90 Annales Universitatis Paedagogicae Cracoviensis Studia Naturae, 3 (supplement): 90–97, 2018, ISSN 2543-8832 DOI: 10.24917/25438832.3supp.12 Francesco Vizzarri1*, Marisa Palazzo M1, Donato Casamassima1, Carlo Corino2, Sara Chiapparini2, Lubo- mir Ondruska3, Nikola Knizatova4, Martin Massanyi4, Filip Tirpak4, Peter Massanyi4 1Department of Agricultural and Environmental Science, University of Bari Aldo Moro, Bari, Italy, *francesco.vizzarri@uniba.it 2Department of Health, Animal Science and Food Safety, University of Milano, Milano, Italy 3National Agricultural and Food Centre, Nitra, Slovak Republic 4Department of Animal Physiology, Slovak University of Agriculture, Nitra, Slovak Republic Dietary supplementation with algae and polyphenols in male rabbits: effects on semen quality traits Introduction In animal production, poor reproductive performance currently a�ects livestock pro- ductivity. Several e�orts toward overcoming this challenge (poor reproductive per- formance) have culminated in identifying oxidative stress as the main reason because animals’ productivity is impaired either directly or indirectly by di�erent unfavour- able conditions (Rahal et al., 2014). Oxidative stress has been widely reported as the mechanism behind many pathological development and disease conditions, including reproductive ine�ciency (Sikiru et al., 2018). In rabbit production, this aspect is a very important parameter for reproductive performance evaluation, because it determines pro�tability as well as products yield, and it is one of the major factor determining en- terprise productivity and production objectives. In male organisms, pro-oxidant con- dition a�ects both seminal qualities and reproductive functions of the spermatozoa. High levels of reactive oxygen substances are also reported to induce oxidative damage of DNA in the sperm plasma membrane, mitochondrion, and nuclear genome. �e danger in this damage is not associated with poor reproductive performance alone, but also with cancer development and inherited infertility in the o�-springs (Aitken, Krausz, 2001). In recent years, many studies have been focused on natural substances that can a�ect the health of animals and challenge the new animal welfare prospective. �e present research is based on the claim that exogenous supplementation of antioxidants is a proven tool of reducing oxidative stress associated with reproduc- 91 D ietary supplem entation w ith algae and polyphenols in m ale rabbits: effects on sem en quality traits tive performance and the need to discover di�erent sources of antioxidants capable of improving reproductive activities. A mix of di�erent extracts was used as a dietary supplement, and it consists exclusively of natural products. Its main components are polyphenols from terrestrial and marine origins and plant polysaccharides. �e e�ect of this supplement on reproduction has not been reviewed in the past, and that is a reason why we decided to test its e�ect on the reproductive potential of male rabbits. �e aim of the present study is to determine e�ects of natural mix extracts during a 120-day in vivo experiment on selected reproductive traits of male rabbits. Material and methods Animals and experimental design �e trial lasted 120 days and was conducted in the Animal Production Research Cen- tre in Nitra, Slovak Republic, on 14 adult New Zealand White rabbit bucks. All ex- perimental procedures and the management of animals were conducted in accord- ance with European Community guidelines n. 86/609/EEC regarding the protection of animals for experimental purposes. �e tested animals (aged 15 ± 3 months) were divided into three homogeneous groups, and the body weights were recorded at the beginning and the end of experiment: the control (CON; n = 5) was fed with commer- cial feed, the �rst experimental group (T1; n = 4) received a 0.3% feed additive mix, and the second experimental group (T2; n = 5) received a 0.6% feed additive mix. �e mix extract supplement, containing mainly polyphenols from algae and chestnut tan- nin extracts, was analysed using HPLC-DAD according to Russo et al. (2017) and the following natural compounds were determined as the most prominent: neochlorogen- ic acid, elaigic acid, syringic acid, cynaroside, and rutin. �e natural extract was pro- duced and provided by Lombarda Trading SRL (Casale Belvedere, Cremona, Italy). �e ingredients and chemical composition of diets are reported in table 1. �e semen samples were collected on day 0 (basal), and days 30, 60, 90, and 120 of the feeding period with the help of an arti�cial vagina. �e obtained semen samples were diluted with physiological solution in the ratio 1:5. A�er processing, the samples were incubated at the temperature of 37°C and were analysed immediately in tripli- cate. Each of prepared samples was evaluated using a Computer Assisted Semen Ana- lyzer (CASA) system – Sperm Vision (Minitub, Tiefenbach, Germany) equipped with a microscope (Olympus BX 51, Japan) to assess the spermatozoa motility (Massanyi et al., 2008). Each sample was placed into Makler Counting Chamber (depth 10 μm, Se�–Medical Instruments, Germany). Using the rabbit speci�c set up, the following parameters were evaluated: spermatozoa concentration (CONC, 106/mL), total motile spermatozoa (%, motility > 5 μm/s), and progressive motile spermatozoa (%, motility > 20 μm/s). Fr an ce sc o V iz za rr i, M ar is a P al az zo M , D on at o C as am as si m a, C ar lo C or in o, S ar a C hi ap pa rin i, Lu bo m ir O nd ru sk a, N ik ol a K ni za to va , M ar tin M as sa ny i, Fi lip T irp ak , P et er M as sa ny i 92 Tab. 1. Ingredients and chemical composition of the diets [g/kg] Ingredients Experimental diet CON T1 T2 Maize 282 281 280 Alfalfa hay 305 305 305 Sun�ower meal 135 135 135 Palm seed oil 8 8 8 Soybean oil 7 7 7 Wheat 80 80 80 Cane molasses 20 20 20 Carob bean meal 90 90 90 Oat 53 53 53 Calcium carbonate 7 7 7 Sodium Chloride 3 3 3 Dicalcium phosphate 2 2 2 Methionine (99%) 2.5 2.5 2.5 Lysine (78.5%) 1.6 1.6 1.6 Choline (75%) 1.4 1.4 1.4 Vitamin and mineral premix* 2.5 2.5 2.5 Experimental supplement** 0.00 3 6 Chemical composition*** Crude protein 184.0 183.6 183.5 Ether extract 35.7 35.5 35.5 Crude �bre 187.0 186.8 187.0 Ash 86.0 85.7 85.8 Nitrogen free extract 507.0 507.1 506.9 NDF 302.1 301.5 301.7 ADF 195.8 195.4 195.3 ADL 39.9 39.5 39.5 Notes: *Supplied per kg diet: 13.500 I.U. vitamin A (trans-retinyl acetate); 800 I.U. vitamin D3 (chole- calciferol); 35 mg vitamin E (α-tocopherol min 91%), 35 mg copper (cupric sulphate pentahydrate), 150 mg aminoside sulphate; ** quantities of plant extract, T1 – experimental group fed 0.3% of natural mix supplement and T2 – experimental group fed 0.6% of natural mix supplement; *** analyses determined in triplicate �e superoxide dismutase levels (SOD), which catalyses the dismutation of su- peroxide radical reaction in hydrogen peroxide and molecular oxygen, together with glutathione peroxidase (GPx) determination, were determined using a commercial colorimetric kit-assay provided by Randox (Randox Laboratories Ltd., United King- dom). SOD activity was expressed in units per milligram of protein [U/mg], and GPx activity was expressed in units per gram of protein [U/g]. �e ferric reducing an- tioxidant power (FRAP) test, developed by Benzie and Strain (1996), measures the antioxidant capacity of plasma and is used to assess the ability to reduce the ferric iron complex in an acidic environment. One unit FRAP is expressed in mmol/ml and D ietary supplem entation w ith algae and polyphenols in m ale rabbits: effects on sem en quality traits 93 indicates the number of moles of ferric ion (Fe3+) reduced to ferrous ion (Fe2+) from one mol of tested antioxidants. Statistical analysis Obtained data was statistically analysed with the help of the PC program Excel and a  commercially available statistics package SAS 8.0 (SAS Institute Inc., USA) using Student’s t-test and Sche�e’s test. Statistical signi�cance was indicated by p values of less than 0.05, 0.01, and 0.001. Tab. 2. Semen characteristics (x ± SD) of control (CON, n = 5) and experimental (T1, n = 4; T2, n = 5) buck rabbits Items Dietary treatment1 CON T1 T2 P-value2 Concentration [10 6 × ml-1] Basal 0.656±0.270 0.695±0.428 0.728±0.532 n.s. 30d 0.638±0.280 0.635±0.602 0.607±0.490 n.s. 60d 0.647±0.1754 0.699±0.345 0.871±0.387 n.s. 90d 0.640±0.245 0.538±0.307 0.586±0.461 n.s. 120d 0.663±0.251 0.685±0.123 0.609±0.213 n.s. Motility [%] Basal 79.640±2.240 81.630±2.760 82.959±4.040 n.s. 30d 84.760±2.694 85.840±9.272 77.700±12.740 n.s. 60d 85.090±7.526 87.210±8.245 89.380±8.023 n.s. 90d 82.840±4.511 87.630±4.567 77.330±12.710 n.s. 120d 92.100±4.209 88.670±5.257 85.680±7.959 n.s. Progressive motility [%] Basal 66.440±3.130 66.530±13.690 62.490±12.940 n.s. 30d 74.510±4.948 69.940±18.980 62.420±14.260 n.s. 60d 74.280±12.600 79.070±13.890 81.280±11.370 n.s. 90d 70.890±4.401 77.160±4.913 64.390±18.440 n.s. 120d 84.610±6.610 82.040±6.592 76.020±14.470 n.s. Note: 1(CON) – Control group fed with commercial feed; T1 – experimental group fed 0.3% of natural mix supplement; T2 – experimental group fed 0.6% of natural mix supplement; 2p – value: n.s. = not signi�cant Results and discussion �e dietary supplementation with the natural extracts mix did not cause any changes in the animal body weights, and it did not induce any evident clinical signs in rabbits over the 120-days of the experimental period. �e concentration of spermatozoa was not signi�cantly di�erent between experimental groups and the control group a�er 4 months of dietary treatment (Tab. 1). Mourvaki et al. (2010) also found no e�ect with use of �axseed dietary supplementation on the volume and spermatozoa concen- Fr an ce sc o V iz za rr i, M ar is a P al az zo M , D on at o C as am as si m a, C ar lo C or in o, S ar a C hi ap pa rin i, Lu bo m ir O nd ru sk a, N ik ol a K ni za to va , M ar tin M as sa ny i, Fi lip T irp ak , P et er M as sa ny i 94 tration in rabbit. However, Okab et al. (2013), feeding rabbit bucks with dried seaweed (2%), showed a signi�cant decrease in spermatozoa concentration, the percentage of live spermatozoa, and ejaculate volume. �e spermatozoa motility parameters (mo- tility and progressive motility; Tab. 1) were not signi�cantly di�erent between the control group and experimental group with natural extracts mix supplementation. Controversially, Yousef et al. (2003) observed an improvement of spermatozoa motil- ity parameters a�er the dietary supplementation of ascorbic acid and vitamin E, alone and in combination, in male rabbits. Since a lack of e�ects has been observed in our experimental study, further research is needed in order to test di�erent doses of natu- ral extracts mix supplements. Tab. 3. Antioxidant seminal plasma markers (x ± SD) of control (CON, n = 5) and experimental (T1, n = 4; T2, n = 5) buck rabbits Items Dietary treatment1 CON T1 T2 p–value2 SOD [U × mg-1 TP] Basal 0.408±0.085 0.502±0.117 0.477±0.091 n.s. 30d 0.422±0.097 0.664±0.575 0.353±0.059 n.s. 60d 0.296±0.194 0.443±0.268 0.346±0.150 n.s. 90d 0.381±0.123 0.296±0.102 0.421±0.088 n.s. 120d 0.264±0.114 0.429±0.240 0.311±0.094 n.s. GPx [U × g-1 TP] Basal 42.517±10.225 40.120±0.311 39.370±2.221 n.s. 30d 44.630±15.370 36.330±0.476 30.370±9.601 n.s. 60d 23.280±12.330 41.760±28.010 35.790±16.640 n.s. 90d 32.960±12.300 32.750±14.000 44.420±15.620 n.s. 120d 26.600±11.2501 65.580±19.3102 30.530±4.5251 ** FRAP [µmol Fe2+ × g-1 TP] Basal 85.125±29.119 93.225±17.455 89.541±7.853 n.s. 30d 97.420±48.960 112.800±18.400 60.600±12.100 n.s. 60d 75.590±54.340 106.300±64.980 69.750±26.350 n.s. 90d 59.240±15.330 73.330±15.540 68.020±28.520 n.s. 120d 69.440±18.5101 103.000±36.1002 67.110±7.5271 * Note: 1(CON) – Control group fed with commercial feed; T1 – experimental group fed 0.3% of natural mix supplement; T2 – experimental group fed 0.6% of natural mix supplement; 2 p–value: n.s. = not sig- ni�cant; * (p < 0.05); ** (p < 0.01) Tables 2–3 contain data regarding the tested oxidative markers in seminal plasma. At the end of the dietary treatment (a�er 120 days), all three parameters (SOD, GPx and FRAP) were positively altered, although the statistical signi�cance was reached only for GPx (p < 0.01) and FRAP (p < 0.05) values. In fact, group T1 data showed the high- est content of the two parameters when compared with group CON. In literature, it has also been reported that SOD activity survey in seminal plasma could be a useful tool D ietary supplem entation w ith algae and polyphenols in m ale rabbits: effects on sem en quality traits 95 for determining sperm fertilization potential and could improve the diagnosis of male infertility (Shiva et al., 2011). In general, antioxidant dietary supplementation leads to an improvement of the antioxidant markers pro�le in the seminal plasma, in particu- lar, when algae-based feed additive is supplemented in the diet (Murphy et al., 2017). Conclusion Since oxidative stress is considered a biochemical process negatively a�ecting repro- duction, the reduction of its e�ects is highly important for the promotion of animal welfare. �e use of polyphenols and tannins in rabbit diets is a source of natural an- tioxidants, and we can conclude that supplementation of 0.3% of natural mix did not signi�cantly negatively a�ect any of the studied reproductive parameters of male rab- bits, but we have found some improvement in several antioxidant parameters. Acknowledgment �is work was funded by the Slovak Research and Development Agency Grants no. VEGA 1/0539/18, VEGA 1/0760/15, APVV-16-0289 and APVV-15-0544. References Aitken, R.J., Krausz, C. (2001). Oxidative stress, DNA damage and the Y chromosome. Reproduction, 122, 497–506. Benzie, F.F., Strain, J.J. (1996). �e ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Analytical Biochemestry, 239, 70–76. DOI: 10.1006/abio.1996.0292 Massanyi, P., Chrenek, P., Lukac, N., Makarevich, A.V., Ostro, A., Zivcak J., Bulla, J. (2008). Comparison of di�erent evaluation chambers for analysis of rabbit spermatozoa motility parameters using CASA system. Slovak Journal of Animal Science, 41(2), 60–66. Mourvaki, E., Cardinali, R., Dal Bosco, A., Corazzi, L., Castellini, C. (2010). E�ects of �axseed dietary supplementation on sperm qualità and on lipid composition of sperm subfractions and prostatic granules in rabbit. Science Direct, 73, 629–637. DOI: 10.1016/j.theriogenology.2009.10.019 Murphy, E.M., Stanton, C., Brien, C.O., Murphy, C., Holden, S., Murphy, R.P., Varley, P., Boland, M.P., Fair, S. (2017). �e e�ect of dietary supplementation of algae rich in docosahexaenoic acid on boar fertility. �eriogenology, 90, 78–87. DOI: 10.1016/j.theriogenology.2016.11.008 Okab, A.B, Samara, E.M., Abdoun, K.A., Rafay, J., Ondruska, L., Parkanyi, V., Pivko, J., Ayoub, M.A., Al- Haidary, A.A., Aljumaah, R.S., Massanyi, P., Lukac, N. (2013). E�ects of dietary seaweed (Ulva lac- tuca) supplementation on the reproductive performance of buck and doe rabbits. Journal of Applied Animal Research, 41(3), 347–355. DOI: 10.1080/09712119.2013.783479 Rahal, A., Kumar, A., Singh, V., Yadav, B., Tiwari, R., Chakraborty, S., Dhama, K. (2014). Oxidative stress, prooxidants, and antioxidants: �e interplay. BioMed Research International, ID 761264. DOI: 10.1155/2014/761264 Russo, R., Pucci, L., Giorgetti, L., Árvay, J., Vizzarri, F., Longo, V., Pozzo, L. (2017). Polyphenolic char- acterisation of plant mixture (Lisosan® Reduction) and its hypocholesterolaemic e�ect in high fat diet-fed mice. Natural Product Research DOI: 10.1080/14786419.2017.1402328 SAS user’s guide: Statistics. Version 8 Edition. Cary (2003). NC: SAS Institute. Shiva, M., Gautam, A.K., Verma, Y., Shivgotra, V., Doshi H., Kumar, S. (2011). Association between Fr an ce sc o V iz za rr i, M ar is a P al az zo M , D on at o C as am as si m a, C ar lo C or in o, S ar a C hi ap pa rin i, Lu bo m ir O nd ru sk a, N ik ol a K ni za to va , M ar tin M as sa ny i, Fi lip T irp ak , P et er M as sa ny i 96 sperm quality, oxidative stress, and seminal antioxidant activity. Clinical Biochemistry, 44, 319–324. DOI: 10.1016/j.clinbiochem.2010.11.009 Sikiru, A.B., Alemede, I.C., Egena, S.S.A., Ijaiya, A.T. (2018). Oxidative stress and reproductive ine�cien- cies: �e science, evidences, and solutions. Agricultural Extension Journal, 2(1), 17–26. Yousef, M.I., Abdallah, G.A., Kamel, K.I. (2003). E�ect of ascorbic acid and Vitamin E supplementa- tion on semen quality and biochemical parameters of male rabbits. Animal Reproduction Science, 76, 99–111. DOI: 10.1016/S0378-4320(02)00226-9 Abstract In recent years, many studies have been focused on natural substances that can a�ect the health of animals. A mix of di�erent extracts was used as a dietary supplement, and it consisted exclusively of natural products. Its main components were polyphenols from terrestrial and marine origins and plant polysaccharides. �e e�ect of this supplement on reproduction has not been reviewed in the past, which is why its e�ect on the reproduction potential of male rabbits was tested. �e aim of the present study is to determine the e�ects of the natural mix during a 120-day in vivo experiment on selected reproductive traits of male rabbits. Natural mix was supplemented in two di�erent concentrations (T1 – 0.3% and T2 – 0.6%) with the basal ingredi- ents of the conventional rabbit feed in pellet form. In our experiments, emphasis was placed on both the spermatozoa concentration and its motility parameters as well as on the properties of seminal plasma and antioxidant activity. �e dietary supplementation with the natural extracts mix positively altered the quality traits of rabbit spermatozoa, but these changes were statistically not signi�cant. In experimental group T1, a signi�cant increase of GPx and FRAP content, both regarding the antioxidant markers pro�le in seminal plasma, was recorded. We can conclude that the supplementation of 0.3% of natural mix did not signi�cant- ly negatively a�ect any of the studied reproductive parameters of male rabbits, but some improvement in several antioxidant parameters was found. Key words: extract, rabbit, spermatozoa, mobility, seminal plasma, antioxidants Received: [2018.06.01] Accepted: [2018.11.22] Suplementacja diety algami i polifenolami u samca królika: wpływ na cechy jakościowe nasienia Streszczenie W ostatnich latach wiele badań dotyczy substancji naturalnych, które mogą wpływać na zdrowie zwierząt. Jako suplement diety wykorzystano mieszankę różnych ekstraktów, składającą się wyłącznie z naturalnych produktów. Jej głównymi składnikami były polifenole pochodzenia lądowego i morskiego oraz polisachary- dy roślinne. Oddziaływanie tych suplementów na reprodukcję nie zostało w przeszłości poddane analizie, co było powodem dla którego zbadano ich wpływ na potencjał reprodukcyjny samców królików. Celem ni- niejszego badania było określenie wpływu naturalnej mieszanki podawanej podczas 120-dniowego ekspe- rymentu in vivo na wybrane cechy reprodukcyjne samców królika. Naturalną mieszankę w dwóch różnych stężeniach (T1 – 0,3% i T2 – 0,6%) uzupełniono podstawowymi składnikami tradycyjnego pokarmu dla królików w postaci śrutu. W doświadczeniach nacisk położono, zarówno na koncentrację plemników, jak i na ich parametry ruchowe, a także na właściwości plazmy nasiennej oraz aktywność przeciwutleniającą. Suplementacja diety mieszaniną naturalnych ekstraktów pozytywnie zmieniła cechy jakościowe plemników królika, ale zmiany te nie były statystycznie istotne. W grupie doświadczalnej T1 odnotowano istotny wzrost zawartości, zarówno GPx, jak i FRAP, pod względem pro�lu markerów antyoksydacyjnych w plazmie nasie- nia. Można zatem stwierdzić, że suplementacja 0,3% naturalnej mieszanki nie wpłynęła znacząco negatyw- nie na żaden z badanych parametrów reprodukcyjnych samców królików, a nawet odnotowano tu pewną poprawę kilku parametrów antyoksydacyjnych. Słowa kluczowe: ekstrakt, królik, plemniki, ruchliwość, plazma nasienna, przeciwutleniacze D ietary supplem entation w ith algae and polyphenols in m ale rabbits: effects on sem en quality traits 97 Information on the authors Francesco Vizzarri http://orcid.org/0000-0003-1316-7937 Fellow researcher at the University of Bari Aldo Moro, researching animal welfare and the quality of animial production. Marisa Palazzo http://orcid.org/0000-0002-9116-9420 Technician at the University of Molise, researching animal welfare and the quality of animal production. Donato Casamassima http://orcid.org/0000-0002-3079-4249 Full Professor at the University of Molise, researching animal welfare and animal production. Carlo Corino http://orcid.org/0000-0001-8146-0583 Full Professor at the University of Milano, researching animal nutrition. Sara Chiapparini http://orcid.org/0000-0002-6484-3352 PhD Student at the University of Milano, researching animal nutrition. Lubomir Ondruska http://orcid.org/0000-0002-8460-3137 Director of Institute of Small Farm Animals, researching breeding, genetics, nutrition, controlled repro- duction, technology, biotechnical methods, and their innovations in rabbit husbandry. Nikola Knizatova PhD Student at the University of Agriculture in Nitra, researching animal physiology. Martin Massanyi PhD Student at the University of Agriculture in Nitra, researching animal physiology. Filip Tirpak http://orcid.org/0000-0001-9293-9055 PhD Student at the University of Agriculture in Nitra, researching animal physiology. Peter Massanyi http://orcid.org/0000-0002-4216-0948 Full Professor at the Slovak University of Agriculture in Nitra, researching animal physiology.