Effect of organic selenium on Turkey semen

Animal Reproduction Science 100(2007)311–317Effect of organic selenium on Turkey semenquality during liquid storageS.G.Dimitrov a ,V .K.Atanasov a ,∗,P.F.Surai b ,S.A.Denev aaTrakia University,6000Stara Zagora,Bulgaria b Scottish Agricultural College,Edinburgh,Scotland,UK Received 29March 2006;received in revised form 17July 2006;accepted 20July 2006Available online 28August 2006AbstractThe aim of the present study was to evaluate the effect of dietary organic selenium on the turkey semen during storage.Twenty males (BUT,Big 6,40weeks of age)were divided into control (n =10)and experi-mental group (n =10).The turkeys in the both groups were fed with a commercial diet containing 0.1ppm Se in the form of sodium selenite.The experimental birds were additionally supplied with 0.3ppm organic Se in the form Sel-Plex TM (Alltech,Inc.).After 30days of feeding,the semen samples were collected twice a week for the 3weeks of the study and diluted 1+1(v/v)with TUR-2diluent,and stored in a water bath (+10to 15◦C)for 6h.The percentage of motile spermatozoa,the sperm viability (live/dead spermatozoa),total lipids,phospholipids and total cholesterol were assessed in fresh and stored semen.The fertilizing ability of semen was assessed by artificial insemination of 30hens per group with dose containing 200×106spermatozoa weekly.After 6h of semen storage,the motility of spermatozoa decreased significantly in the control group (by 8.7relative percent,P <0.05)and only by four relative percent (P >0.05)in experimental group reflecting a protective effect of dietary Se supplementation.The proportion of live spermatozoa was higher in fresh semen and significantly lower in stored semen.The positive effect of Se supplementation was observed on the lipid composition of stored semen:the concentration of the total lipids and phospho-lipids in the seminal plasma from control group significantly increased,while in the experimental group remained constant.Better semen integrity in the experimental group was associated with an improved fer-tilizing ability of spermatozoa:the fertility rate of stored spermatozoa in the control group was 88%,while in the experimental group was 90.5%.©2006Elsevier B.V .All rights reserved.Keywords:Turkeys;Selenium;Semen;Storage∗Corresponding author.Tel.:+35942699313;fax:+35942670942.E-mail address:vka@mail.bg (V .K.Atanasov).0378-4320/$–see front matter ©2006Elsevier B.V .All rights reserved.doi:10.1016/j.anireprosci.2006.07.007312S.G.Dimitrov et al./Animal Reproduction Science100(2007)311–3171.IntroductionSelenium is an essential element playing an important role in animal reproduction(Surai,2002). Indeed there are at least25selenoproteins in human and animal body(Kryukov et al.,2003).Those selenoproteins participate in regulation of various physiological functions including antioxidant protection,redox regulation of gene expression,thyroid metabolism and sperm structure integrity maintenance.In fact,recently it has been shown that sperm capsular selenoprotein which is known for many years to play an important structural role in spermatozoa to be a form of glutathione peroxidase(GSH-Px),which was converted from enzymatic role to structural part of spermatozoa.Data on the effect of Se on avian semen are extremely limited.In fact,GSH-Px has been found to be expressed in chicken seminal plasma and spermatozoa(Surai et al.,1998,1998b).There are species-specific differences in activity and distribution of GSH-Px in avian semen.For example, in seminal plasma total GSH-Px activity was the highest in turkey and lowest in duck and goose (Surai et al.,1998).In spermatozoa,on the other hand,the highest GSH-Px activities were found for goose and duck and much lower GSH-Px activity was recorded for guinea fowl,turkey or chicken.If selenium is limiting in the diet(which is the case in many countries in the world),then dietary supplementation of this trace element should have a beneficial effect on the antioxidant defense in various tissues including sperm.This was confirmed in our studies.Inclusion of Se in the diet of male chickens significantly increased Se-GSH-Px activity in the liver,testes,spermatozoa and seminal plasma(Surai et al.,1998c).As a result,a significant decrease in the sperm’s and tissue susceptibility to lipid peroxidation was observed.This protective effect was more expressed in stored semen as compared to fresh.In this respect,it is extremely important that an inducible form of the enzyme(Se-GSH-Px)represents more than75%of the total enzymatic activity in chicken spermatozoa and more than60%in the testes and liver of cockerels.The aim of the present study was to evaluate the effect of dietary organic selenium on the turkey semen during storage.2.Materials and methods2.1.BirdsTwenty males(BUT,Big6,40weeks of age)were divided into control(n=10)and experimental group(n=10).The turkeys in the both groups were fed with diet containing12.5–13.4MJ and 10%crude protein and0.1ppm Se in the form of sodium selenite.The experimental birds were additionally supplied with0.3ppm organic Se in the form Sel-Plex TM(Alltech,Inc.).2.2.Semen collection and treatmentAfter30days of feeding,the semen was collected twice a week for the3weeks of the study by abdominal massage(Burrows and Quin,1937).Pooled semen samples were diluted1+1(v/v) with TUR-2diluent(Dimitrov,1997).The diluted semen was stored in a water bath(+10to15◦C) for6h in still position.2.3.Semen quality testThe percentage of motile spermatozoa was evaluated subjectively at0and6h in semen diluted in TUR-2under brightfield microscopy using the“hanging drop”method(Wishart and Wilson, 1997)on a scale ranging from0to100%.S.G.Dimitrov et al./Animal Reproduction Science100(2007)311–317313 The viability(live/dead spermatozoa)was performed using eosin–nigrosin smears(Bakst and Cecil,1997)under light microscope.2.4.Lipid analysesThe spermatozoa were separated from seminal plasma by centrifugation(500×g for10min). The supernatant was centrifuged again under same conditions,giving a second supernatant,which was then centrifuged at3000×g for30min.Thefinal supernatant was considered“seminal plasma”and frozen(−20◦C)until further analyses(Blesbois et al.,1997).After rapid thawing the obtained supernatants were used for the colourimetric determination of the total lipids(Zollner and Kirsch,1962),total phospholipids(Kates,1970)and total cholesterol (Watson,1960).2.5.Artificial inseminationThe fertilizing ability of semen was assessed by artificial insemination of30hens per group for three successive weeks(total609eggs)with dose containing200×106spermatozoa weekly.The fertility rate was determinated by egg candling after7days of incubation.2.6.Statistical analysesThe data were statistically analyzed by Student’s t-test(Stat.Soft.Inc.,1994).3.ResultsThe results indicate that before storage semen quality was similar in control and experimental groups(Table1).However,after6h of storage,the motility of spermatozoa decreased significantly in the control group(by8.7relative percent,P<0.05)and only by3.95relative percent(P>0.05) in experimental group reflecting a protective effect of dietary Se supplementation.As one could expect,the proportion of live spermatozoa was higher in fresh semen and significantly lower in stored semen.As a result the percentage of dead spermatozoa was lower in fresh semen and sig-nificantly increased after storage.The positive effect of Se supplementation was observed on the lipid composition of stored semen(Fig.1).In fact,during in vitro storage,the concentration of the total lipids,phospholipids and cholesterol in the seminal plasma from control group significantly increased(by36.30mg%,8.93mg%and0.268mmol/l,respectively,P<0.001)(Figs.2and3). The protective effect of Se was associated with a decreased release of total lipids and phospho-lipids from spermatozoa to seminal plasma during semen storage.But the level increase of these Table1Parameters of fresh and liquid storage turkey sperm(mean±S.E.)Sperm parameters Control group Experimental group0h6h0h6hMotility(%)71.55±2.5A65.32±1.5B71.73±7.069.01±2.8 Live spermatozoa(%)77.56±4.4A68.86±5.4B77.66±4.7A69.04±3.6B Dead spermatozoa(%)23.4±3.9A31.14±2.1B22.34±3.3A30.96±2.3B Different alphabets within row indicate a statistical differences between means(P<0.05).314S.G.Dimitrov et al./Animal Reproduction Science100(2007)311–317Fig.1.Total lipids levels in seminal plasma of fresh(0h)and stored(6h)turkey semen.***P<0.001.Fig.2.Phospholipids levels in seminal plasma of fresh(0h)and stored(6h)turkey semen.***P<0.001.Fig.3.Cholesterol levels in seminal plasma of fresh(0h)and stored(6h)turkey semen.***P<0.001.S.G.Dimitrov et al./Animal Reproduction Science100(2007)311–317315Fig.4.Fertility after artificial insemination of hens with diluted and stored(+10to15◦C/6h)turkey semen. parameters in seminal plasma of the experimental group was not significant(P>0.05).Better semen integrity in the experimental group was associated with an improved fertilizing ability of spermatozoa.Indeed,the fertility rate of stored spermatozoa in the control group was88%,while in the experimental group was90.5%(Fig.4).4.DiscussionThe mainfinding of this work is a protective effect of Se dietary supplementation on the turkey semen quality during storage.Indeed,semen obtained from turkey males supplemented with organic selenium was characterised better resistance to storage.This was reflected by better motility after semen storage.Furthermore,on the biochemical level,protective effect of Se was related to decrease release of total lipids and phospholipids from spermatozoa to seminal plasma during semen storage.Indeed,lipid peroxidation is considered as an important factor decreasing semen quality during storage(Surai,2002).In fact,investigation of lipid peroxidation in turkey semen was carried out in Ukraine(Surai,1983,1984).In particular an induced(by Fe2+)lipid peroxidation in turkey semen was used to assess semen susceptibility to lipid peroxidation,since the initial level of peroxides in fresh or even stored semen was shown to be comparatively low.This work was further developed by Cecil and Bakst(1993)who showed that during aerobic storage of turkey spermatozoa,lipid peroxidation was time and temperature-dependent.The authors also suggested that turkey spermatozoa are more sensitive to lipid peroxidation than semen from other species.It seems likely that lipid peroxidation in semen is also age-dependent.For example,Donoghue and Donoghue(1997)reported that MDA concentrations were10-fold higher in semen from older turkey males(56weeks of age)than for younger ones(30weeks of age).Furthermore in vitro storage did not cause lipid peroxidation in sperm obtained during thefirst half of the reproductive period but MDA levels significantly increased in sperm obtained during the second half of this period(Douard et al.,2003).The molecular mechanisms of lipid peroxidation in avian semen have received little attention. Recently it has been shown that during sperm storage,lipid peroxidation is associated with a significant decrease in PUFA concentration in spermatozoa.In particular,the main PUFA in the chicken semen(22:4n−6)was most susceptible to peroxidation.Its proportion in the phospholipid fraction was significantly decreased as a result of incubation of chicken sperm for12h at20◦C (Surai et al.,1998b).The inclusion of a promoter of lipid peroxidation(Fe2+)in the incubation316S.G.Dimitrov et al./Animal Reproduction Science100(2007)311–317medium further increased the rate of lipid peroxidation,significantly decreasing the proportions of not only22:4n−6,but also of20:4n−6,22:5n−3and22:6n−3in the phospholipid frac-tion of the spermatozoa.The confirmation of the suggestion that the loss of PUFA was due to peroxidation came from the data showing simultaneous accumulation of TBARS in the semen (Surai et al.,1998b).Recently,it has been shown that the total lipid content,the proportion of total phospholipids,and the levels of phosphatidylcholine(PC),phosphatidylethanolamine(PE) and sphingomyelin(Sph)were significantly decreased in chicken semen during in vitro storage and this was associated with a reduction in the proportion of motile,viable and morphologically normal cells(Blesbois et al.,1999).Similarly,in turkey spermatozoa incubated at37◦C in the presence of exogenous Fe2+,a significant decrease in PS(by47%)and PE(by35%),the two most unsaturated fractions of avian spermatozoa,was observed(Surai et al.,1998a;Maldjian et al.,1998).Storage of diluted turkey semen for48h at4◦C was also associated with a decrease in total phospholipid content and of PC and,to lesser extent,of Sph,phosphatidylserine(PS)and phosphatidylinositol(PI)(Douard et al.,2000).It is interesting to note that H2O2and organic hydroperoxides had toxic effects on avian sperm motility(Surai et al.,1998b).Recently,it has been shown that inclusion of organic selenium in the form of Sel-Plex into the cockerel diet was associated with a significant two-fold increase Se concentration in the semen(Pappas et al.,2005).Selenium was mainly concentrated in spermatozoa.Furthermore, inclusion of Se into the cockerel diet was associated with increased GSH-Px activity and decreased spermatozoa susceptibility to lipid peroxidation(Surai et al.,1998).In fact,replacing sodium selenite in the diet of cockerels by Sel-Plex was shown to have a beneficial effect in terms of fertility.In particular,Edens(2002)showed that,when cockerels were fed on a basal diet containing0.28ppm Se without additional dietary supplementation of this trace element,the percentage of normal spermatozoa was only57.9%and two major abnormalities seen were bent midpiece(18.7%)and corkscrew head(15.4%).When this diet was supplemented with an additional0.2ppm Se in the form of selenite,the percentage of normal spermatozoa increased to89.4%and abnormalities in the form of bent midpiece and corkscrew head were decreased down to6.2and1.8%respectively.However,when organic selenium was included in the cockerel’s diet in the same amount,semen quality was further improved and those abnormalities decreased down to0.7and0.2%and the percentage of normal spermatozoa increased up to98.7%.These results clearly showed that the form of dietary Se supplementation is a crucial factor of its efficiency,with organic selenium being much more effective in comparison to selenite.Therefore Se deficiency is associated with midpiece damage to spermatozoa(Surai, 2002).It is clear that the midpiece of spermatozoa of the Se-deficient male is broken.In such conditions sperm motility and fertilising capacity would be compromised.Additional data from Edens(2002)indicated that selenomethionine(0.3ppm from21weeks of age)in the diet of Hubbard roosters improved semen quality to a greater extent than achieved by selenite at the same dose.The sperm quality index significantly increased as well as the percentage of normal spermatozoa.At the same time,the proportions of various abnormalities in semen decreased. There was also a positive effect on fertility,which was improved by0.56–1.03%by Sel-Plex dietary supplementation.These experimental results confirmed the importance of selenium in maintaining chicken semen quality and specifically showed the advantages of organic selenium. 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