AbstractThe aim of this work was to evaluate whether the residual amount of γ-glutamyl-transferase (GGT) and alkaline phosphatase (ALP) in bovine sperm after freezing/thawing is correlated with fertility parameters, including blastocyst rates after in vitro fertilisation (IVF). The enzyme activities were determined in both spermatozoa and supernatant after centrifugation. While ALP was only correlated with sperm viability, GGT activity was correlated with sperm motility (rs = .4; p < .05) both in sperm and supernatant. Interestingly, GGT activity was also correlated with cleavage (rs = .5; p < .05 and .8; p < .01, for sperm and supernatant respectively) and blastocyst (rs = .6 and .9, for sperm and supernatant respectively; p < .01) rates obtained after IVF. These results suggest that GGT could play an important role in the protection of sperm against oxidative stress and could be considered a reliable marker to assess frozen/thawed sperm quality in bovine

Bianca Gasparrini

Francesca Ciani

Giuseppe Vassalotti

Lucia Boccia

Luigi Zicarelli

Maria Elena Pero

Pietro Lombardi

Valentina Longobardi

Italian Journal of Animal Science

English

The aim of this work was to evaluate whether the residual amount of γ-glutamyl-transferase (GGT) and alkaline phosphatase (ALP) in bovine sperm after freezing/thawing is correlated with fertility parameters, including blastocyst rates after in vitro fertilisation (IVF). The enzyme activities were determined in both spermatozoa and supernatant after centrifugation. While ALP was only correlated with sperm viability, GGT activity was correlated with sperm motility (rs = .4; p < .05) both in sperm and supernatant. Interestingly, GGT activity was also correlated with cleavage (rs = .5; p < .05 and .8; p < .01, for sperm and supernatant respectively) and blastocyst (rs = .6 and .9, for sperm and supernatant respectively; p < .01) rates obtained after IVF. These results suggest that GGT could play an important role in the protection of sperm against oxidative stress and could be considered a reliable marker to assess frozen/thawed sperm quality in bovine

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Influence of γ-glutamyltransferase and alkaline phosphatase activity on in vitro fertilisation of bovine frozen/thawed semen

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Influence of γ-glutamyltransferase and alkaline phosphatase activity on <i>in vitro</i> fertilisation of bovine frozen/thawed semen

PERO, MARIA ELENA

LOMBARDI, PIETRO

LONGOBARDI, VALENTINA

BOCCIA, LUCIA

VASSALOTTI, GIUSEPPE

ZICARELLI, LUIGI

CIANI, FRANCESCA

GASPARRINI, BIANCA

Archivio della ricerca - Università degli studi di Napoli Federico II

Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=tjas20Download by: [Cornell University Library] Date: 05 July 2017, At: 01:21Italian Journal of Animal ScienceISSN: (Print) 1828-051X (Online) Journal homepage: http://www.tandfonline.com/loi/tjas20Influence of γ-glutamyltransferase and alkalinephosphatase activity on in vitro fertilisation ofbovine frozen/thawed semenMaria Elena Pero, Pietro Lombardi, Valentina Longobardi, Lucia Boccia,Giuseppe Vassalotti, Luigi Zicarelli, Francesca Ciani & Bianca GasparriniTo cite this article: Maria Elena Pero, Pietro Lombardi, Valentina Longobardi, Lucia Boccia,Giuseppe Vassalotti, Luigi Zicarelli, Francesca Ciani & Bianca Gasparrini (2017): Influence ofγ-glutamyltransferase and alkaline phosphatase activity on in vitro fertilisation of bovine frozen/thawed semen, Italian Journal of Animal Science, DOI: 10.1080/1828051X.2017.1290509To link to this article:  http://dx.doi.org/10.1080/1828051X.2017.1290509© 2017 The Author(s). Published by InformaUK Limited, trading as Taylor & FrancisGroup.Published online: 22 Feb 2017.Submit your article to this journal Article views: 107View related articles View Crossmark dataSHORT COMMUNICATIONInfluence of c-glutamyltransferase and alkaline phosphatase activity onin vitro fertilisation of bovine frozen/thawed semenMaria Elena Pero, Pietro Lombardi, Valentina Longobardi , Lucia Boccia, Giuseppe Vassalotti,Luigi Zicarelli, Francesca Ciani and Bianca GasparriniDipartimento di Medicina Veterinaria e Produzioni Animali, University of Naples Federico II, Napoli, ItalyABSTRACTThe aim of this work was to evaluate whether the residual amount of c-glutamyl-transferase(GGT) and alkaline phosphatase (ALP) in bovine sperm after freezing/thawing is correlated withfertility parameters, including blastocyst rates after in vitro fertilisation (IVF). The enzyme activitieswere determined in both spermatozoa and supernatant after centrifugation. While ALP was onlycorrelated with sperm viability, GGT activity was correlated with sperm motility (rs¼ .4; p< .05)both in sperm and supernatant. Interestingly, GGT activity was also correlated with cleavage(rs¼ .5; p< .05 and .8; p< .01, for sperm and supernatant respectively) and blastocyst (rs¼ .6 and.9, for sperm and supernatant respectively; p< .01) rates obtained after IVF. These results suggestthat GGT could play an important role in the protection of sperm against oxidative stress andcould be considered a reliable marker to assess frozen/thawed sperm quality in bovine.ARTICLE HISTORYReceived 15 November 2016Revised 16 January 2017Accepted 19 January 2017KEYWORDSEnzyme determination;fertility test; in vitrofertilisationIntroductionThe great variability in fertility among bulls stronglyaffects the efficiency of Artificial Insemination andin vitro embryo production. Laboratory tests, routinelyperformed in a Semen Collection Center, do notenable the determination a priori of fertility. A moreaccurate estimation of semen fertility is currentlyachieved by increasing the number of tests performedin the laboratory. The identification of male fertilitymarkers is, therefore, fundamental to improve theselection of males for breeding.In cattle, fertility-associated proteins such as osteo-pontin (Cancel et al. 1997) and lipocalin-type prosta-glandin (PG) D synthase (Gerena et al. 1998) havebeen identified. It has been reported that lacticdehydrogenase may represent an accurate marker forsemen quality in stallions (Pesch et al. 2006).Furthermore, older studies showed that alkaline phos-phatase is suitable for differentiation of oligo- andazoospermia in stallions (Turner & McDonnell 2003).In standard in vitro fertilisation (IVF) procedure fro-zen-thawed sperm is commonly used but it is knownthat freezing may determine several types of spermdamages including enzyme leakage. Pace andGraham (1970) demonstrated that the residual amountof glutamic oxaloacetic transaminase (GOT) after freez-ing was correlated with in vivo bull fertility. To ourknowledge, no information are available about theinfluence of residual concentrations of gamma glu-tamyl transferase (GGT) and alkaline phosphatase (ALP)after freezing/thawing and bovine semen fertility.Therefore, the aim of this work was to investigatewhether the concentration of GGT and ALP in frozen/thawed bovine sperm is correlated to various fertilityparameters, including the in vitro fertilising capability,evaluated in terms of cleavage and blastocyst ratesafter IVF.Materials and methodsThe study was carried out on frozen/thawed semenfrom eight adult fertile bulls (three ejaculates/bull)housed at the Semen Collection Center “CO.FA”(Cremona, Italy). After thawing, the total and progres-sive motility were evaluated by using CASA. In add-ition to this, sperm were fixed and stained withTrypan blue-Giemsa as reported by Boccia et al.(2007). Sperm cells were microscopically evaluated atCONTACT Dr Valentina Longobardi longobardivalentina@gmail.com Department of Veterinary Medicine and Animal Production, University ofNaples Federico II, Via Delpino 1, Naples, 80137, Italy 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), whichpermits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.ITALIAN JOURNAL OF ANIMAL SCIENCE, 2017http://dx.doi.org/10.1080/1828051X.2017.129050940magnification (Nikon Diaphot 300), to assess theincidence of viable cells with intact acrosome.Furthermore, IVF was carried out on abattoir-derived cumulus-oocyte-complexes that were in vitromatured, fertilised and cultured according to ourstandard procedure (Sattar et al. 2011). In particular,frozen-thawed sperm from each ejaculate were sepa-rated by Percoll gradients and used at the concentra-tion of 1 106 sperm/mL to inseminate on average225 oocytes, over 4 replicates. Cleavage and blastocystrates were recorded on day 7 (day 0¼ IVF).Frozen semen straws were thawed in a water bathat 37 C for 30 s and centrifuged at 1000g for 10minto separate sperm cells from the supernatant. Spermcells were lysed in CHAPS lysis buffer (150 nM KCl,50mM HEPES, 0.1% CHAPS, protease inhibitor, pH 7.4(protease inhibitor kit, Thermo Scientific) and centri-fuged using 1.5mL microcentrifuge tubes (USAScientific). The activity of GGT and ALP was measuredin supernatant and in spermatozoa with a kinetic pro-cedure using Spinreact Reagents (Spinreact, SA,St. Esteve de Bas Girona, Spain) and the absorbanceread with a spectrophotometer (Thermo Scientific,BIOMED 6, Waltham, MA) (Pero et al. 2010). In order toeliminate the differences in sperm concentrationamong samples, the values were corrected for thedilution factor used, to give the final concentration of5 107 sperm/straw for each sample. The concentra-tion of enzyme activity was expressed in U/L.Results and discussionAs depicted in Table 1, differences in enzyme concen-tration between spermatozoa and supernatant wereanalysed by the Mann–Whitney Rank Sum Test.Correlations between different parameters were ana-lysed by the Spearman rank correlation coefficient (rs).The average cleavage and blastocyst rates (over 4 rep-licates) per each ejaculate were correlated with thecorresponding enzyme levels per straw.All fertility parameters showed a certain degree ofvariation but the greatest variation was recorded forblastocyst rate (ranging from 3.3% to 50.9%). Asalready demonstrated by Roussel and Stallcup (1966) ahigh variability in enzymatic activity was also recordedamong straws and the concentrations of GGT and ALPin the supernatant were higher (p< .001 and p< .05,respectively) than in sperm cells (Table 1).The GGT activity in both cells and supernatant wassignificantly correlated with total motility (rs¼ .45,p< .05 and rs¼ .41, p< .05; respectively), cleavage(rs¼ .47, p< .05 and rs¼ .80, p< .0001; respectively)and blastocyst (rs¼ .60, p< .01 and rs¼ .87, p< .0001;respectively) rates. The ALP activity both in sperm cells(rs¼ .43; p<.05) and supernatant (rs¼ .51; p< .05) wasonly correlated with sperm viability. Since the highvariability and the limited number of samples mayaffect the power of statistical analysis, in Table 2 dataare presented in a descriptive manner to show therelationship between enzyme activity and in vitro fertil-ity for each bull.The high concentrations of GGT and ALP found inboth sperm cells and supernatant confirm that theenzymes play a role in sperm function and suggestthat their activity may be also critical to guarantee fro-zen/thawed sperm quality. It is possible to speculatethat GGT plays an important role in the protection ofspermatozoa from oxidative stress because of the posi-tive correlation found in this study between GGT activ-ity and sperm motility. These results are in agreementwith previous studies carried out in stallion (Peschet al. 2006; Dogan et al. 2009). It was also reported apositive correlation among GGT and cleavage andblastocyst rates after IVF, supporting the hypothesisthat this enzyme could be considered a fertility markerfor frozen/thawed bovine semen. Moreover, it isTable 1. Enzymatic activities (U/L) in semen samples.Sperm cells SupernatantMean ± SE Min Max Mean ± SE Min MaxGGT 575.2 ± 50.1A 80.0 1032.1 2884.7 ± 262.8B 727.6 5229.8ALP 1739.6 ± 177.0a 639.8 3959.1 2779.4 ± 380.7b 714.1 8732.7a,bValues with different superscripts are significantly different; p< .05.A,BValues with different superscripts are significantly different; p< .001.Table 2. Percentages of motility, viability, cleavage, blastocysts and GGT and ALP activities in sperm cells (SC) and surnatant (S)in bovine frozen-thawed semen samples (means of the 3 ejaculates per 8 bull).BullsMotilityMean ± SEViabilityMean ± SECleavageMean ± SEBlastocystsMean ± SEGGT (SC)Mean ± SEALP (SC)Mean ± SEGGT (S)Mean ± SEALP (S)Mean ± SE1 46.3 ± 2.7 70.1 ± 3.5 67.0 ± 4.2 20.8 ± 6.7 34.5 ± 8.4 136.6 ± 45.5 165.9 ± 32.9 232.0 ± 50.32 37.0 ± 3.8 81.2 ± 2.3 76.7 ± 2.9 31.0 ± 2.8 27.4 ± 1.8 76.3 ± 10.0 109.6 ± 10.5 121.9 ± 33.43 43.3 ± 4.5 74.2 ± 9.0 72.8 ± 5.8 25.4 ± 4.1 34.7 ± 5.3 118.6 ± 12.6 106.7 ± 14.7 250.6 ± 76.94 47.3 ± 3.2 78.1 ± 3.1 70.7 ± 5.7 29.0 ± 7.5 25.2 ± 8.7 128.9 ± 12.1 103.5 ± 5.3 94.6 ± 23.65 36.7 ± 3.4 75.3 ± 3.8 71.2 ± 7.3 18.2 ± 7.4 20.2 ± 3.6 81.6 ± 14.8 68.0 ± 4.1 165.9 ± 38.36 36.3 ± 3.8 59.7 ± 5.6 71.1 ± 1.0 24.5 ± 1.3 19.1 ± 2.5 56.1 ± 14.9 75.5 ± 22.8 44.5 ± 8.37 42.3 ± 3.7 76.3 ± 1.7 68.7 ± 2.0 22.2 ± 6.1 28.2 ± 8.0 77.1 ± 27.8 128.4 ± 37.1 240.8 ± 91.78 38.3 ± 5.2 68.2 ± 3.8 72.5 ± 3.4 25.9 ± 0.2 28.0 ± 6.8 51.9 ± 7.7 93.7 ± 19.8 73.0 ± 25.02 M. E. PERO ET AL.known that GGT is involved in the transfer of aminoacids across the cellular membrane and in glutathionemetabolism by transferring the glutamyl moiety to avariety of acceptor molecules including water, certainL-amino acids, and peptides, leaving the cysteineproduct to preserve intracellular homeostasis of oxida-tive stress (Alvarez & Storey 1989). It has been sug-gested that GGT also play an important role in thetransfer of the glutathione residues to reactive groupson the sperm surface (Agrawal & Vanha-Perttula 1988).Therefore, the beneficial effect observed on spermmay be due to GGT protective activity against oxida-tive stress. However, it would be worth investigatingin future studies on the relation between GGT activ-ities and oxidative stress parameters.A relation between ALP and different fertilityparameters has been reported in fresh semen in otherspecies (Turner & McDonnell 2003). On the contrary,our results showed that ALP activity in bovine frozen/thawed semen is only correlated with sperm viability,whereas no relationship was found with cleavage andblastocyst rates after IVF. This suggests that in cattlethis enzyme should not be considered a suitable toolto predict frozen-thawed sperm fertility.ConclusionsIn conclusion, it was demonstrated that the residualGGT activity after freezing is correlated with the invitro fertilising ability of bovine sperm. Indeed, meas-urement of GGT in frozen-thawed bovine sperm is areliable method to predict the embryo output afterIVF. Therefore, because of the availability of fast andsimple tests for enzyme assay, measurement of GGTcould represent a useful tool for a fast determinationof frozen/thawed sperm quality, before enrolling bullsin IVF programmes.AcknowledgementsThe authors thank Semen Collection Center “CO.FA”(Cremona, Italy) for their availability over the duration of thisstudy.Disclosure statementThe authors report no conflicts of interest. The authors aloneare responsible for the content and writing this article.ORCIDValentina Longobardi http://orcid.org/0000-0001-6560-3572ReferencesAgrawal YP, Vanha-Perttula T. 1988. Glutathione, L-glutamicacid and gamma-glutamyltransferase in the bull repro-ductive tissue. Int J Androl. 11:123–131.Alvarez JG, Storey BT. 1989. Role of glutathione peroxidasein protecting mammalian spermatozoa from loss of motil-ity caused by spontaneous lipid peroxidation. Gamete Res.23:77–90.Boccia L, Di Palo R, De Rosa A, Attanasio L, Mariotti E,Gasparrini B. 2007. Evaluation of buffalo semen by Trypanblue/Giemsa staining and related fertility in vitro. Ital JAnim Sci. 6 (Suppl.2):739–742.Cancel AM, Chapman DA, Killian GJ. 1997. Osteopontin is the55-kilodalton fertility-associated protein in Holstein bullseminal plasma. Biol Reprod. 57:1293–1301.Dogan I, Polat U, Nur Z. 2009. Correlations betweenseminal plasma enzyme activities and semen parame-ters in seminal fluid of Arabian horses. IJVR.10:119–124.Gerena RL, Irikura D, Urade Y, Eguchi N, Chapman DA, KillianGJ. 1998. Identification of a fertility-associated protein inbull seminal plasma as lipocalin-type prostaglandin D syn-thase. Biol Reprod. 58:826–833.Pace MM, Graham FF. 1970. The release of glutamic axalo-acetic transaminase from bovine spermatozoa as a testmethod of assessing semen quality and fertility. BiolReprod. 3:140–146.Pero ME, Pelagalli A, Lombardi P, Avallone L. 2010.Glutathione concentration and gamma-glutamyltransferaseactivity in water buffalo colostrum. J Anim Physiol AnimNutr. 94:549–551.Pesch S, Bergmann M, Bostedt H. 2006. Determination ofsome enzymes and macro- and microelements in stallionseminal plasma and their correlations to semen quality.Theriogenology. 66:307–313.Roussel JD, Stallcup OT. 1966. Relationships between phos-phatase activity and other characteristics in bull semen. JReprod Fertil. 12:423–429.Sattar A, Rubessa M, Di Francesco S, Longobardi V, DiPalo R, Zicarelli L, Campanile G, Gasparrini B. 2011. Theinfluence of gamete co-incubation length on the invitro fertility and sex ratio of bovine bulls with differ-ent penetration speed. Reprod Domest Anim.46:1090–1097.Turner RM, McDonnell SM. 2003. Alkaline phosphatase installion semen: characterization and clinical applications.Theriogenology. 60:1–10.ITALIAN JOURNAL OF ANIMAL SCIENCE 3

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Influence of γ-glutamyltransferase and alkaline phosphatase activity on in vitro fertilisation of bovine frozen/thawed semen

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