Sage (Salvia Officinalis) and Fennel (Foeniculum Vulgare) Improve Cryopreserved Boar Epididymal Semen Quality Study

CryoLetters 36 (2), 83-90 (2015) © CryoLetters, [email protected]

SAGE (SALVIA OFFICINALIS) AND FENNEL (FOENICULUM VULGARE) IMPROVE CRYOPRESERVED BOAR EPIDIDYMAL SEMEN QUALITY STUDY

A.Montón1, L. Gil1, C. Malo1*, M. Olaciregui1, N. González1, I. de Blas2

1Department of Animal Pathology, Obstetrics and Reproduction Area, Faculty of Veterinary, University of Zaragoza, Zaragoza, Spain. 2Department of Animal Pathology, Infectious Diseases Area, Faculty of Veterinary, University of Zaragoza, Zaragoza, Spain. *Corresponding author email: [email protected] Tel: 976761544 ; Fax: 976761612 .

Abstract

The aim of this study was to evaluate the effect of fennel and sage extracts and the influence of the egg yolk source (fresh or pasteurized) on the success of freezing boar epididymal spermatozoa. In experiment 1, epididymal sperm was recovered by flushing and cryopreserved in a lactose-egg yolk solution supplemented with various concentrations (10, 5 and 2.5 g/L) of sage or fennel. Sperm quality was evaluated (motility, viability, HOST and acrosome integrity) at 0h and 2 h after thawing. Fennel 10 g/L and sage 5 g/L and control (no extracts) were selected for experiment 2 which also compared fresh or pasteurized egg yolk in the freezing extender and measured DNA integrity of the frozen sperm. Results showed that the interaction between fennel and sage antioxidants with fresh egg yolk significantly improved post thaw sperm quality and protected boar epididymal spermatozoa from cryopreservation damage as a result of oxidative stress. Keywords: Cryopreservation; natural antioxidant; boar; epididymis; spermatozoa

INTRODUCTION and these PUFA´s are one of the main Sperm cryopreservation is by far the molecules involved in lipid peroxidation most important technique in male reproductive (LPO) by reactive oxygen substances (ROS). technology. Investigations carried out by The latter negatively affect boar sperm several research groups, in many antioxidant functions [6, 16]. There is an inverse substances have been added to relationship between this phenomenon and in cryopreservation extenders, have improved vivo or in vitro sperm motility [2, 18] post-thaw semen quality. The collection of The antioxidant system of semen has epididymal sperm allows the rescue of sperm been described as a defense against LPO and is from individuals, which have died of natural important for the maintenance of sperm causes. Moreover, epididymal sperm is a good motility and viability [3, 7, 9]. Although pro- alternative to fresh ejaculates due to its greater and anti-oxidants are balanced in the ejaculate, resistance to cryopreservation [27, 39]. The the processes of freezing and thawing produce boar sperm membrane has a high concentration physical and chemical stress on the sperm of polyunsaturated fatty acids (PUFA) [1, 12, membrane, which impairs spermquality, 24, 33, 36], which render it particularly viability and fertilizing capacity [4]. This sensitive to cold shock damage. Sperm phenomenon is associated with an increase in pathology is largely based on PUFA alteration the concentration of free radicals and

83 peroxidation of membrane phospholipids [11, and its derivatives as its most important 17, 23]. antioxidant compounds. Indeed, the existence Typically, freezing media for boar of these compounds presumably makes it semen contains fresh egg yolk supplemented powerful against LPO [13]. Although it has with other agents, stabilizers, cryoprotectants been used in upgrading antioxidant potential in and antioxidants to protect cells from cell cultures, such as hepatocytes [25] or PC12 temperature decrease [43]. The use of fresh cells [19], there are no references to its use in egg introduces a microbial contamination risk sperm cryopreservation. [8, 9]. To reduce this risk, the use of The aim of this study was to determine pasteurized egg yolk would decrease the the protective effect of fennel and sage extracts possibility of transferring pathogens into the and their interaction with natural or storage environment of biological systems. pasteurized egg yolk on boar epididymal However, with the exceptation of some cryopreserved sperm quality. ruminant species,there are few references related to its use in semen biotechnology, only with ruminant species [31, 42]. MATERIAL AND METHODS Although egg yolk contains Reagents and media antioxidants to prevent oxidative damage, the Unless otherwise indicated, all high percentage of PUFA and phospholipids in chemicals were from Sigma–Aldrich Co. the sperm membrane and the removal of (Alcohobendas, Madrid, Spain). Fresh eggs antioxidants during sperm processing [12] were collected from the farm of the Faculty of make the addition of other antioxidants Veterinary (Zaragoza, Spain). Pasteurized egg- essential, in order to offset the excess yolk was obatained from Oreka ® (Azkoitia, formation of free radicals (FR) and to reduce Guipúzcoa, Spain). Fennel seeds (Soria the damaging effects of LPO in frozen semen. Natural, Soria, Spain) and sage shoots (La Flor Many natural antioxidant compounds derived del Pirineo, Manresa, Spain) were commercial from plant sources have been identified as a natural herbs. barrier to free radicals [45]. Recently, interest has increased in the food and drug industry to Experimental extenders search for natural antioxidants alternatives that In experiment 1, lactose–egg yolk would be less harmful to health [46]. These extender containing 20% (v:v) egg yolk, 64% antioxidants may protect the cell from free of 11% L-lactose solution (290 mM), 16% of radicals, delaying the process of chronic pure water and 100 g/mL of kanamycin diseases and preventing deterioration of foods sulphate was used as base extender. Seven [22, 37]. extenders were designed differing in the Fennel (Foeniculum vulgare) is a presence of fennel at different concentrations potential source of antioxidants [35]. It [2.5 g (F2.5); 5 g (F5); 10 g (F10)] or sage [2.5 contains rosmarinic (which shows antioxidant g (S2.5); 5 g (S5); 10 g (S10)] or nothing (no activity similar to that of caffeic acid or extract, control)(Table 1). Fennel and sage quercetin [32]) and flavonoids [40] (which act extenders were prepared by first soaking dry as scavengers of free radicals). Malo et al. [29] commercial herbs in100mL of 100º C water. tested the reduction in the formation of Sage leaves and fennel fruits at different malondialdehyde (MDA) in porcine thawed concentrations (2.5, 5 and 10 g/L) were added ejaculated semen, demonstrated improved to the water and maintained for 10 minutes at progressive motility, indicating that fennel this temperature. Once the water had cooled to antioxidants were able to inhibit LPO. Our 25º C, the solution was filtered to remove the present work sought to assess if similar results leaves. The resulting solution had a pH could be found in epididymal sperm. approximately 7±0.2 and an osmolarity of Sage (Salvia officinalis) is a approximately of 300 mmol 1-1. Sperm Mediterranean plant whose dried leaves are dilution was carried out in a two step used as a spice for fatty food preservation by procedure, fraction 1 (F1) and fraction 2 (F2), reducing LPO [41]. It is considered the most which contained glycerol (9%) and Orvus Es powerful source of antioxidants among plant Paste (OEP; Minitube) (1.5%). F10, 5S and the herbs [28], with rosmarinic acid, carnosic acid control were further studied in experiment 2,

84 varying the source of egg yolk (fresh or for every evaluated parameter three freezing pasteurized) (Table 2). replicates were performed, three straws were The determination of the concentration studied for each replicate and 200 spermatozoa of rosmarinic acid and flavonoids in the plant were evaluated for each straw. Progressive extracts was performed using the High motility (PM) was measured by means of a Performance Liquid Chromatography (HPLC) computer assisted semen analysis (CASA) technique of Soria Natural S.A. (Soria, Spain) system (ISAS®;PROISER; Valencia; Spain). laboratories. For each evaluation, a 5 μL sperm sample was placed in a Makler counting chamber and five Collection, dilution and freezing of boar fields were analysed (300 spermatozoa per epidydimis spermatozoa sample). Teste from Duroc boars aged 1 and 3 Sperm viability was evaluated with years, were obtained at the local eosin-nigrosin [14]. The semen sample was slaughterhouse of Zuera (Zaragoza, Spain) and diluted 1:1 with stain solution (5% eosin, 10% then transported to the laboratory at room nigrosin in a citrate solution) and smeared on a temperature. Sperm were collected using slide. Live spermatozoa remained unstained. retrograde flushing. The caude epididymides The percentage of live (unstained) were dissected and cannulated with a 25 G spermatozoa was expressed as viability. In needle connected to 10 mL syringe containing addition, percentage of acrosomal intact 2 mL of BTS at 22º C. Then manual pressure integrity was evaluated under a phase contrast was applied by a syringe and spermatozoa microscope after a 1:10 dilution in buffered were collected in a Petri dish in order to obtain 2% glutaraldehyde solution [38]. Membrane a heterospermic mixture from three boars for functional integrity was further assessed by the each cryopreservation. The recovered sperm hypoosmotic swelling test (HOST) [20]. The mass was placed in 5 mL of BTS warmed to technique consisted of incubating 30 μL of room temperature. diluted semen with 100 μl of BTS Cryopreservation was carried out using hypoosmotic solution (100 mOsm/Kg) at 37°C the straw freezing procedure of Westendorf et for 15 min. The samples were then fixed in 2% al. [43], modified by Carvajal et al. [10]. The glutaraldehyde in buffer. The proportion of sperm were centrifuged at 1,500g for 3 spermatozoa with swollen tails was considered minutes and the pellet was diluted to 1,500 x as HOST positive. One hundred cells were 106 spermatozoa/mL in F1. The sperm counted by sample for each parameter. suspension was cooled to 5°C within 2 h and then diluted with F2 to yield a final sperm DNA integrity concentration of 1,000 x 106 spermatozoa/mL We followed the protocol of Garcia et and a glycerol concentration of 3%. Samples al [15]. Thawed samples were centrifuged for were loaded into 0.5 mL straws and frozen in a 10 min at 1000g. The supernatant was commercial controlled rate programmable removed and the pellet was resuspended in freezer (IceCube 14S; SY-LAB; Minitüb cold methanol. After 15 minutes, samples were Iberica S.L.). The freezing rate protocol centrifuged again. The supernatant was adopted was 6°C/min from 5°C to (-5 °C), removed and the pellet was resuspended in an 40°C from (-5°C) to (-80°C), held for 30 s and acridine orange solution (0.2 mg/mL). Stained finally 70°C/min to 150°C. Straws were then samples were observed by fluorescence plunged into liquid nitrogen (-196 °C) for microscopy. Sperm with damaged DNA storage. stained due to the affinity of acridine orange fragmented genetic material whereas spem Semen quality with intact dna stained green. Frozen semen samples were thawed at 37°C for 21 seconds and then resuspended in Experimental Design BTS (1:3, v:v; 37 °C) [10] to assess sperm Experiment 1 was designed to compare motility, viability, acrosome integrity and different freezing extenders according to sage response to the hypoosmotic swelling test and fennel concentrations. Rosmarinic acid (HOST). Semen samples were evaluated after and flavonoids content were determined in the thawing at 0 and 2 h incubation at 37°C, and plant extracts. Nine pairs of testis were used.

Three straws were tested per trial evaluating acrosome integrity but differences were sperm motility, viability, acrosome integrity observed after two hours of incubation at 37º and response to the hypoosmotic swelling test C. According to the results of the prefreezing (HOST) pre-freezing and at 0 and 2 h viability, and the viability and the acrosome incubation at 37°C after thawing. Control, F10 integrity after two hours of incubation, we and S5 were selected for the following selected fennel 10 g/L and sage 5 g/L for experiment. Experiment 2 was designed to experiment 2. assess the protective action of fresh and pasteurized egg yolk in semen Experiment 2 cryopreservation. Nine pairs of testicles were Those extenders which were made used. Three straws were tested per trial. The with fresh egg yolk showed better results than same parameters as in Experiment 1 were those based on pasteurized egg yolk after evaluated as well as DNA fragmentation after thawing, not only in classical parameters thawing. (motility, viability, HOST test and acrosome . integrity) but also in the DNA fragmentation Statistical analysis test. Results of classical parameters are We used the statistical package SPSS summarized in Table 4. version 15.0 for Windows. Comparison of The fennel 10 g/L with fresh egg yolk means were carried out using ANOVA or extender improved progressive motility both at Kruskal-Wallis tests (depending on normality thawing (50.00±3.651) and after incubation at of variables tested with Kolmogorov Smirnov), 37° C (24.33±2.201). Sperm in both extenders and differences between groups were checked (sage 5g/l and fennel 10 g/L) showed high with the Duncan post hoc test (for normally values of viability after thawing (60.67±2.155 distributed variables) or two-by-two groups and 68.33±1.874, respectively) and acrosome comparison with Mann-Whitney test (for not integrity (66.00±1.770 and 70.00±1.544, normally distributed variables). With regards respectively) and after two hours of incubation to DNA integrity, confidence intervals for (50.83±2.926 and 54.33±1.520, respectively). frequencies of qualitative variables were No significant differences were observed in the calculated with the Wilson Score method. results of the HOST test. Finally, with regards to the percentage RESULTS of spermatozoa with intact DNA, sage 5 g/L- fresh egg yolk and fennel 10 g/L-fresh egg Experiment 1 yolk showed highly significant differences Results of the natural antioxidant (P<0,001) compared with the control (70% concentrations measured by HPLC from fennel (IC95%= 63,32% - 75,93%) and 65% and sage extracts showed that the (IC95%= 58,16% - 71,27%) vs 50% (IC95%= concentration of rosmarinic acid was higher in 43,14% - 56,86%)) respectively) and the the sage extract (23.49 mg/100g) than in the pasteurized egg yolk media (Ley-PY 30.5% fennel extract (1.7 mg/100g). Furthermore, the (IC95%= 24,54%- 37,20%); F10-PY 33.5% concentration of the general content of (IC95%= 27,32% - 40,30%); S5-PY 29% flavonoids measured in fennel extract was 1.39 (IC95%=23,15%-35,64%). mg/100g. Results of progressive motility (PM), DISCUSSION viability, HOST test and acrosome integrity are summarized in Table 3. Significant differences In the first experiment the addition of fennel or in progressive motility pre-freezing sage extracts at different concentrations demonstrated a beneficial effect of sage 2.5 improved the viability and the acrosome g/L and 5 g/L of fennel, compared with the integrity of boar epididymal sperm two hours control. There were no significant differences after incubation. There were not significant in sperm viability immediately after thawing differences in acrosome integrity immediately whereas two hours later sperm containing after thawing. This differed from results medium and high concentrations of sage, were reported for other natural antioxidants such as significantly better. Immediately after thawing, rosemary, on boar fresh ejaculate [30]. there were no significant differences in

However, we achieved highly significant LPO and DNA fragmentation index (DFI). differences in the second experiment. DNA stability was demonstrated to be The maximum concentration was important to fertilization in vivo [5] Therefore, selected in the case of fennel, what agrees with combination of antioxidants from fresh egg Oktay et al. [35] who asserted the antioxidant yolk and sage or fennel extracts would appear power of fennel increases with the to enhance DNA integrity of frozen boar concentration of its natural antioxidants. epididymal sperm. In the second experiment, sperm in Salvia officinalis is being included in extenders which were made with fresh egg the preservation of in vitro cell systems due to yolk and natural antioxidants from fennel or its antioxidant power. Extracts applied in sage extracts showed better results than those cultures of mouse and rat hepatocytes of which were based on pasteurized egg yolk. improved liver antioxidant potential [25] by Similarly way, Olaciregui et al. [34] found that increasing the activity of glutathione reductase all extenders performed with pasteurized egg and glutathione-S-transferase. Moreover, cell yolk showed worse results in contrast with destruction mediated by beta-peptide in those media made with fresh egg yolk. Alzheimer disease was reduced after 24 hours Yamamoto & Omori [44] proved that although of contact with sage extracts that mainly egg yolk contains antioxidants to prevent contained rosmarinic acid [19]. The use of oxidative damage, the high percentage of sage requires more research in its action on PUFA and phospholipids on boar sperm cryopreserved sperm quality and DNA membrane makes the addition of other fragmentation since there are no references antioxidants essential to decrease the excess related to its use in semen cryopreservation. formation of ROS. Thus, it is noteworthy that In conclusion, we can assert a positive the extender containing fennel 10 g/L or sage 5 effect associated with the interaction between g/L and fresh egg yolk improved the fresh egg yolk and natural antioxidants from progressive motility and the acrosome integrity fennel or sage extracts, improving all the of sperm immediately after thawing and 2 parameters evaluated in this study. Fennel 10 hours later, and improved viability after g/L attained the most significant improvement thawing, with respect to the control. As shown of semen quality after thawing. Pasteurized in Table 4, the fennel 10 g/L with fresh egg egg yolk was not a good substitute for fresh yolk extender improved progressive motility egg yolk when used under the conditions of both at thawing and after incubation at 37 ° C; this experiment. More studies are required for it also increased viability at thawing and its use with boar epididymal cryopreserved acrosome integrity just afterthawing and after sperm quality in order to reduce two hours of incubation. Additionally, the microbiological risks. Also further studies extender based on sage 5 g/L and fresh egg involving pure components of fennel and sage, yolk improved post-thaw acrosomal integrity. individually or in combination, are required to Therefore, the combination of the natural find the best antioxidant combination for antioxidants from plant sources and fresh egg freezing boar epididymal sperm. yolk is likely to be an essential point for Acknowledgements: This research was made enhancing the results provided by the LEY possible by the financial support of project control [29,30]. Sage reduced progressive UZ2011-BIO-03 (University of Zaragoza) and motility after two hours of incubation as the Government of Aragon (DGA). We are compared to the control. also grateful to the Toxicology Area of the In addition, in the extenders where natural Faculty of Veterinary (University of Zaragoza) antioxidants from fresh egg yolk and sage or and Carlos Carricajo (Soria Natural S.A., fennel extracts interacted, the DNA integrity Soria, Spain) for scientific support. was maintained at significantly better level.

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