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%.