Salivary cortisol concentrations changes in horses during daily routine

Article Details: Received: 2020-10-20 | Accepted: 2020-11-27 | Available online: 2021-01-31https://doi.org/10.15414/afz.2021.24.mi-prap.1-4AbstractThe aim of the study was to observe the salivary concentrations of cortisol in horses during daily routine over the course of four weeks lasting experiment in three different stages of day to monitor the changing values of cortisol in horse saliva during the day. Saliva was obtained from 12 Slovak warmblood horses – 1 stallion, 6 mares and 5 geldings. In the experiment we focused purely on changes of salivary cortisol concentrations over the course of the daily routine.The results shown us multiple significant changes between individual sample collections and thus, we can state that according to our results the highest concentration of cortisol in horse saliva is in the morning and it decreases throughout the day with lowest measured concentrations being in the final sample collection of the day (at 22:00). These changes had no visible effect on horses´ organism throughout entirety of the experiment and were caused due to horses daily rhythm.Keywords: horse, cortisol, saliva, rest, daily routineReferencesBohák, Zs, Szabó, F, Beckers, J-F, Melo de Sousa, N, Kutasi, O., Nagy, K, Szenci, O. 2013. Monitoring the circadian rhythm of serum and salivary cortisol concentrations in the horse. Domest. Anim. Endocrinol. 45:38-42. https://doi.org/10.1016/j.domaniend.2013.04.001Contreras-Aguilar, MD, Lamy, E, Escribano, D, Cerón, JJ, Tecles, F, Quiles, AJ, Hevia, ML. 2020. Changes in salivary analytes of horses due to circadian rhythm and season: A Pilot Study. Animals. 10(9):1486. https://doi.org/10.3390/ani10091486Halo, M, Hollý, A, Mlyneková, E, Polyaková, L, Horný, M, Kovalčík, E. 2009. Influence feeding and training on the metabolic profil sport horses. J Cent. Eur. Agric. 10(4):411-416.Halo, M, Strapák, P, Mlyneková, E, Kovalčík, E, Horný, M. 2008. Influence stres on the training process of the horses. J. Cent. Eur. Agric. 9(1):217-224.Ille, N, von Lewinski, M, Erber, M, Wulf, M, Aurich, J, Mostl, E, Aurich, C. 2013. Effects of the level of experience of horses and their riders on cortisol release, heart rate and heart rate variability during a jumping course. Anim. Welf. 22:457-465. 10.7120/09627286.22.4.457Irvine, CHG, Alexander, SL. 1994. Factors affecting the circadian rhythm in plasma cortisol concentrations in the horse. Domest. Anim. Endocrinol. 11(2):227-238. https://doi.org/10.1016/0739-7240(94)90030-2Kang, O-D, Lee W-S. 2016. Changes in salivary cortisol concentration in horses during different types of exercise. Asian-Australas J Anim Sci. 29(5):747-752. 10.5713/ajas.16.0009Kang, O-D, Yun Y-M. 2016. Influence of Horse and Rider on Stress during Horse-riding Lesson Program. Asian Australas J Anim Sci. 29(6):895-900. 10.5713/ajas.15.1068Leal, BB, Alves, GES, Douglas, RH, Bringel, B, Young, RJ, Haddad, JPA, Viana, WS, Faleiros, RR. 2011. Cortisol circadian rhythm ratio: A simple method to detect stressed horses at higher risk of colic? Equine Vet. J. 31:188-190. https://doi.org/10.1016/j.jevs.2011.02.005Massányi, P, Stawarz, R, Halo, M, Formicki, G, Lukac, N, Cupka, P, Schwarcz, P, Kovacik, A, Tusimova, E, Kovacik, J. 2014. Blood concentration of copper, cadmium, zinc and lead in the horses and its relation to hematological and biochemical parameters. J. Environ. Sci. Health A. 49:973-979. 10.1080/10934529.2014.894322 Peeters, M, Sulon J, Beckers J-F, Ledoux D, Vandenheede M. 2011. Comparison between blood serum and salivary cortisol concentrations in horses using an adrenocorticotropic hormone challenge. Equine Vet J. 43(4):487-493. 10.1111/j.2042-3306.2010.00294.x Schmidt, A, Mostl, E, Wehnert, Ch, Aurich, J, Muller, J, Aurich, Ch. 2010. Corisol release and heart rate variability in horses during road transport. Hormones Behaviour. 57: 209-215. 10.1016/j.yhbeh.2009.11.003Strzelec, K, Kankofer, M, Piertzak, S. 2011. Cortisol concentration in the saliva of horses subjected to different kinds of exercise. Act Vet. Brno. 80:101-105. 10.2754/avb201180010101Stull, CL, Morrow, J, Aldridge, BA, Stott, JL, McGlone, JJ. 2008. Immunophysiological responses of horses to 12-hour rest during 24 hours of road transport. Vet. Rec. 162:609-614. 10.1136/vr.162.19.609Van der Kolk, JH, Nachreiner, RF, Schott HC, Refsal KR, Zanella AJ. 2001. Salivary and plasma concentration of cortisol in normal horses and horses with Cushing´s disease. Equine Vet J. 33(2):211-213. 10.1111/j.2042-3306.2001.tb00604.

Nutritional indicators in the technological process of sausage processing

Received: 2020-09-29 Accepted: 2021-02-08 Available online: 2021-02-28https://doi.org/10.15414/afz.2021.24.mi-apa.15-20According to biological and nutritional value, meat and meat products are among the most important components of humannutrition. The risk of meat contamination is a great concern from the point of view of food safety, and especially human health. Theaim of this study was the determination of nutritional values in meat samples of fresh and smoked sausage. From a technologicalpoint of view, the water content was the highest in meat samples and continually decreased in the samples that underwentprocessing. The water content of the meat samples was 68.2%. In the samples of unsmoked and smoked sausages, the measuredvalues were slightly lower. In the samples of unsmoked sausages, the water content was 63.1%. As the water content decreased,the fat content of the sausages increased. The protein content has not changed significantly in the production process. In the meatthe value of proteins was 19.07 g 100 g-1 and in the samples of smoked sausages the result was 18.78 g 100 g-1. The content ofessential fatty acids was the highest in meat samples. This value decreased in unsmoked as well as smoked sausages. Cholesterollevels were rising over the course of the experiment. Results of this study clearly show difference in technological parametersrelated to technological process.Keywords: meat, sausage, technological process, nutritional indicatorsReferencesAngelovičová, M. et al. (2016). Comparison of fatty acid profile in the chicken meat after feeding with narasin, nicarbazin andsalinomycin sodium and phyto-additive substances. Journal of Environmental Science and Health, Part B, 51(6), 374–382. https://doi.org/10.1080/03601234.2016.1142320ČUBOŇ, J. et al. (2012). Hodnotenie surovín a potravín živočíšneho pôvodu. Nitra : Slovak University of Agriculture, 381 p.ČUBOŇ, J. et al. (2019) Protein degradation and fat oxidation changes in salted meat processing. Journal of Microbiology,Biotechnology and Food Sciences, 9(6), 376–379. https://doi.org/10.15414/jmbfs.2019.9.special.376-379COLE, L. J. et al. (2020). A critical analysis of the potential for EU Common Agricultural Policy measures to support wild pollinatorson farmland. Journal of Applied Ecology, 57(4), 681–694. https://doi.org/10.1111/1365-2664.13572 DEBRECENI, O. et al. (2016). Comparison the physicochemical quality indicators of Musculus longissimus Dorsi from MangalitsaBreed and their crossbreeds. Journal of Central European Agriculture, 17(4), 1253–1263. https://doi.org/10.5513/jcea01/17.4.1840DELGADO, C. L. (2003). Rising consumption of meat and milk in developing countries has created a new food revolution. TheJournal of Nutrition, 133(11), 3907S–3910S. https://doi.org/10.1093/jn/133.11.3907SGRIFFITHS, P.; DE HASSETH, J. A. (2007). Fourier transform infrared spectrometry. 2nd ed., Wiley-Blackwell.GÓMEZ, I. et al. (2020). The effects of processing and preservation technologies on meat quality: Sensory and nutritionalaspects. Foods, 9, 1416. https://doi.org/10.3390/foods9101416GRUSAK, M. A. et al. (1999). Improving the nutrient composition of plants to enhance human nutrition and health. AnnualReview of Plant Biology, 50(1), 133–161. https://doi.org/10.1146/annurev.arplant.50.1.133GUPTA, V. et al. (2017). Lifestyle, Stress, and Disorders. Basic and Applied Aspects of Biotechnology. Springer : Singapore, pp. 475–486. https://doi.org/10.1007/978-981-10-0875-7_22Haščík, P. et al. (2019a). Spracovanie hydiny a minoritných živočíšnych produktov. Nitra : Slovak University of Agriculture, 176 p.HAŠČÍK, P. et al. (2019b). The profile of fatty acids in chicken’s meat after humic acid and phytobiotics application. Journalof Microbiology, Biotechnology and Food Sciences, 9(6), 439–444. https://doi.org/10.15414/jmbfs.2019.9.special.439-444HIRD, S. J. et al. (2014). Liquid chromatography-mass spectrometry for the determination of chemical contaminants in food.TrAC Trends in Analytical Chemistry, 59, 59–72. https://doi.org/10.1016/j.trac.2014.04.005IMRICH, I. et al. (2020). Comparison of the physico-chemical meat quality of the breeds Mangalitsa and Large white with regardto the slaughter weight. Potravinarstvo Slovak Journal of Food Sciences, 14, 135–141. https://doi.org/10.5219/1334JIMÉNEZ-COLMENERO, F. et al. (2001). Healthier meat and meat products: Their role as functional foods. Meat Science, 59, 5–13.https://doi.org/10.1016/S0309-1740(01)00053-5KROČKO, M. et al. (2016). Effect of spices commercial mixture with GDL on the quality of fermented dry-cured sausages. SlovakJournal of Food Sciences, 10(1), 295–299. https://doi.org/10.5219/603LÍPOVÁ, P. et al. (2019). Efect of intramuscular fat content on physical-chemical parameters of pork from Mangalitsa and theircrossbreed. Potravinarstvo Slovak Journal of Food Sciences, 13(1), 422–428. https://doi.org/10.5219/1095ORZECHOWSKA, B. et al. (2008). Relationships between muscle fibre characteristics and physico-chemical properties oflongissimus lumborum muscle and growth rate in pig fatteners of three breeds. Animal Science Papers and Reports, 26(4), 277–285.PREZIOSI, P. et al. (1998). Effects of supplementation with a combination of antioxidant vitamins and trace elements, atnutritional doses, on biochemical indicators and markers of the antioxidant system in adult subjects. Journal of the AmericanCollege of Nutrition, 17(3), 244–249. https://doi.org/10.1080/07315724.1998.10718754SHARMA, M. et al. (2009). Occupational lifestyle diseases: An emerging issue. Indian Journal of Occupational and EnvironmentalMedicine, 13(3), 109. https://doi.org/10.4103/0019-5278.58912STEINHAUSEROVÁ, I. et al. (2015) Hygiene and sanitation in meat production. Meat, 4, 7–13.STOLL-KLEEMANN, S. et al. (2017). Reducing meat consumption in developed and transition countries to counter climatechange and biodiversity loss: a review of influence factors. Regional Environmental Change, 17(5), 1261–1277. https://doi.org/10.1007/s10113-016-1057-5ZAJÁC, P. et al. (2015). Analysis of texturometric properties of selected traditional and commercial sausage. PotravinarstvoSlovak Journal of Food Sciences, 9(1), 458–467. https://doi.org/10.5219/47

High taurine concentrations negatively effect stallion spermatozoa parameters in vitro

Article Details: Received: 2020-09-29 | Accepted: 2020-11-27 | Available online: 2021-01-31https://doi.org/10.15414/afz.2021.24.mi-prap.15-19Over the past decades natural substances are widely used in the maintaining of spermatozoa viability. The target of present study was to evaluate the effect of various taurine concentrations on stallion spermatozoa during 37°C cultivation. Fresh semen was collected from 10 breeding stallions. The experimental groups were supplemented with six different concentration of taurine (in mg/ml): A – 2.5, B – 5, C – 7.5, D – 10, E – 15, F – 20 and compared to control (CON – 0). Spermatozoa motility was assessed using the Computer Assisted Semen Analyzer (CASA) system in 6 time periods (0, 1, 2, 3, 4 and 5 hours). The MTT test was used for detection of viability. For measuring antioxidant activity FRAP and TOS methods were used. Significantly negative effect was observed in the samples with the highest concentration of taurine (20 mg/ml). Spermatozoa viability was not significantly affected in analysed concentrations of taurine. Significant higher antioxidant activity was detected in the sample with the highest taurine concentration. Data clearly showed negative effects of high taurine concentrations on stallion spermatozoa.Keywords: taurine, CASA, antioxidant activity, spermatozoa, stallion References  Benzie, I. F. and Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Analytical Biochemistry, 239(1), 70-76. https://doi.org/10.1006/abio.1996.0292Bucak, M. N. et al. (2007). The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen: Microscopic and oxidative stress parameters after freeze–thawing process. Theriogenology, 67(5), 1060-1067. https://doi.org/10.1016/j.theriogenology.2006.12.004Erel, O. (2005). A new automated colorimetric method for measuring total oxidant status. Clinical Biochemistry, 38(12), 1103-1111. https://doi.org/10.1016/j.clinbiochem.2005.08.008Halo, M. and Tirpák, F. (2018) Stallion fertility - the basis of successful reproduction. Svet koní. 18.Halo Jr., M. et al. (2019) Time and dose-dependent effects of Viscum album quercus on rabbit spermatozoa motility and viability in vitro. Physiological Research, 68(6), 955-972. https://doi.org/10.33549/physiolres.934223 Ijaz, A. and Ducharme, R. (1995). Effect of various extenders and taurine on survival of stallion sperm cooled to 5 C. Theriogenology, 44(7), 1039-1050. https://doi.org/10.1016/0093-691x(95)00290-o Jambor, T. et al. (2017) In vitro effect of 4-nonylphenol on human chorionic gonadotropin (hCG) stimulated hormone secretion, cell viability and reactive oxygen species generation in mice Leydig cells. Environmental Pollution, 222, 219–225. https://doi.org/10.1016/j.envpol.2016.12.053 O'flaherty, L. et al. (1997) Intestinal taurine transport: a review. European Journal of Clinical Investigation, 27(11), 873-880. https://doi.org/10.1046/j.1365-2362.1997.2000747.xReddy, N. S. S. et al. (2010). Effects of adding taurine and trehalose to a tris-based egg yolk extender on buffalo (Bubalus bubalis) sperm quality following cryopreservation. Animal Reproduction Science, 119(3-4), 183-190. https://doi.org/10.1016/j.anireprosci.2010.01.012 Slanina, T. et al. (2018) Effect of taurine on turkey (Meleagris gallopavo) spermatozoa viability and motility. Czech Journal of Animal Science, 63(4),127-135. https://doi.org/10.17221/79/2017-CJASStephens, T. D. et al. (2013) Effects of pentoxifylline, caffeine, and taurine on post-thaw motility and longevity of equine frozen semen. Journal of Equine Veterinary Science, 33(8), 615-621. https://doi.org/10.1016/j.jevs.2012.10.004Tirpák, F. et al. (2017) Low taurine concentrations possitively affect rabbit spermatozoa properties in later time intervals. Journal of Microbiology, Biotechnology and Food Sciences, 7, 128-131. https://doi.org/10.15414/jmbfs.2017.7.2.128-13

Effect of nickel and zinc peroral administration on meat quality of rabbits

The aim of our study was to determine the effect of single nickel administration as well as co–administration with zinc on meat quality parameters. In the experiment 45 [25 female (5 per group) and 20 male (4 per group)] rabbits of broiler line Californian were involved. Animals were divided to five groups: K (n=9) – control; P1 (n=9) – received 17.5 g NiCl2.100 kg-1 feeding dose (FD); P2 (n=9) – received 35 g NiCl2.100 kg-1 FD; P3 (n = 9) – received 17.5 g NiCl2.100 kg-1FD and 30 g ZnCl2.100 kg-1 FD and finally groups P4 (n = 9) – received 35 g NiCl2.100 kg-1 FD and 30 g ZnCl2.100 kg-1 FD. Animals were fed ad libitum using KKV1 feeding mixture with or without nickel and zinc addition for 90 days. Meat quality was analyzed from a sample of musculus biceps femoris for the content of water in muscle, content of proteins, fat, energy, electric conductivity, pH, colour and content of bounded water. The value of pH was detected by injection analysis. The content of water, proteins and fat was analyzed using Infratech 1265 Meat Analyzer. Meat colour was detected by spectrophotometer (Specol 11) and the meat ability to bind water by pressure method. In meat quality any significant differences were found among all groups

Impact of Seminal Chemical Elements on the Oxidative Balance in Bovine Seminal Plasma and Spermatozoa

Mutual relationships between selected chemical elements (Na, K, Fe, Cu, Mg, and Zn), basic motility characteristics (motility and progressive motility), and markers of the oxidative balance (superoxide dismutase, catalase, glutathione, albumin, and malondialdehyde) were investigated in bovine seminal plasma and spermatozoa. Computer assisted sperm analysis was used to assess the motility parameters; mineral concentrations were determined by the voltammetric method and flame absorption spectrophotometry; antioxidants and malondialdehyde were evaluated by UV/VIS spectrophotometry. Concentrations of chemical elements in both seminal fractions were in the following descending order: Na > K > Zn > Mg > Fe > Cu. Higher amounts of all minerals and nonenzymatic antioxidants were detected in the seminal plasma ( < 0.01; < 0.001), while higher MDA concentration and activity of enzymatic antioxidants were recorded in the cell lysates ( < 0.01; < 0.001). Na, Fe, Cu, Mg, and Zn were positively correlated with the motility and antioxidant parameters ( < 0.05; < 0.01; < 0.001). Inversely, K exhibited the positive associations with malondialdehyde ( < 0.05). This study demonstrates that most chemical elements are integral components of bovine semen and are needed for the protection against oxidative stress development

Effects of dietary supplementation of nickel and nickel-zinc on femoral bone structure in rabbits

<p>Abstract</p> <p>Background</p> <p>Nickel (Ni) and zinc (Zn) are trace elements present at low concentrations in agroecosystems. Nickel, however, may have toxic effects on living organisms and is often considered as a contaminant. This study reports the effect of peroral administrated Ni or a combination of Ni and Zn on femoral bone structure in rabbits.</p> <p>Methods</p> <p>One month-old female rabbits were divided into three groups of five animals each. Group 1 rabbits were fed a granular feed mixture with addition of 35 g NiCl₂per 100 kg of mixture for 90 days. In group 2, animals were fed a mixture containing 35 g NiCl₂and 30 g ZnCl₂per 100 kg of mixture. Group 3 without administration of additional Ni or Zn served as control. After the 90-day experimental period, femoral length, femoral weight and histological structure of the femur were analyzed and compared.</p> <p>Results</p> <p>The results did not indicate a statistically significant difference in either femoral length or weight between the two experimental groups and the control group. Also, differences in qualitative histological characteristics of the femora among rabbits from the three groups were absent, except for a fewer number of secondary osteons found in the animals of groups 1 and 2. However, values for vascular canal parameters of primary osteons were significantly lower in group 1 than in the control one. Peroral administration of a combination of Ni and Zn (group 2) led to a significant decreased size of the secondary osteons.</p> <p>Conclusions</p> <p>The study indicates that dietary supplementation of Ni (35 g NiCl₂per 100 kg of feed mixture) and Ni-Zn combination (35 g NiCl₂and 30 g ZnCl₂per 100 kg of the mixture) affects the microstructure of compact bone tissue in young rabbits.</p

Effects of Cadmium, Lead, and Mercury on the Structure and Function of Reproductive Organs

Reproductive organs are essential not only for the life of an individual but also for the survival and development of the species. The response of reproductive organs to toxic substances differs from that of other target organs, and they may serve as an ideal &ldquo;barometer&rdquo; for the deleterious effects of environmental pollution on animal and human health. The incidence of infertility, cancers, and associated maladies has increased in the last fifty years or more, while various anthropogenic activities have released into the environment numerous toxic substances, including cadmium, lead, and mercury. Data from epidemiological studies suggested that environmental exposure to cadmium, lead, and mercury may have produced reproductive and developmental toxicity. The present review focused on experimental studies using rats, mice, avian, and rabbits to demonstrate unambiguously effects of cadmium, lead, or mercury on the structure and function of reproductive organs. In addition, relevant human studies are discussed. The experimental studies reviewed have indicated that the testis and ovary are particularly sensitive to cadmium, lead, and mercury because these organs are distinguished by an intense cellular activity, where vital processes of spermatogenesis, oogenesis, and folliculogenesis occur. In ovaries, manifestation of toxicity induced by cadmium, lead, or mercury included decreased follicular growth, occurrence of follicular atresia, degeneration of the corpus luteum, and alterations in cycle. In testes, toxic effects following exposure to cadmium, lead, or mercury included alterations of seminiferous tubules, testicular stroma, and decrease of spermatozoa count, motility and viability, and aberrant spermatozoa morphology

Evidence for Ovarian and Testicular Toxicities of Cadmium and Detoxification by Natural Substances

Cadmium (Cd) is an environmental toxicant, capable of reducing mitochondrial ATP production and promoting the formation of reactive oxygen species (ROS) with resultant oxidative stress conditions. The ovary and testis are the primary gonads in which female gametes (oocytes) and male gametes (spermatozoa), estrogen and testosterone are produced. These organs are particularly susceptible to Cd cytotoxicity due to their high metabolic activities and high energy demands. In this review, epidemiological and experimental studies examining Cd toxicities in gonads are highlighted together with studies using zinc (Zn), selenium (Se), and natural substances to reduce the effects of Cd on follicular genesis and spermatogenesis. Higher blood concentrations of Cd ([Cd]b) were associated with longer time-to-pregnancy in a prospective cohort study. Cd excretion rate (ECd) as low as 0.8 &mu;g/g creatinine was associated with reduced spermatozoa vitality, while Zn and Se may protect against spermatozoa quality decline accompanying Cd exposure. ECd &gt; 0.68 &micro;g/g creatinine were associated with an increased risk of premature ovarian failure by 2.5-fold, while [Cd]b &ge; 0.34 &micro;g/L were associated with a 2.5-fold increase in the risk of infertility in women. Of concern, urinary excretion of Cd at 0.68 and 0.8 &mu;g/g creatinine found to be associated with fecundity are respectively 13% and 15% of the conventional threshold limit for Cd-induced kidney tubular effects of 5.24 &mu;g/g creatinine. These findings suggest that toxicity of Cd in primary reproductive organs occurs at relatively low body burden, thereby arguing for minimization of exposure and environmental pollution by Cd and its transfer to the food web

The effect of training load stress on salivary cortisol concentrations, health parameters and hematological parameters in horses

The performance of sport horses is conditioned not only by the quality of its gene pool, but also by a large number of external factors. The most dominant being nutrition, quality of breeding, level of zootechnical care and the quality of the sports rider and coach. Important factor is the process of individuals’ adaptation to the training load occurring during the training itself. This study was focused on the analysis of salivary cortisol levels as well as hematological and biochemical blood parameters in relation to load to which the tested horses were subjected. In the study 14 horses of sport breeds were analyzed a all tested horses were in the same (medium) level of training load. Tested horses underwent following stages of workload – transportation, jumping training, parkour competition, treadmill training, riding training, shoeing and lunging of various intensity. Saliva samples were obtained using a tampon on a string which was inserted into horse's oral cavity, chewed by the horse and placed in a sterile tube with a closable lid. Afterwards, the samples were then stored in deep-freezing boxes at temperature of −80 °C. The EIA cortisol kit was used in this study. The absorbance was read at the wavelength of 450 nm against a reference wavelength of 620–630 nm or a blank sample. Blood samples were obtained at the beginning of the experiment, after half a year of running the experiment and at the end of the experiment from v. jugularis. Hematological analysis were carried out using automatic hematologic analyser and multiple parameters were observed. Analysis of biochemical parameters in blood serum were realized using commercial DiaSys kits and semiautomatic biochemical spectrophotometer. Sodium, potassium and chlorides were measured using automatic analyzer EasyLytePlus. In all monitored forms of exercise (transportation, jumping training, parkour competition, treadmill training, riding training, horse shoeing, lunging), an increase in cortisol concentrations immediately after the exercise was recorded, but only spotted statistically significant differences were found during the transportation of monitored horses. The levels of blood parameters were within the reference range during the experiment period. From a comprehensive evaluation of the results, it can be stated that there were no visible health changes to the horses that underwent the experimental load and that manipulation with horses is an important factor that has effect on horses’ stress response. In general, the results of this study show no visible impact of training and/or load on the health status of horses over entire 12-month duration of the experiment