Effects of feeding omega-3-fatty acids on fatty acid ...

Animal Reproduction Science 160 (2015) 97–104

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Effects of feeding omega-3-fatty acids on fatty acid composition and quality of bovine sperm and on antioxidative capacity of bovine seminal plasma Hakan Gürler a,∗ , Oguz Calisici a,b , Duygu Calisici a,b , Heinrich Bollwein c a b c

Clinic for Cattle, University of Veterinary Medicine Hannover, Germany Süt Kardesler A.S. Animal Breeding Center, Izmir, Turkey Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Switzerland

a r t i c l e

i n f o

Article history: Received 7 June 2015 Received in revised form 29 July 2015 Accepted 30 July 2015 Available online 5 August 2015 Keywords: Bulls Fatty acids Seminal plasma Total antioxidant capacity Lipid peroxidation

a b s t r a c t The aim of the present study was to examine the effects of feeding alpha-linolenic (ALA) acid on fatty acid composition and quality of bovine sperm and on antioxidative capacity of seminal plasma. Nine bulls (ALA bulls) were fed with 800 g rumen-resistant linseed oil with a content of 50% linolenic acid and eight bulls with 400 g palmitic acid (PA bulls). Sperm quality was evaluated for plasma membrane and acrosome intact sperm (PMAI), the amount of membrane lipid peroxidation (LPO), and the percentage of sperm with a high DNA fragmentation index (DFI). Fatty acid content of sperm was determined using gas chromatography. Total antioxidant capacity, glutathione peroxidase, and superoxide dismutase activity were determined in seminal plasma. Feeding ALA increased (P < 0.05) the docosahexaenoic acid (DHA) content in bulls whereas in PA bulls did not change. PMAI increased after cryopreservation in ALA bulls as well as in PA bulls during the experiment period (P < 0.005). LPO of sperm directly after thawing did not change during the study period in ALA group, but decreased in PA group (P < 0.006). After 3 h of incubation LPO increased in the ALA group (P < 0.02), while LPO did not differ between phases within groups. In conclusion, feeding of neither saturated nor polyunsaturated fatty acids affect the antioxidant levels in seminal plasma. Both saturated as well as polyunsaturated fatty acids had positive effects on quality of cryopreserved bovine sperm, although the content of docosahexaenoic acid in sperm membranes increased only in ALA bulls. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In mammalian spermatozoa, long-chain polyunsaturated fatty acids of the n-3 family, in particular docosahexaenoic acid (DHA; C22:6n-3), are predominant (Petit et al., 2007; Zachut et al., 2011). These fatty acids

∗ Corresponding author at: Institute of Reproductive Biology, University of Veterinary Medicine Hannover, Bünteweg 2, D-30559 Hannover, Germany. Tel.: +49 511 856 7184; fax: +49 511 953 8504. E-mail address: [email protected] (H. Gürler). http://dx.doi.org/10.1016/j.anireprosci.2015.07.010 0378-4320/© 2015 Elsevier B.V. All rights reserved.

are important for sperm membrane integrity, sperm motility and viability. The proportion of omega-3-PUFAs in sperm and seminal fluid decreases with age in bulls. This contributes to rising susceptibility of spermatozoa to cryoinjury (Argov-Argaman et al., 2013). The lipid composition of the plasma membrane is also a major determinant of motility and freezability of sperm among species (Hammerstedt et al., 1990; Parks and Lynch, 1992; Hammerstedt, 1993). Furthermore, it was noticed that lower contents of C20 and C22 PUFAs in the sperm of older bulls (Kelso et al., 1997b) was related to a decrease in the ability to fertilize oocytes. It is suggested by

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H. Gürler et al. / Animal Reproduction Science 160 (2015) 97–104

Argov-Argaman et al. (2013) that such alterations might affect the semen’s capacity to successfully undergo the cryopreservation procedures, which are widely used in intensive reproduction management. Animals cannot synthesize n-6 or n-3 fatty acids de novo as they lack the appropriate fatty acid desaturase enzymes and should obtain them from dietary sources (Wathes et al., 2007). Furthermore, fatty acids in the diets are known to affect fatty acid composition of sperm in human (Conquer et al., 2000) and a variety of farm animals (Kelso et al., 1997a,b; Zaniboni et al., 2006; Brinsko et al., 2005; Harrison and Abhyankar, 2005). The decrease in PUFA content was related to decreased antioxidant levels (GPx and SOD) in seminal plasma of aging bulls during reproductive period (Kelso et al., 1997b). Somatic cells contain several antioxidants, GPx, SOD, and catalase in their cytoplasm. As sperm are devoid of most of this cytoplasm, the main antioxidant source in semen is the seminal plasma (Bilodeau et al., 2000). For example, it was shown that GPx and SOD in seminal plasma play important roles in the inhibition of LPO (Kelso et al., 1996). Previous reports have shown that enzymatic and non-enzymatic antioxidants had positive effects on sperm freezability (Bilodeau et al., 2001; Fernandez-Santos et al., 2007; Bansal and Bilaspuri, 2008). An important positive effect of PUFA in sperm function has been attributed to its effect on fluidity of the plasma membrane (Gholami et al., 2010). In the last fifteen years, in various feeding experiments DHA or its precursors have been supplied to change the fatty acid composition of sperm membrane in order to improve sperm quality and fertility. Several different studies revealed that the fatty acid profile of sperm membrane can be modified with diet and, thus, improvement in sperm quality was demonstrated in a variety of livestock species including chicken (Zaniboni et al., 2006), turkey (Blesbois et al., 2004), boar (Maldjian et al., 2005); buffalo (Adeel et al., 2009) and stallion (Brinsko et al., 2005). However, diets that contain more PUFAs are associated with impaired antioxidant capacity of animal tissues, blood, and semen (Surai et al., 2000a; Castellini et al., 2003). Feeding bulls with a commercial nutriceutical product that contained 25% n-3 (eicosapentaenoic acid [EPA] and DHA) for 12 weeks increased the percentage of live sperm during the supplementation period and improved the motility and progressive motility in fresh but not in frozen sperm (Gholami et al., 2010). After feeding of more PUFA, elevated LPO of sperm and blood were noticed (Surai et al., 2000a; Castellini et al., 2003). Therefore, it has been recommended that the dietary supplementation of PUFAs has to comprise additional antioxidants (Surai et al., 2000a; Zanini et al., 2003; Cerolini et al., 2005). A recent study by Adeel et al. (2009) showed that the feeding of sunflower oil and sunflower seed (rich in PUFA) improves motility and plasma membrane integrity of post-thawed buffalo sperm. However, until now there is no evidence for the transport of PUFAs from the diet to spermatozoa and its effect on seminal levels of antioxidants in Holstein Friesian bulls. Thus, the aim of the present study was to characterize the effects of feeding alpha-linolenic (ALA) acid on fatty acid composition, sperm quality and antioxidative capacity of seminal plasma in Holstein Friesian bulls.

2. Material and methods 2.1. Bulls Two consecutive experimental periods (P1 and P2) were carried out with the same fat supplementations. There were twenty bulls in both experimental periods (18 Holstein Friesian bulls and 2 Red Holstein bulls). The first experimental period (P1; n = 10) was conducted between August and October 2007 and the second period (P2; n = 10) was carried out between December 2007 and March 2008. The bulls were housed in an AI station (Masterrind GmbH; Verden, Germany). Two bulls from the first treatment period and one bull from the second treatment period had to be excluded due to general illness not related to fat supplementation. Nine bulls in the ALA group were 3.2 ± 1.0 years and bulls in the PA group were 3.7 ± 0.8 years old. 2.2. Dietary supplementation of bulls The bulls in both groups were fed with a basal diet consisting of hay-silage (15 kg/day), concentrate and 300 g/day of a mineral feed. The basal ration of the ALA group was supplemented with 800 g coated alpha-linolenic acid per day (net alpha-linolenic acid = 400 g). The fat supplement contained at least 70% PUFA (oleic acid: 10–22%, linoleic acid: 12–18%, alpha linolenic acid: 56–71%). The ration of the PA group was supplemented with 400 g palmitic acid to achieve the same energy density for both groups. This supplement was a fractioned palm fat with a high percentage of palmitic acid (palmitic acid: minimum 75%, stearic acid: minimum 10%, myristic acid: ca. 1.5%, oleic acid: ca. 10%, linoleic acid: ca. 2%, eicosanoic acid (C20:0): ca. 0.5%). Both fat supplements were fed in two equal portions twice a day. 2.3. Semen collection, dilution and freezing Semen was collected twice a week for 16 weeks (4 weeks before and 12 weeks after the start of the dietary treatment) using an artificial vagina. Concentration (Z2TM COULTER COUNTER® Cell and Particle Counter, Beckman Coulter GmbH, Krefeld, Germany), volume and total sperm number of each ejaculate were determined directly after collection. Progressive motility was estimated using microscopy at 37 ◦ C at 200× magnification. A portion of each ejaculate with a total sperm number of 450 × 106 was diluted to a final concentration of 60 × 106 cells/mL using a Tris-egg yolk based extender. Five milliliters of each ejaculate were kept undiluted for the separation of seminal plasma. After the dilution of ejaculates at 20 ◦ C, they were packaged in 30 French straws. Ten straws were used for sperm analyses before cooling and after cryopreservation. Twenty straws were cooled slowly to 5 ◦ C over a period of 180 min and frozen to −110 ◦ C within 390 s on racks in a freezer (Model K, Hede Nielsen, Horsens, Denmark). Frozen samples were placed directly into liquid nitrogen and stored at least 24 h until analysis.


2.4. Fatty acid extraction and analysis Fatty acid composition of sperm was determined in shock frozen sperm at trial weeks 1 and 16. After the collection, the ejaculates were centrifuged (2500 × g, 10 min, 4 ◦ C) to separate sperm from the seminal plasma. The sperm pellets were stored (nine months) until analysis at −20 ◦ C. The extraction of lipids from sperm was performed with chloroform/methanol (1:2, v/v) according to Bligh and Dyer (1959). After extraction, fatty acids were separated in form of their methylester derivatives using the method described by Lepage and Roy (1986). The identification of fatty acid methyl esters was performed by gas chromatography on a capillary column (Zebron ZB-WAX plus; Phenomenex Inc, Torrance, CA, USA) 30 mm × 0.25 mm (0.25 ␮m film thickness). The separation of fatty acids was carried out using the following temperature program: 120 ◦ C for 2 min, increase to 190 ◦ C (25 ◦ C/min) and to 250 ◦ C (10 ◦ C/min). The latter temperature was kept for 30 min. Peak values were detected by flame ionization. The percentage of fatty acids was calculated by comparison of the total fatty acid peak area with that of the fatty acid standard (heptadecanoic acid). 2.5. Handling of seminal plasma and measurement of TAC, GPx and SOD Each ejaculate (5 mL) was centrifuged at 3000 × g for 10 min at 4 ◦ C and the supernatant was stored at −20 ◦ C at least 24 h until analysis. Samples of seminal plasma were measured within 6 months. Before measurement, frozen samples of seminal plasma were thawed at room temperature and diluted 100-fold with phosphate buffer solution (PBS; 50 mM/l, pH 7.4) and immediately assessed for total antioxidant capacity (TAC) and glutathione peroxidase (GPx) activity. The samples were diluted further 10-fold with PBS for determination of superoxide dismutase (SOD). TAC, GPx and SOD were measured from samples once before feeding (4 weeks) and in the last four weeks of feeding (from 9 to 12 weeks of feeding). 2.5.1. Instrumentation and automated measurement of TAC The TAC-peroxyl radical assay (Kollettis et al., 1999) was modified for use in an automated 96-well microplate reader and was performed via a fully automatic multiple pipetting system (Tecan Genesis AWS 200/8, Tecan AG, Switzerland). This system automates the pipetting, providing accurate and consistent intervals between pipetting and measuring, as well as rapid sample evaluations. In the kinetic mode of the microplate reader, 30 ␮L HRP and 30 ␮L signal reagent were added to 230 ␮L of PBS in a disposable 96-well microplate. The microplate was measured for 240 s in order to equilibrate the desired amount of chemiluminescence output to 50,000 relative chemiluminescence units (RLU). After the equilibration measurement, 10 ␮L of diluted seminal plasma was added to the signal reagent and the chemiluminescence was measured. Ten microliter of Trolox were added simultaneously as a standard solution at concentrations between 3 and 75 ␮M for the

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calibration. The pipetting was conducted with four needles of the robotic system; these needles were consistently washed after each pipetting procedure. Therefore, there was a consistent interval of 24 s between each four wells of the disposable microplate. Because the suppression of chemiluminescence started immediately after the addition of samples or trolox, this time difference was calculated for each of the four wells and added to the recovery time of chemiluminescence. Four repeated measurements were performed for each sample and the mean value was calculated. Results were expressed in ␮M of Trolox equivalents. 2.5.2. Superoxide dismutase (SOD) assay The SOD-like activity of seminal plasma was determined as described by Ewing and Janero (1995). For this assay, a working solution in PBS containing 0.1 mM ethylene-diamine-tetra-acetic-acid (EDTA), 62 ␮M nitro blue tetrazolium, 78 ␮M nicotinamide adenine dinucleotide hydrogen (NADH) was prepared immediately before use. The seminal plasma, diluted 1000-fold, was pipetted into a 96-well-microplate containing 200 ␮L working solution. A standard curve was constructed using SOD (SOD from bovine liver, 4800 U/mg). The reaction was initiated by adding 25 ␮L phenazine methosulfate (PMS) solution (33 ␮M PMS, prepared in PBS). Addition of PMS was conducted in the microplate reader. Change of the probe without any sample solution was used as a blank control. The absorbance was measured at 560 nm. The concentration of solutions in the microplate were 50 mM PBS containing 0.1 mM EDTA, 50 ␮M NBT, 78 ␮M NADH, and 3.3 ␮M PMS (final concentrations in 250 ␮L total volume). 2.5.3. Glutathione peroxidase (GPx) assay Total GPx activity was determined by using an indirect coupled method of Günzler and Flohe (1985). The method was modified for the use in an automated pipetting system. The assay mixture contained 150 ␮L potassium phosphate buffer (0.1 M, pH 7.0; 1 mM EDTA), 25 ␮L sample, 25 ␮L glutathione reductase (GR; 2.4 U/mL in 0.1 M PBS pH 7.0) and Glutathione (GSH; 10 mM in distillated water). This mixture was pre-incubated at 37 ◦ C for 10 min. Twenty five microliter of nicotinamide adenine dinucleotide phosphate (NADPH; 1.5 mM in 0.1% NaHCO3 solution) were added to the mixture. The hydroperoxide-independent NADPH oxidation was monitored during 3 min by the microplate reader at 340 nm. Thereafter, 25 ␮L t-butyl hydroperoxide (TBHP; 12 mM) were added. The change in absorbance of the reaction mixture was determined during 5 min at 340 min. The oxidation of NADPH to NADP+ was accompanied by a decrease in absorbance. The rate of the decrease in absorbance is directly proportional to GPx activity in the sample. Units of enzyme activity were calculated using the extinction coefficient of 6.22 mM−1 cm−1 for NADPH. 2.6. Flow cytometric analyses Flow cytometry was performed with an Epics XL-MLC flow cytometer (Beckman Coulter, Fullerton, California, USA). Data were analyzed using EXPO32 ADC XL 4 ColorTM software (Beckman Coulter, Fullerton, California, USA). All measurements for plasma membrane and acrosome

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integrity and LPO were done before cooling and immediately after thawing. In addition, LPO was evaluated after 3 h incubation at 37 ◦ C. Sperm DNA integrity was quantified by using the sperm chromatin structure assay (SCSATM ) on cryopreserved samples. Straws were thawed by placing them in a 37 ◦ C water bath for 30 s. Sperm samples were diluted to a concentration of 5 × 106 sperm/mL with Tyrode’s medium (100 mM NaCl, 3.1 mM KCl, 2.0 mM CaCl2 , 0.4 mM MgCl2 , 0.3 mM Na2 HPO4 , 1 mM Na-pyruvate, 21.6 mM Na-lactate, 24.9 mM NaHCO3 , 10 mM Hepes, 0.05 g/L penicillin, 0.5 g/L PVA, 0.05 g/L PVP; pH: 7.4; 320 mOsm) for analyses by using FITC-PNA/PI and BODIPY assays. The green fluorescence emissions of FITC-PNA (fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin), C-11 BODIPY581/591 (4,4-Difluoro-5-(4-phenyl-1,3butadienyl)-4-bora-3a,4adiaza-s-inda-cene-3-undecanoic acid) and acridine orange (AO) were collected by a 530/30 nm filter (FL1) and the red fluorescence of AO and PI (propidium iodid) by a 650 nm long pass filter (FL3). After FITC-PNA/PI and LPO measurements, the collected data for 10,000 events were stored as list mode files and analyzed with EXPO 32 AnalysisTM software. AO was used for SCSA analysis as described by Evenson and Jost (2000a). Data handling for SCSATM was performed with the Data Analysis Software DAS Version 4.19 (Beisker, 1994). 2.6.1. Plasma membrane and acrosome integrity The percentage plasma membrane and acrosome intact sperm (PMAI) was evaluated by FITC-PNA/PI assay (Fischer et al., 2010). Five microliters of FITC-PNA (100 ␮g/mL) and 3 ␮L PI (2.99 mM) were added to 492 ␮L of diluted sperm suspension in Tyrode’s medium. This procedure was carried out on extended sperm before and after cryopreservation. Sperm samples were incubated at 37 ◦ C for 15 min and remixed just before measurement. 2.6.2. Lipid peroxidation (LPO) Lipid peroxidation was evaluated on diluted sperm before cooling and on thawed sperm using C11BODIPY581/591 as described previously (Aitken et al., 2007; Neild et al., 2005). Bodipy was added to the sperm suspension at a final concentration of 5 ␮M. PI was added to sperm suspensions (at a final concentration of 2.99 ␮M) 15 min prior to being analyzed by using flow cytometry. This procedure was carried out before cryopreservation (LPOb) and 0 h (LPO0 ha) as well as 3 h after thawing (LPO3 ha). Thereafter, sperm samples were mixed and incubated at 37 ◦ C for 15 min and re-mixed just before measurement. PI-positive sperm (dead) were gated out of analyses and the values of LPO were calculated on the live sperm population. 2.6.3. Sperm chromatin structure assay (SCSA) The percentage of sperm showing a high degree of DNA fragmentation (% DFI), DNA fragmentation index (DFI), and the standard deviation of DFI (SD-DFI) were determined by SCSA as described by Evenson and Jost (2000b). Thawed sperm samples were diluted to a concentration of 2 × 106 sperm/mL with TNE buffer (pH 7.4, 0.01 M

Tris–HCl, 0.15 M NaCl, 1 mM EDTA). The sperm suspension (200 ␮L) was treated with 400 ␮L acid-detergent solution (pH 1.2, 0.08 N HCl, 0.15 M NaCl, 0.1% Triton X-100) for 30 s, and then stained with 1.2 mL (6 mg/L) purified AO in a phosphate–citrate buffer (0.2 M Na2 HPO4 , 0.1 M citric acid, 0.15 M NaCl, 1 mM EDTA, pH 6.0). Samples were examined after 3 min of incubation. Each sample was examined twice and mean values were used for further analyses.

2.7. Statistical analysis Statistical analyses were carried out using the software packages StatView 5.0 and SAS (SAS Institute Inc., Cary, NC, USA). For the statistical analyses of time dependent changes and differences between both animal groups in sperm quality during the study period, for all sperm quality parameters mean values each out of four weeks were calculated. Levels of antioxidants were investigated once before feeding and last four weeks of feeding. Normal distribution of data was proven by Kolmogorov–Smirnov test. Values of antioxidants and sperm quality parameters were expressed as means ± standard deviations (SD). Means of percentages of fatty acids from two ejaculates at trial weeks 1 and 16 were calculated. One-way analysis of variance (ANOVA) followed by Fisher’s LSD test (least significant difference test) was conducted to investigate the effects of diets on sperm quality, antioxidant levels and fatty acids compositions. From the data of the pre-feeding period, Pearson’s correlation coefficients were calculated to compare the mean values of antioxidants in seminal plasma (TAC, GPx, and SOD) and the percentages of fatty acids in sperm membranes. P values ≤0.05 were considered as significant and 0.05 < P ≤ 0.01 was considered as tendencial.

3. Results 3.1. Content of fatty acids in sperm membrane Before feeding of bulls, DHA was the unique detectable omega-3 fatty acid in the plasma membrane of sperm with a portion of 40.1% of all fatty acids. The portions of the saturated fatty acids palmitic acid (C16:0), myristic acid (C14:0) and stearic acid (C18:0) were 22.4%, 12.1% and 8.6%, respectively. Oleic acid (C18:1 n-9) was the only found monounsaturated fatty acid (6.0%). Linoleic acid (C18:2 n6) accounted for 3.5%. Furthermore, eicosatrienoic (C20:3 n-6) and ecosadienoic acid (C20:2 n-6) accounted for 3.3% and 1.1%, respectively. After feeding with ALA, the DHA level of sperm increased (P < 0.03). No change (P > 0.05) in DHA levels occurred after the supplementation of PO. The same (P > 0.05) alterations in DHA level were detected during both feeding periods, P1 and P2. Therefore, only the averages of the data of the trials P1 and P2 are shown (Table 3). There was a non significant decrease of palmitic acid in ALA bulls (4.2%) and a non significant increase of this fatty acid in PA bulls (4.7%). The feeding of PO led to a higher (P < 0.05) portion of palmitic acid compared to the supplementation of ALA.


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Table 1 Effects of diets containing palmitic acid (PO) or alpha-linolenic acid (ALA) on sperm concentration (concentration), volume of the ejaculate (volume), total sperm number, plasma membrane integrity (PMAI), DNA fragmentation index (DFI), and standard deviation of DNA fragmentation index (SD-DFI) of bovine sperm samples. Measurements of PMAI and LPO were performed before (b) and immediately after thawing (0 ha). In addition, LPO was measured after 3 h incubation at 37 ◦ C (LPO3 ha). The DFI, %DFI and SD-DFI were determined after thawing of samples. Values are means ± SD from 8 consecutive ejaculates (twice weekly for 4 weeks) of PO (n = 8) and ALA (n = 9) bulls before (4 weeks) and during feeding. PO Diet week Concentration [109 /mL] Volume [mL] Total sperm number [109 /mL] PMAIb [%] PMAI0 ha [%] LPOb LPO0 ha LPO3 ha DFI %DFI [%] SD-DFI

ALA

Before

1–4

5–8

9–12

Before

1–4

5–8

9–12

1.5 ± 0.3

1.5 ± 0.3

1.5 ± 0.3

1.5 ± 0.3

1.5 ± 0.3

1.4 ± 0.3

1.4 ± 0.3

1.4 ± 0.3

7.7 ± 1.8 11.3 ± 3.5

7.3 ± 1.7 11.0 ± 3.4

7.9 ± 1.7 11.8 ± 3.7

7.2 ± 1.7 10.4 ± 3.3

7.1 ± 1.4 10.4 ± 2.9

7.4 ± 1.8 10.2 ± 3.4

7.6 ± 1.5 10.6 ± 2.7

7.1 ± 1.7 9.7 ± 3.2

83.6 33.0 2.1 2.7 16.0 200.1 3.2 18.1

± ± ± ± ± ± ± ±

5.0 13.3 2.3 4.7 10.9 6.0 1.8 5.9

85.0 44.1 3.2 3.2 17.8 198.8 3.9 18.5

± ± ± ± ± ± ± ±

4.2 14.9* 2.6* 1.0 5.4 7.6 1.6 3.5

85.3 ± 4.4 42.0 ± 15.0* 1.4 ± 2.1 2.2 ± 3.2 17.3 ± 7.7 198.9 ± .8.0 3.6 ± 1.9 16.8 ± 4.5

84.8 44.8 1.9 2.6 18.0 202.3 4.5 19.2

± ± ± ± ± ± ± ±

4.4 12.0* 1.6 2.2 5.3 6.1 1.9 4.9

79.7 28.6 2.9 2.9 15.6 200.1 3.7 18.5

± ± ± ± ± ± ± ±

8.3 10.5 2.3 4.7 8.9 6.0 1.9 3.5

82.6 39.3 3.4 2.2 19.9 198.8 3.6 18.5

± ± ± ± ± ± ± ±

7.3 13.6* 2.7 2.8 5.0* 8.0 1.7 3.3

83.3 41.0 2.6 2.8 19.5 198.9 3.3 16.7

± ± ± ± ± ± ± ±

6.8 11.8* 1.9 2.4 5.2* 6.8 1.6 3.6

82.6 41.6 2.6 2.8 20.3 202.7 4.6 17.9

± ± ± ± ± ± ± ±

7.4 13.7* 3.9 2.5 4.6* 6.0 2.2 4.1

Values during the feeding period with asterisk (* ) differ (P < 0.05) compared to the values before the feeding period.

3.2. Sperm number and quality Sperm concentration, ejaculate volume, total sperm number of ejaculate did not change (P > 0.05) in ALA- and PA bulls during the experimental period. The PMAIb values stayed constant (P < 0.05) during the study period, while PMAIa increased (P < 0.005) by 11% in ALA bulls and by 9% in PA group during the study period (Table 1). LPO of sperm before cryopreservation didn’t change in the ALA group, but decreased in the PA group (P < 0.01) LPO of sperm directly after thawing showed same pattern as before cryopreservation and did not change during the study period in the ALA group, but decreased in the PA group (P < 0.006). After 3 h of incubation LPO increased in the ALA group (P < 0.02), while LPO did not differ (P > 0.05) between phases within groups and between groups within phases (P > 0.05). The %DFI-, DFI, and SD-DFI values were not affected (P > 0.05) by the feeding of PO and ALA.

3.3. Effect of feeding ALA on antioxidant levels of seminal plasma In both groups the levels of TAC, SOD and GPx did not change (P > 0.05) during the feeding of PO and ALA (P > 0.05). The values of TAC, SOD and GPx did not differ (P > 0.05) between PO - and ALA bulls before as well as during the feeding period (Table 2).

3.4. Correlation between fatty acids and antioxidants There were negative correlations (P < 0.05) between the values of TAC and percentages of palmitic (C16:0) and myristic (C14:0) acid (P < 0.05). Levels of SOD in seminal plasma were in tendency associated (P = 0.09) with DHA (C22:6 (n-3)) in the sperm plasma membrane and GPx was in tendency associated (P = 0.06) with C18:2 (n-6). There were no (P > 0.05) correlations between all other antioxidant levels in seminal plasma and the relative contents

of all other fatty acids in the sperm plasma membrane (Table 3).

4. Discussion In the present study, no alterations in levels of antioxidants in seminal plasma were detected by feeding of PA or ALA. Therefore, we suppose that amounts of antioxidants in the diet of bulls are well-balanced. Previous studies have shown that increasing PUFA amounts in the diet of different species may change antioxidant status of semen (Surai et al., 2000b; Rooke et al., 2001; Castellini et al., 2003; Cerolini et al., 2005; Zaniboni et al., 2006). Castellini et al. (2003) reported in rabbit bucks that supplementation of long chain fatty acids modified the fatty acid profile and concurrently reduced the total antioxidant capacity of seminal plasma determined by using Oxy-adsorbent spectrophotometric assay. They found that supplementation of neither vitamin E nor vitamin C restored oxidative stability of semen in animals showing a rise of oxidative stress after feeding of fatty acids. Improvements of sperm quality were observed only after adding a combination of both antioxidants, vitamin E as well as vitamin C to the diet. Zaniboni et al. (2006) even noticed that fish oil decreased susceptibility to peroxidation of sperm in turkey, but only by feeding adequate vitamin E supplementation. These findings, i.e., positive effects of fat supplementations only by combining it with antioxidants, may be explained by the fact that sperm contain high levels of PUFA. The presence of high levels of PUFA requires efficient antioxidant levels to protect sperm against lipid peroxidation (Aitken and Baker, 2004). In contrast to the above mentioned studies, there are also reports which showed positive effects of fats on sperm quality without additional antioxidant supplementation in stallion (Brinsko et al., 2005) and in chicken (Kelso et al., 1997a). The reason for this contradictory result may be the fact that in the different studies various diets were used, which contained different fatty acids. As the various types of fatty acids show different

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Table 2 Effects of diets containing palmitic acid (PO) or alpha-linolenic acid (ALA) on levels of total antioxidant capacity (TAC = ␮mol/L), glutathione peroxidase (GPx = nmol NADPH oxidized/min/mg of protein) and superoxide dismutase (SOD = U/mL) in bovine seminal plasma. Values are means ± SD from 8 consecutive ejaculates (twice weekly for 4 weeks) of PO (n = 8) and ALA (n = 9) bulls before feeding and after 9 weeks (mean of weeks 9–12) of feeding. PO

TAC GPx SOD

ALA

before feeding

after feeding

before feeding

after feeding

570 ± 284 65.5 ± 20.8 11025 ± 733

503 ± 209 65.6 ± 23.3 10835 ± 810

554 ± 195 66.4 ± 16.9 10616 ± 1084

653 ± 207 64.4 ± 19.0 10769 ± 1504

Table 3 Relationships between antioxidants in seminal plasma (TAC = ␮mol/L, GPx = nmol NADPH oxidized/min/mg of protein and SOD = U/mL) and relative contents of fatty acids in plasma membrane of sperm (n = 17). Fatty acid

C22:6 (n-3)

C22:0

C20:2

C18:2 (n-6)

C18:1 (n-9)

C18:0

C16:0

C14:0

TAC GPx SOD

0.26 0.04 0.42a

−0.42 0.31 −0.03

0.02 0.13 0.39

−0.37 0.47a −0.11

0.35 −0.37 −0.11

0.29 −0.46 −0.10

−0.59* 0.38 −0.39

−0.56* 0.27 −0.36

* a

P < 0.05. 0.05 < P ≤ 0.10.

effects on sperm membranes they require different amounts of antioxidants (Bongalhardo et al., 2009). Furthermore, the above mentioned studies were carried out in different species which show varying antioxidant levels. Bovine seminal plasma contains higher antioxidant levels compared to equine (Ball, 2008) and porcine seminal plasma (Ochsendorf and Fuchs, 1997). Antioxidants in seminal plasma play an essential role to maintain oxidative stability (Surai et al., 1997; Surai et al., 2000a; Agarwal et al., 2005; Kasimanickam et al., 2006). In a recent study comparing antioxidants and fatty acid contents in human sperm, Tavilani et al. (2008) found that there is a negative relationship between SOD activity and levels of palmitic acid in normospermic samples. They showed non-significant positive correlations between SOD activity and DHA levels in sperm which is in accordance with our results. In addition, it is known that the ratio of PUFA/saturated fatty acids in human sperm is an important indicator of normospermic sperm. It was higher than that in asthenozoospermic sperm (Tavilani et al., 2006). In our study, there were significant negative correlations between TAC levels in bull seminal plasma and levels of saturated fatty acids (SFA; palmitic and myristic acid) of sperm. Phospholipids of mammalian sperm are important substrates for respiration and particularly palmitic and myristic acid within phospholipids can be oxidized for energy production (Hartree and Mann, 1961; Scott and Dawson, 1968). We assume that the antioxidants of seminal plasma are not used for the protection of SFA in sperm membrane. There are studies on many species including stallion (Brinsko et al., 2005), human (Conquer et al., 2000), fowl (Blesbois et al., 2004), boars (Rooke et al., 2001) and rabbit (Castellini et al., 2003) showing positive effects of feeding PUFAs on sperm quality, but until now the mechanism how long chain PUFAs affect sperm quality is not quite clear (Cerolini et al., 2005; Kelso et al., 1997a, Surai, 2000b). Our report is the first to show that the supplementation of ALA increases the DHA content in bull sperm. However, there was no specific effect of DHA on sperm quality before cryopreservation as well as after thawing. This finding is in accordance with those of Paulenz et al. (1999) who showed in boars that dietary supplementation

of n-3 PUFAs by feeding of cod liver oil increased the DHA in sperm membranes but did not improve the tolerance to cold shock and freezing. In contrast, Rooke et al. (2001) observed an increase of boar sperm quality after feeding tuna oil. Similarly, Brinsko et al. (2005) noticed that feeding stallions DHA rich diets would improve freezeability of sperm. It is noteworthy here to mention that, content of PUFA and levels of antioxidants differ between species. Bovine semen shows a higher DHA content (Parks and Lynch, 1992) and higher GPx (Bilodeau et al., 2000) levels compared to equine (Saaranen et al., 1989; Ball et al., 2000) and porcine semen (Saaranen et al., 1989). The latter two species have higher levels of docosapentaenoic acid [DPA; 20:5 (n-6)] (Parks and Lynch, 1992) in sperm and lower GPx levels in seminal plasma compared to bulls. Such a species difference in content of fatty acids and antioxidants could be a reason for different effects of PUFA supplementation on sperm quality. One limitation of our study was the lack of a control group without any fat supplementation. On the other hand, we carried out the same fat supplementations at two different times and achieved the same results. The interesting findings of this study were the positive effects of both fat sources on post thawing sperm plasma membraneand acrosomal integrity. Diets enriched with n-3 or n-6 fatty acids affected sperm production in different species like in chicken (Surai et al., 2000b) or in stallion (Brinsko et al., 2005). Many of them, for example in stallion (Brinsko et al., 2005) and boar (Rooke and Speake, 2001), resulted in an improvement of sperm quality (Surai et al., 2000b; Rooke and Speake, 2001). However, discordant results have been reported (Paulenz et al., 1999). In summary, the content of antioxidants in seminal plasma was not affected by the additional feeding of fatty acids, neither in saturated nor polyunsaturated forms. However, both types of fatty acids led to an increase of sperm quality after cryopreservation. References Adeel, M., Ijaz, A., Aleem, M., Rehman, H., Yousaf, M.S., Jabbar, M.A., 2009. Improvement of liquid and frozen-thawed semen quality of Nili-Ravi

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