Kačániová M., Baláži A., Vašíček J., Vozaf J., Jurčík R., Ďuračka M., Žiarovská J., Kováč J., Chrenek P. The Impact of Bacteriocenoses on Sperm Vitality, Immunological and Oxidative Characteristics of Ram Ejaculates: Does the Breed Play a Role? Animals. 2022;12:54. doi: 10.3390/ani12010054. PubMed DOI PMC

Tvrdá E., Bučko O., Rojková K., Ďuračka M., Kunová S., Kováč J., Benko F., Kačániová M. The Efficiency of Selected Extenders against Bacterial Contamination of Boar Semen in a Swine Breeding Facility in Western Slovakia. Animals. 2021;11:3320. doi: 10.3390/ani11113320. PubMed DOI PMC

Ďuračka M., Belić L., Tokárová K., Žiarovská L., Kačániová M., Lukáč N., Tvrdá E. Bacterial communities in bovine ejaculates and their impact on the semen quality. Syst. Biol. Reprod. Med. 2021;67:438–449. doi: 10.1080/19396368.2021.1958028. PubMed DOI

Shafeeque C.M., Singh R.P., Sharma S.K., Mohan J., Sastry K.V., Kolluri G., Saxena V.K., Tyagi J.S., Kataria J.M., Azeez P.A. Development of a new method for sperm RNA purification in the chicken. Anim. Reprod. Sci. 2014;149:259–265. doi: 10.1016/j.anireprosci.2014.06.032. PubMed DOI

Boone M.A., Hughes B.L. Contamination of semen and its effect on avian fertility. Poult. Sci. 1970;49:402–404. doi: 10.3382/ps.0490402. PubMed DOI

da Silva M., Dombre C., Brionne A., Monget P., Chessé M., De Pauw M., Mills M., Combes-Soia L., Labas V., Guyot N., et al. The unique features of proteins depicting the chicken amniotic fluid. Mol. Cell. Proteomics. 2019;18:S174–S190. doi: 10.1074/mcp.RA117.000459. PubMed DOI PMC

Silphaduang U., Hincke M.T., Nys Y., Mine Y. Antimicrobial proteins in chicken reproductive system. Biochem. Biophys. Res. Commun. 2006;340:648–655. doi: 10.1016/j.bbrc.2005.12.054. PubMed DOI

Schulz M., Sánchez R., Soto L., Risopatrón J., Villegas J. Effect of Escherichia coli and Its Soluble Factors on Mitochondrial Membrane Potential, Phosphatidylserine Translocation, Viability, and Motility of Human Spermatozoa. Fertil. Steril. 2010;94:619–623. doi: 10.1016/j.fertnstert.2009.01.140. PubMed DOI

Dutta S., Sengupta P., Izuka E., Menuba I., Jegasothy R., Nwagha U. Staphylococcal Infections and Infertility: Mechanisms and Management. Mol. Cell. Biochem. 2020;474:57–72. doi: 10.1007/s11010-020-03833-4. PubMed DOI

He B., Guo H., Gong Y., Zhao R. Lipopolysaccharide-induced mitochondrial dysfunction in boar sperm is mediated by activation of oxidative phosphorylation. Theriogenology. 2017;87:1–8. doi: 10.1016/j.theriogenology.2016.07.030. PubMed DOI

Qiang H., Jiang M.S., Lin J.Y., He W.M. Influence of enterococci on human sperm membrane in vitro. Asian J. Androl. 2007;9:77–81. doi: 10.1111/j.1745-7262.2007.00219.x. PubMed DOI

Zhu X., Shi D., Li X., Gong W., Wu F., Guo X., Xiao H., Liu L., Zhou H. TLR signalling affects sperm mitochondrial function and motility via phosphatidylinositol 3-kinase and glycogen synthase kinase-3α. Cell. Signal. 2016;28:148–156. doi: 10.1016/j.cellsig.2015.12.002. PubMed DOI

Song T., Shi Y., Wang Y., Qazi I.H., Angel C., Zhang M. Implication of polyhistidine, a novel apoptosis inhibitor, in inhibiting lipopolysaccharide-induced apoptosis in boar sperm. Animals. 2019;9:719. doi: 10.3390/ani9100719. PubMed DOI PMC

Silva E.J.R., Ribeiro C.M., Mirim A.F.M., Silva A.A.S., Romano R.M., Hallak J., Avellar M.C.W. Lipopolysaccharide and lipotheicoic acid differentially modulate epididymal cytokine and chemokine profiles and sperm parameters in experimental acute epididymitis. Sci. Rep. 2018;8:103. doi: 10.1038/s41598-017-17944-4. PubMed DOI PMC

Lesniak W., Pecoraro V.L., Schacht J. Ternary complexes of gentamicin with iron and lipid catalyze formation of reactive oxygen species. Chem. Res. Toxicol. 2005;18:357–364. doi: 10.1021/tx0496946. PubMed DOI

Davis R.J. Signal transduction by the JNK group of MAP kinases. Cell. 2000;103:239–252. doi: 10.1016/S0092-8674(00)00116-1. PubMed DOI

Santiago-Moreno J., Esteso M.C., Villaverde-Morcillo S., Toledano-Déaz A., Castaño C., Velázquez R., López-Sebastián A., Goya A.L., Martínez J.G. Recent advances in bird sperm morphometric analysis and its role in male gamete characterization and reproduction technologies. Asian J. Androl. 2016;18:882–888. doi: 10.4103/1008-682X.188660. PubMed DOI PMC

Cerolini S., Zaniboni L., Maldjian A., Gliozzi T. Effect of docosahexaenoic acid and alpha-tocopherol enrichment in chicken sperm on semen quality, sperm lipid composition and susceptibility to peroxidation. Theriogenology. 2006;66:877–886. doi: 10.1016/j.theriogenology.2006.02.022. PubMed DOI

Tvrdá E., Benko F., Ďuračka M. Oxidative Stress as an Underlying Mechanism of Bacteria-Inflicted Damage to Male Gametes. Oxygen. 2022;2:547–569. doi: 10.3390/oxygen2040036. DOI

Barbonetti A., Vassallo M.R., Costanzo M., Battista N., Maccarrone M., Francavilla S., Francavilla F. Involvement of cannabinoid receptor-1 activation in mitochondrial depolarizing effect of lipopolysaccharide in human spermatozoa. Andrology. 2014;2:502–509. doi: 10.1111/j.2047-2927.2014.00210.x. PubMed DOI

Moskovtsev S.I., Mullen J.B.M., Lecker I., Jarvi K., White J., Roberts M., Lo K.G. Frequency and severity of sperm DNA damage in patients with confirmed cases of male infertility of different aetiologies. Reprod. Biomed. Online. 2010;20:759–763. doi: 10.1016/j.rbmo.2010.03.002. PubMed DOI

Anel-Lopez L., Riesco M.F., Montes-Garrido R., Neila-Montero M., Boixo J.C., Chamorro C., Ortega-Ferrusola C., Carvajal A., Altonaga J.R., de Paz P., et al. Comparing the Effect of Different Antibiotics in Frozen-Thawed Ram Sperm: Is It Possible to Avoid Their Addition? Front. Vet. Sci. 2021;2:656937. doi: 10.3389/fvets.2021.656937. PubMed DOI PMC

Tímermans A., Vázquez R., Otero F., Gosálvez J., Johnston S., Fernández J.L. Antibiotic toxicity on human spermatozoa assessed using the sperm DNA fragmentation dynamic assay. Andrologia. 2022;54:e14328. doi: 10.1111/and.14328. PubMed DOI

Al-Kass Z., Eriksson E., Bagge E., Wallgren M., Morrell J.M. Microbiota of semen from stallions in Sweden identified by MALDI-TOF. Vet. Anim. Sci. 2020;10:100143. doi: 10.1016/j.vas.2020.100143. PubMed DOI PMC

Burrows W.H., Quinn J.P. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 1937;16:19–24. doi: 10.3382/ps.0160019. DOI

Kačániová M., Terentjeva M., Štefániková J., Žiarovská J., Savitskaya T., Grinshpan D., Kowalczewski P.Ł., Vukovic N., Tvrdá E. Chemical Composition and Antimicrobial Activity of Selected Essential Oils against Staphylococcus spp. Isolated from Human Semen. Antibiotics. 2020;9:765. doi: 10.3390/antibiotics9110765. PubMed DOI PMC