http://dx.doi.org/10.1590/1678-4162-10415 Arq. Bras. Med. Vet. Zootec., v.71, n.2, p.489-499, 2019 Addition of chlorogenic acid and caffeine during the processing of cooled boar semen B.A. Pereira1 https://orcid.org/0000-0002-3614-7886 L.G.P. Rocha1 [Adição de ácido clorogênico e cafeína durante o processamento do sêmen suíno resfriado] https://orcid.org/0000-0002-9082-7372 M.C. Teles1 https://orcid.org/0000-0003-0383-496X B.A. Pereira1, L.G.P. Rocha1, M.C. Teles1, W.E. Silva1, J.A. Barbosa1, S.S. Rabelo1, W.E. Silva1 https://orcid.org/0000-0002-5441-2620 A.S. Uchoa1, J. E. Rodrıguez-Gil2, L.J. Pereira1, M.G. Zangeronimo1* J.A. Barbosa1 https://orcid.org/0000-0001-8983-3890 S.S. Rabelo1 https://orcid.org/0000-0002-0000-0284 1 Universidade Federal de Lavras ˗ Lavras, MG A.S. Uchoa1 2 https://orcid.org/0000-0003-0838-2528 Universitat Autónoma de Barcelona, Bellaterra (Cerdanyola del Valles) ˗ Espanha J. E. Rodrıguez-Gil2 https://orcid.org/0000-0002-1112-9884 L.J. Pereira1 ABSTRACT https://orcid.org/0000-0002-0502-2554 M.G. Zangeronimo1*  https://orcid.org/0000-0002-3530-9345 A study was conducted to evaluate the effect of chlorogenic acid (ChA) added pre-cooling and its combination with caffeine added during warming on cooled-stored boar semen parameters. Ten ejaculates were diluted in commercial extender with or without 4.5mg/ml ChA and stored at 15°C. After 0, 24 and 72 hours of storage, aliquots of these doses were taken and incubated at 37°C in the presence or absence of 8.0mM caffeine. Semen quality was evaluated after 10 and 120 minutes of incubation. The ChA increased (P <0.01) the sperm motility, viability, acrosomal integrity and the percentage of spermatozoa with high mitochondrial activity (PMHA), however, decreased (P <0.01) the malondialdehyde (MDA) concentration. Caffeine increased (P <0.05) the sperm motility, viability, PMHA and the MDA concentration and reduced (P <0.05) the acrosome integrity. When associated (ChA+caffeine), there was an increase (P <0.05) in sperm motility and viability, PMHA and acrosome integrity. The addition of ChA to the dilution medium improves the quality of the swine inseminating doses. The addition of caffeine during re-warming is only recommended when the semen is stored for prolonged periods (72h), and the inseminating dose should be used immediately after its addition. Keywords: antioxidant, boar semen storage, metabolic activator, methylxanthine, polyphenol RESUMO O objetivo deste estudo foi avaliar os efeitos da adição de ácido clorogênico (ChA) antes do resfriamento e sua combinação com cafeína adicionada durante o reaquecimento sobre a qualidade do sêmen suíno resfriado. Dez ejaculados foram diluídos em diluidor comercial com adição ou não de 4,5mg/mL de ChA e armazenados a 15°C. Após zero, 24 e 72 horas de armazenamento, 10mL foram retirados e incubados a 37°C na presença ou ausência de 8,0mM de cafeína. A qualidade seminal foi avaliada após 10 e 120 minutos de incubação. O ChA aumentou (P<0,01) a motilidade, a viabilidade, a integridade acrosomal e a porcentagem de espermatozoides com alta atividade mitocondrial (PMHA), entretanto diminuiu (P<0,01) a concentração de malondialdeído (MDA). A cafeína aumentou (P<0,05) a motilidade, a viabilidade, a PMHA e a concentração de MDA e reduziu a integridade acrossomal. Quando associados (ChA+cafeína), houve aumento (P<0,05) na motilidade, na PMHA, na viabilidade e na integridade acrossomal. Conclui-se que a adição de ChA ao meio de diluição melhora a qualidade das doses inseminantes de suínos. A adição de cafeína durante o reaquecimento só é recomendada ao sêmen adicionado de ChA quando esse for armazenado por períodos prolongados (72h), devendo a dose inseminante ser utilizada imediatamente após sua adição. Palavras-chave: antioxidante, sêmen suíno armazenado, ativador metabólico, metilxantina, polifenol Recebido em 6 de dezembro de 2017 Aceito em 15 de maio de 2018 *Autor para correspondência (corresponding author) E-mail: zangeronimo@dmv.ufla.br Pereira et al. INTRODUCTION motility, straightness, and linearity of sperm movement (Yamaguchi e Funahashi, 2012; Currently, artificial insemination is widely Yamaguchi et al., 2013). It is known that practiced in swine production using fresh caffeine positively affects sperm function by seminal doses or stored at 15°C for usual periods upregulating glycolysis via a buildup of cyclic of 24 to 48h (Pinart et al., 2013). Although the adenosine monophosphate (cAMP), which fertility of boar semen can be maintained under generates adenosine triphosphate (ATP), used to these conditions, cooling for longer periods power sperm movement (Lardy et al., 1971). results in decreased sperm quality. This occurs Rhemrev et al. (2001) suggested that the motility mainly due to excessive production of reactive of the spermatozoa was related to their fertilizing oxygen species (ROS) by spermatozoa, which in capacity and the sperm motility was dependent turn cause oxidative damage in sperm cell on mitochondrial function. membranes compromising viability and sperm function (Waterhouse et al., 2004). The lipid However, one undesirable side effect linked with peroxidation of cell membrane induced by ROS the utilization of metabolic activators is a alters the structure and function of the sperm, concomitant increase of sperm ROS production, generates DNA damage (Schulte et al., 2010) which would act against sperm function. Thus, and reduces intracellular ATP levels and sperm an effective technique to utilize metabolic motility (Aitken et al., 2012). activators to improve boar semen conservation would include a system that allows us to avoid To address these problems, addition of the undesirable increase in ROS. A possibility antioxidants in semen extenders have been used, would be the simultaneous utilization of a ensuring better quality of the cooled semen metabolic activator, like caffeine, together with (Martin-Hidalgo et al., 2011; Mendez et al., an antioxidant, like ChA. In this manner, the 2013). The chlorogenic acid (ChA) is a antioxidant effect of the ChA could counteract polyphenol known for its antioxidant properties the caffeine-caused increase in ROS. Taking this in vitro (Rice-Evans et al., 1996) that has been into consideration, the main objective of this associated with suppressing the activation of study was to determine if combined use of ChA, transcription factors induced by ROS (Xu et al., in the dilution media, and caffeine, during re- 2010). Recently, a study has proved the warming of inseminating doses, could be useful efficiency of ChA in improving the quality of the to improve the quality of boar semen stored at cooled boar semen (Pereira et al., 2018). Thus, 15°C until 72 hours. the addition of antioxidants could have a beneficial effect on increasing the useful storage MATERIALS AND METHODS time of boar semen. All procedures were approved by the Ethics There are other substances that can improve Committee on Animal Use of UFLA with stored boar semen quality. For instance, protocol number 027/13. Ten ejaculates from metabolic activators, such as caffeine, have also three fertility proven pure breed boars (Pietrain, been used in this sense. Caffeine is a Duroc and Large White) were used. The animals methylxanthine capable of improving the were 1.0-1.5 years of age and were kept in motility of spermatozoa and mitochondrial individual pens (3.0m long, 2.0m wide and activity (Nabavi et al., 2013), resulting in an 1.30m high) received water ad libitum and 3.0kg increase in fertilization rates (Numabe et al., of feed divided into two daily rations. Animals 2001), when added immediately prior of were housed at the Experimental Swine Center; insemination. In swine, there is evidence that the Department of Animal Science, Federal addition of caffeine during the semen re- University of Lavras (UFLA; Lavras; Minas warming of cooled boar semen reduces the Gerais, Brazil). sperm viability, however, increases in the vigour of spermatozoa (Nunes, 2012). The reduction of Ejaculates were obtained using the gloved hand sperm viability was attributed by the authors to method during routine collection on the farm. the higher metabolic activity of the cells. In After collection, ejaculates were immediately frozen-thawed boar semen, the use of caffeine in sent to the Laboratory of Swine Breeding the thawing solution improved the progressive (Department of Physiology and Pharmacology, 490 Arq. Bras. Med. Vet. Zootec., v.71, n.2, p.489-499, 2019 Addition of chlorogenic... UFLA). Initial subjective observations on Darmstadt, Germany). The pH of all samples motility and vigour were carried out on a drop of remained between 7 and 8, which is considered semen placed between a slide and coverslip ideal for sperm survival (Johnson et al., 2000). previously heated to 37ºC under phase microscope (Nikon Eclipse E200-LED, Nikon, Ninety minutes after this process, all four doses Tokyo, Japan) at 100×. For analysis of sperm were stored at 15°C in a refrigerator. Before morphology 200μL of fresh semen was addition storage, a 10ml aliquot of each insemination dose in 1000μL of formal saline solution, and a drop was heated in test tubes in a water bath at 37°C. was evaluated under a phase microscope One aliquot from a ChA-added dose and another (Olympus CX31, Olympus Corporation, aliquot from a negative control were Hamburg, Germany) at 1000×. Sperm supplemented with caffeine (8.0mM) in a concentration was evaluated in a Neubauer cell concentration determined in previous study counter chamber after dilution of 10μL fresh (Nunes, 2012). For this, 15.33mg of caffeine semen in 1000μL of formal saline solution, and (pure caffeine, 533, Isofar, Rio de Janeiro, the number of spermatozoa/ml was determinate Brazil) was diluted in 1.0ml of BTS at 37°C, under a phase microscope at (Olympus CX31, which was added into 9.0ml of the inseminate Olympus Corporation, Hamburg, Germany) dose. The other two 10ml aliquots were added to 400×. Only the rich fraction of motile ejaculates 1.0ml of BTS without caffeine and they were was used for processing of insemination doses. used as controls for the caffeine effect. After 10 and 120min of incubation, all samples were All ejaculates fulfilled the minimum evaluated to determine the percentage of mobile requirements for use in artificial insemination: and viable cells, integrity of sperm acrosome, >70% total motility, <20% proximal cytoplasmic mitochondrial activity of the cells and droplets and total number of spermatozoa concentration of glucose to determine the >200×106 per ml (Silva et al., 2011). After the glucose uptake during this time. Furthermore, sperm concentration was calculated, the semen after 60min of incubation samples were volume that contained 1.5 billion sperm was collected to determine the concentration of diluted in BTS extender previously prepared and malondialdehyde (MDA). The same incubation heated to 37°C with or without addition of ChA. procedure was carried out after 24 and 72h of From each ejaculate, four insemination doses storage, with the exception of glucose intake that were processed and distributed in a randomized was evaluated only at 0 and 72h of storage. block design (blocked by ejaculate) in a 2 x 2 factorial scheme (with or without ChA, with or To evaluate sperm motility, a drop of semen was without caffeine) in a split plot in time placed between a slide and a cover slip (incubation time of semen) with ten repetitions previously heated to 37°C. Observations were that were representative of the ejaculate. made using a phase-contrast microscope (Nikon Eclipse E200-LED, Nikon, Tokyo, Japan) with For this, immediately before the processing of 100x magnification. Two trained persons insemination doses, 180mg of ChA (Chlorogenic subjectively evaluated 10 fields, to determine the acid crystalline, C3878, Sigma-Aldrich Co., Ltd., percentage of motile sperm. Analyses were done St. Louis, MO, USA) was diluted in 5.0ml of blind and in triplicate. The vigour was evaluated BTS extender (Beltsville Thawing Solution®, using a system scale from 0 to 5, where 0 Minitube, Porto Alegre, Brazil) at 37°C using an represents the absence of movement and 5 is ultrasonic sonicator (QR 300W, Ultronique, maximum intensity. Indaiatuba, Brazil). This solution was then added to the volume of semen that contained 1.5 billion Sperm membrane integrity was measured by a sperm. After, BTS at 37°C were then added until smear of drop of semen mixed with a drop of the final volume of 40ml, so that each eosin-nigrosin (Blom, 1950). The cells were insemination dose contained 4.5mg of ChA per evaluated under optical microscope (Olympus ml of semen (Pereira et al., 2018). Two CX31, Olympus Corporation, Hamburg, inseminate doses were processed with ChA and Germany) at 400×. The values were expressed by two without this substance. Then, the pH of the percentage of cells with intact membranes in doses was measured using pH-indicator strips relation to the total number of cells counted (200 (109535, Merck Millipore Corporation, cells). The same process was used to determine Arq. Bras. Med. Vet. Zootec., v.71, n.2, p.489-499, 2019 491 Pereira et al. the acrosomal integrity, however, a single dye of In relation to statistical analysis, the Shapiro POPE (Fast Green - F7258 and Rose Bengal – Wilk test was used to evaluate the normality of 198250, Sigma-Aldrich Co. Ltd., St. Louis, residuals. Data from motility and sperm viability, USA) was used (Pope et al., 1991). The and mitochondrial activity were subjected to mitochondrial oxidative activity was estimated analysis of variance and means within each main by the DAB technique (Hrudka, 1987) in the factor (ChA and caffeine) and time of incubation same phase contrast microscopy at 1000× were compared by the F test, considering α= magnification. Only cells with fully stained 0.05. For vigour, a nonparametric statistical midpieces that indicate high mitochondrial analysis was used and the averages were activity were analyzed. All analyses were compared using the Friedman test. All statistical performed in duplicate in a blinded manner by analysis was performed with the Action 2.3 the same investigator. statistical program. To carry out the biochemical analysis, 1.0ml RESULTS semen aliquots were removed at 10, 60 and 120min of incubation. Samples were centrifuged The ChA improved (P <0.01) the sperm motility in 2.0mL centrifuge tubes at 3360g for 10min. after 120min of incubation of the semen before Supernatants were then transferred to another and at 24 hours of storage, regardless of the centrifuge tube and frozen at −80°C until addition of caffeine (Table 1). With 72 hours of analysis. storage, ChA improved (P <0.01) the sperm motility at both 10 and 120 minutes of The glucose and malondialdehyde (MDA) incubation. Caffeine increased (P <0.05) the concentration in the seminal plasma were sperm motility only in semen stored for 72h at measured by enzyme colorimetric methods 10min of incubation, both in the presence or (Analyze Glucose-PP, Gold Analisa Diagnostica, absence of ChA. Throughout the incubation for Belo Horizonte, Brazil and QuantiChromTM 120 minutes, there was decrease (P <0.05) of TBARS, Assay Kit DTBA-100, Bioassay semen motility, with or without addition of Systems, Hayward, CA, USA, respectively), caffeine and ChA. Although there was following the manufacturer’s instructions. The improvement of the motility in some times of glucose intake by sperm was determined by evaluation, both caffeine and ChA failed to difference between the glucose concentration in maintain sperm motility during 120min of the extracellular medium at 10 and at 120min of incubation. incubation. The MDA concentration was measured at 60min of incutabion. Table 1. Sperm motility of boar semen containing or not chlorogenic acid (CH) and caffeine (CA), evaluated in different times incubation at 37ºC after different times of storage at 15ºC. n= 10 Incubation Chlorogenic Caffeine P= SEM time acid without with CA CH CH x CA - Before storage (diluted semen) - 10 minutes without 81.0 * 82.5 * 1.03 0.23 <0.01 0.24 with 82.0 * 84.5 * 120 minutes without 72.5 A 71.0 A with 79.0 B 81.0 B - 24 hours of storage - 10 minutes without 81.0 * 82.5 * 1.03 0.23 <0.01 0.24 with 82.0 * 84.5 * 120 minutes without 72.5 A 71.0 A with 79.0 B 81.0 B - 72 hours of storage - 10 minutes without 57.5 Aa* 61.5 Ab* 1.00 0.34 <0.01 0.10 with 67.5 Ba* 70.6 Bb* 120 minutes without 47.5 A 46.5 A with 64.0 B 61.5 B * Incubation period differ by F test (P <0.05) ab Within each incubation time, means followed by different upper letters in the column and lower case letters in the line differ by the F-test (P <0.05) 492 Arq. Bras. Med. Vet. Zootec., v.71, n.2, p.489-499, 2019 Addition of chlorogenic... The combination of ChA and caffeine was able increased (P<0.01) the PMHA throughout the to maintain (P<0.01) the percentage of 120min of incubation in all semen samples. mitochondria with high mitochondrial activity Caffeine increased (P <0.01) the PMHA, with (PMHA) during 120min of incubation both in exception in the diluted fresh semen and semen diluted fresh semen and in stored semen for 72h stored for 72h containing ChA at 10min of (Table 2). Alone, ChA was able (P<0.05) to incubation and in the semen stored for 24h, at maintain this characteristic during incubation 120min of incubation. time only in the stored semen for 72h. ChA Table 2. Percentage of mitochondria with high activity of boar semen containing or not chlorogenic acid (CH) and caffeine (CA), evaluated in different times incubation at 37ºC after different times of storage at 15ºC. n= 10 Incubation Chlorogenic Caffeine P= SEM time acid without with CA CH CH x CA - Before storage (diluted semen) - 10 minutes without 38.6 Aa* 40.7 b* 0.40 <0.01 <0.01 0.09 with 40.2 B* 41.2 120 minutes without 36.5 Aa 38.5 Ab with 38.5 Ba 40.8 Bb - 24 hours of storage - 10 minutes without 38.4 Aa 40.9 Ab* 0.49 <0.01 <0.01 0.88 with 41.2 Ba* 43.2 Bb* 120 minutes without 37.6 A 38.7 A with 39.6 B 41.0 B - 72 hours of storage - 10 minutes without 35.4 Aa* 37.4 Ab* 0.47 <0.01 <0.01 0.12 with 37.8 B 39.1 B 120 minutes without 33.1 Aa 35.5 Ab with 36.6 Ba 38.6 Bb * Incubation period differ by F test (P <0.05) ab Within each incubation time, means followed by different upper letters in the column and lower case letters in the line differ by the F-test (P <0.05) Both ChA and caffeine did not influenced stored for 72h, with exception when ChA was (P>0.05) the MDA concentration in the diluted present in the semen. ChA decreased (P<0.01) semen (Table 3). However, caffeine increased the MDA concentration of stored semen. (P<0.01) the MDA concentration of samples Table 3. Malondialdehyde concentration in the boar semen containing or not chlorogenic acid (CH) and caffeine (CA), evaluated after 60min of incubation at 37ºC before and after storage at 15ºC. n= 10 Chlorogenic Caffeine P= SEM acid without with CA CH CH x CA - Before storage (diluted semen) - Without 2.27 2.05 0.10 0.68 0.88 0.55 With 2.10 2.15 - 72 hours of storage - Without 5.17Aa 5.93Ab 0.15 0.01 <0.01 0.66 With 4.33B 4.88B a.b Within storage time, means followed by different upper letters in the column and lower case letters in the line differ by the F-test (P <0.05). The presence of ChA in the extender improved semen without ChA stored for 24h or at 10min of (P <0.01) the sperm viability only on the stored incubation of semen stored for 72h with or semen (24 or 72h), with more expressive results without ChA. The addition of ChA and caffeine in the semen stored for 72h, evaluated at 120min did not able to maintain the sperm viability of incubation (Table 4). Caffeine improved the throughout 120min of incubation. sperm viability only at 120min of incubation of Arq. Bras. Med. Vet. Zootec., v.71, n.2, p.489-499, 2019 493 Pereira et al. ChA improved (P <0.01) the integrity of sperm decreased (P <0.05) the integrity of sperm acrosome in the diluted fresh semen only when acrosome throughout 120min of incubation in all caffeine was added (Table 5). In the semen semen samples. Caffeine decreased this stored for 72h, ChA improved the integrity of characteristic throughout the incubation time of sperm acrosome in all evaluated samples. stored semen and the combination ChA and Caffeine reduced (P <0.05) the integrity of sperm caffeine decreased only in the semen stored for acrosome in some samples without ChA, 72h. however, increased when ChA was present. ChA Table 4. Percentages of sperm viability in the boar semen containing or not chlorogenic acid (CH) and caffeine (CA), evaluated in different times incubation at 37ºC after different times of storage at 15ºC. n= 10 Incubation Chlorogenic Caffeine P= SEM time acid without with CA CH CH x CA - Before storage (diluted semen) - 10 minutes without 92.8 92.7 0.74 0.14 0.22 0.28 with 93.6 94.3 120 minutes without 93.2 91.4 with 93.3 92.3 - 24 hours of storage - 10 minutes without 89.4 A* 88.6 A* 0.70 0.41 <0.01 0.01 with 93.1 B* 91.2 B* 120 minutes without 81.4 Aa 83.7 Ab with 90.0 B 89.1 B - 72 hours of storage - 10 minutes without 57.5 Aa* 61.5 Ab* 1.00 0.34 <0.01 <0.01 with 67.0 Ba* 70.5 Bb* 120 minutes without 47.5 A 46.5 A with 64.0 B 61.5 B * Incubation period differ by F test (P <0.05) ab Within each incubation time, means followed by different upper letters in the column and lower case letters in the line differ by the F-test (P <0.05) Table 5. Percentages of sperm acrosome integrity of boar semen containing or not chlorogenic acid (CH) and caffeine (CA), evaluated in different times incubation at 37ºC after different times of storage at 15ºC. n= 10 Incubation Chlorogenic Caffeine P= SEM time acid without with CA CH CH x CA - Before storage (diluted semen) - 10 minutes without 97.1 a 96.0 Ab 0.32 0.79 <0.01 <0.01 with 97.8 * 98.3 B 120 minutes without 96.3 95.4 A with 96.8 b 98.0 Ba - 24 hours of storage - 10 minutes without 93.8 95.3 * 0.62 0.07 <0.01 0.04 with 95.0 * 95.8 120 minutes without 92.7 91.0 A with 92.5 a 94.3 Bb - 72 hours of storage - 10 minutes without 89.8 A* 90.4 A* 0.41 0.15 <0.01 0.18 with 92.3 B* 91.8 B* 120 minutes without 88.5 Aa 85.8 Ab with 90.2 B 90.6 B * Incubation time differ by F test (P <0.05) a,b Within each incubation time, means followed by different upper letters in the column and lower case letters in the line differ by the F-test (P <0.05) 494 Arq. Bras. Med. Vet. Zootec., v.71, n.2, p.489-499, 2019 Addition of chlorogenic... The vigour was not influenced (P>0.05) by the period when caffeine was added to the semen. presence of both ChA and caffeine (Table 6). No effect of ChA and caffeine was observed in Despite this, there was a decrease (P<0.01) of the glucose intake (Table 7). movement intensity throughout the incubation Table 6. Vigour in scale from 0 (absence of movements) to 5 (high intensity of movements) of boar semen containing or not chlorogenic acid (CH) and caffeine (CA), evaluated in different times incubation at 37ºC after different times of storage at 15ºC. n= 10 Caffeine Incubation time Chlorogenic acid P= without with - Before storage (diluted semen) - 10 minutes without 4.0 4.7* <0.01 with 3.9 4.6* 120 minutes without 3.0 2.8 with 3.0 2.8 - 24 hours of storage - 10 minutes without 3.0 4.2* <0.01 with 3.0 4.1* 120 minutes without 2.6 2.0 with 2.8 2.3 - 72 hours of storage - 10 minutes without 2.6 3.6* <0.01 with 2.8 3.5* 120 minutes without 2.0 1.9 with 2.1 1.9 * Incubation time differ by the Friedman test (P <0.01) Table 7. Glucose intake in the boar semen containing or not chlorogenic acid (CH) and caffeine (CA), evaluated after 60min of incubation at 37ºC before and after storage at 15ºC. n= 10 Caffeine Chlorogenic acid P= without with - Before storage (diluted semen) - without 20.2 15.2 0.14 with 17.0 17.7 - 72 hours of storage - without 14.5 12.0 0.25 with 13.2 10.2 No significance by Frideman test (P >0.05). DISCUSSION effects of caffeine on boar semen quality with a clear mitigation of the side effects linked to Our results clearly indicate that the addition of caffeine addition. The results described are very ChA during storage and refrigeration can be important when taking into account the fact that useful to improve the semen quality of storage one of the biggest current challenges for boar semen at 15°C. Furthermore, our results reproductive technology is the maintenance of also indicate that the presence of ChA has a boar semen quality stored in refrigerated beneficial effect through potentiating the positive conditions for prolonged periods. In addition, the Arq. Bras. Med. Vet. Zootec., v.71, n.2, p.489-499, 2019 495 Pereira et al. evaluation at different incubation times is could be an important mechanism leading to a important to simulate sperm resistance after re- reduction of the oxidative damage of sperm cell warming. Under practical conditions, the use of structures. Furthermore, the low MDA levels semen at different times after re-warming may found in semen stored for 72h in the presence of contribute to the reduction of fertility rate. Thus, ChA indicate that the effect was able to decrease alternatives that can prolong sperm resistance the lipid peroxidation alterations caused by ROS time after re-warming are important. (Gomez-Fernandez et al., 2013; Fraser et al., 2014). Since the integrity of cell structures is Pereira et al. (2018), evaluating different doses crucial to maintaining sperm functionality, the of ChA (0.0, 1.5, 3.0, 4.0 and 6.0mg/ml) reduction of the oxidative stress induced by the concluded that the concentration of 3.2mg/ml of addition of antioxidants would improve the this substance is the best for semen stored for quality of seminal doses (Maia et al., 2009; 24h. However, for semen stored for a longer Wittayarat et al., 2013). In the non-stored semen, period, 6.0mg/ml or more should be used. In the the effects of ChA were not evident, probably present study, the concentration of 4.5mg/mL due to the presence of natural antioxidants in the was considered an intermediary dose, giving seminal plasma, which are able to protect sperm beneficial effects for both semen stored for 24 cells from oxidative damage during short periods and 72h. By other hand, Nunes (2012), of storage (Kowalowka et al., 2008). However, evaluating different doses of caffeine (0, 2, 4, 6 since storage is linked to a great dilution of and 8mM) added in the cooled semen concluded seminal plasma factors, the addition of ChA that the higher dose improves the vigour only in could counteract the loss of the antioxidant effect the semen stored by 48h, but negatively affects because of the dilution of seminal plasma. in lower storage times, probably due to increased metabolic activity of spermatozoa. In this case, The maintenance of sperm function is also the association between a metabolic activator and dependent on the metabolic status of the sperm a membrane protecting substance could bring since cellular activity occurs from the benefits to the use of the cooled sperm semen. consumption of both extra and intracellular energy substrates. In this sense, energy Reduction in sperm quality during storage is production through mitochondrial pathways is of commonly correlated with oxidative damage to the utmost importance in eukaryotic cells. cell structures due to metabolism (Schulte et al., Centering on boar sperm, it is noteworthy that 2010), since preservation temperatures between the total energy yielded by mitochondrial activity 15 and 18°C do not completely stop sperm is a very low percentage (a maximum of 5%) of metabolism. The losses to the seminal quality are total energy production (Marin et al., 2003). more evident after 48h of cooling, reducing the Despite this, the minimal mitochondria-based reproductive performance of the herd (Pinart et energy production is critical for the maintenance al., 2013). The oxidative attack on the sperm of motility and sperm capacitation in swine membranes is able to modify the fluidity of the (Ramio-Lluch et al., 2014). Thus, sperm plasma membrane and interfere with enzymatic mitochondrial activity becomes an important activity and in the function of ion channels evaluation parameter regardless of their intrinsic (Marmunti et al., 2012), thus reducing sperm importance in the production of energy in boar quality. On the other hand, sperm metabolism is sperm. With respect to the seminal metabolic important to guarantee the semen quality (Yeste rate, the addition of caffeine increased sperm et al., 2008). Thus, substances that accelerate mitochondrial activity only after 10min of sperm metabolism combined with substances incubation of semen stored for 72h. This effect with antioxidant properties could have positive of caffeine had been associated with the increase effects on the quality of inseminating doses. of intracellular concentrations of cAMP (Lopez e Alvarino, 2000; Carrington et al., 2011). This is In the present study, we can only speculate about important, since several sperm metabolic the mechanisms by which ChA exerts its positive parameters, such as motility, are closely effects. Other studies have reported the dependent on intracellular cAMP levels (Qu et scavenger ability of polyphenols on sperm al., 2007). function (Martin-Hidalgo et al., 2011; Wittayarat et al., 2013). Thus, the scavenger activity of ChA 496 Arq. Bras. Med. Vet. Zootec., v.71, n.2, p.489-499, 2019 Addition of chlorogenic... On the other hand, increased intracellular In summary, the present study showed that ChA concentrations of cAMP are also associated with has positive effects in sperm motility (mainly elevated overall cell metabolic rates. In the after 120 minutes of incubation), percentage of present study, caffeine was able to increase the mitochondria with maximum activity, MDA number of mitochondria with maximum activity concentration, sperm viability (at 24 and 72h of even in doses that were not supplemented with storage) and acrosome integrity (72h). Caffeine ChA. There is evidence that the phosphorylation increased the sperm motility at 10 minutes of of certain mitochondrial enzymes mediated by incubation of stored semen for 72h, percentage cAMP play a regulatory role in oxidative of mitochondria with maximal activity (0, 24 and phosphorylation (Acin-Perez et al., 2009). 72h) and sperm viability (24 and 72h). On other Therefore, increases in cAMP could increase hand, caffeine increased the MDA concentration mitochondrial activity by stimulating oxidative and reduced the acrosome integrity (both with phosphorylation. In contrast, while the energy 72h of storage) and sperm vigour throughout the generated by mitochondria is essential for sperm 120 minute incubation (0, 24 and 72h). When function, this organelle is the major source of these substances were associated, there was an endogenous ROS production in the cell (Ramio- increase in the sperm motility, sperm viability, Lluch et al., 2014). It is known that ROS can percentage of mitochondria with maximum impair sperm metabolism during storage or activity (all with 72h of storage) and in the incubation. In fact, a significant increase in the acrosome integrity (0 and 24h of storage). The MDA concentration was detected in the present reduction of sperm vigour throughout the study after addition of caffeine in the semen incubation time caused by caffeine adding was stored for 72h. This increase may be linked to the not alleviated by the addition of ChA. These reduction in the PMHA and, consequently, the results suggest that the addition of ChA in the reduction in the vigour after 120min of dilution medium is beneficial for the quality of incubation. The increase of MDA concentration the swine inseminating doses and that the use of may be associated with an increased metabolic caffeine before re-warming is only recommended rate, generated by caffeine’s capability to for the semen stored for prolonged periods (72h). increase the rate of cellular oxidative Although the vigour has been reduced during phosphorylation (Carrington et al., 2011). incubation time when these substances were Despite this evidence of increased sperm used, studies in vivo and in vitro must be metabolic rate, caffeine does not alter glucose conducted to evaluate if this reduction could uptake by sperm, possibly due to consumption of compromise the fertilization capacity of semen. other energy sources, as fructose, from spermatozoa. CONCLUSIONS In the present study, the negative effects of The addition of ChA to the dilution medium caffeine are mitigated with the use of ChA. This improves the quality of the swine inseminating fact indicates that the protective effect against doses. The addition of caffeine during re- sperm membrane damage can be associated with warming is only recommended when the semen the antioxidant activity of ChA. While caffeine is stored for prolonged periods (72h), and the metabolically stimulates mitochondrial activity inseminating dose should be used immediately (Carrington et al., 2011), ChA protects the after its addition. organelle from oxidative damage (Li et al., 2012), promoting good conditions for its ACKNOWLEDGEMENTS performance. (Peña et al. (2003)) also observed the beneficial effects of adding an antioxidant The authors thank CAPES (PNPD Institucional, (vitamin E) to the mitochondrial membrane process number 2457/2011, and Programa potential of thawed boar semen, showing that Pesquisador Visitante Especial - PVE’s, process mitochondria are more sensitive to oxidative number 88881.030399/2013-01), FAPEMIG damage, and antioxidants have a protective effect (PPM-00460-12), CNPq (305478/2015-0 and to this structure. 446288/2014-4), Minitub do Brasil, Fazenda São Paulo and the Postgraduate Program in Veterinary Sciences UFLA for their support. Arq. Bras. Med. Vet. Zootec., v.71, n.2, p.489-499, 2019 497 Pereira et al. REFERENCES LI, S.; BIAN, H.; LIU, Z. et al. Chlorogenic acid protects MSCs against oxidative stress by ACIN-PEREZ, R.; SALAZAR, E.; altering FOXO family genes and activating KAMENETSKY, M. et al. Cyclic AMP intrinsic pathway. Eur. J. 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