Evaluation of viability and apoptosis in horse embryos stored under different conditions at 5 °C

Evaluation of viability and apoptosis in horse embryos stored under different conditions at 5 °C

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  Evaluation of viability and apoptosis in horseembryos stored under different conditions at 5  8 C M. Moussa a,b,* , J.L. Tremoleda c , G. Duchamp a , J-F. Bruyas b ,B. Colenbrander c,d , M.M. Bevers d , P.F. Daels a a Physiology of Reproduction and Behavior, INRA, Nouzilly 37 380, France b  Laboratory of Biotechnology and Pathology of Reproduction, National Veterinary School of Nantes, BP 40706, Nantes 44307, France c  Department of Equine Sciences, Faculty of Veterinary Medicine,Utrecht University, CL Utrecht 3584, The Netherlands d  Department of Farm Animal Health, Faculty of Veterinary Medicine,Utrecht University, CL Utrecht 3584, The Netherlands Received 17 April 2003; received in revised form 11 July 2003; accepted 18 July 2003 Abstract The aim of this study was to evaluate the viability (percentage of dead cells) and the incidence of DNA fragmentation of horse embryos after storage in three different media at 5  8 C for 6 and 24 h.Forty embryos were stored in Emcare Holding Solution 1 for 6 and 24 h, in Hams’F10 or VigroHolding Plus 1 for 24 h at 5  8 C ( n ¼ 9–10 per group) and 10 embryos were evaluated immediatelyafter collection. First, embryos were stained, immediately after collection or following storage, todetect dead cells (DAPI) and, subsequently, DAPI-stained embryos were fixed and stained to detectDNAfragmentation (TUNEL). Finally,allthe fixed embryoswerere-stained withDAPItodeterminethe total number of cells. The percentage of cells stained with both TUNEL and DAPI or TUNEL-onlyorDAPI-onlyweredetermined. Thepercentofdeadcells (DAPI-labelled) perembryo increasedwithduration ofstorage, butno differences were detected between the storagemedia. The percentageof early apoptotic cells (TUNEL þ  /DAPI  ) in fresh and stored embryo for 6 h or 24 h did not differsignificantly ( P > 0 : 05). There was a significant correlation between the percentage of cells labelledby TUNEL and DAPI (  R ¼ 0 : 87) ( P < 0 : 001). These results suggest that cooled storage increasescell death butthisdoes notappear tooccur by inductionofapoptosis and thatDAPIstaining provestobe a quick and reliable method for assessing embryo viability. # 2003 Elsevier Inc. All rights reserved. Keywords:  Equine; Embryo; Viability; Apoptosis; Necrosis; TransportTheriogenology 61 (2004) 921–932 * Corresponding author. Tel.:  þ 33-2-47-42-79-47; fax:  þ 33-2-47-42-77-43. E-mail address:  moussa@tours.inra.fr (M. Moussa).0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved.doi:10.1016/S0093-691X(03)00280-2  1. Introduction Although the use of embryo transfer (ET) has increased steadily over the last twodecades, its development has  fl uctuated in tandem with the  fi nancial health of the equineindustry. Moreover, contributing to its high cost there are several technical problemsrelated to unique biological features of the equids such as synchronisation of donor andrecipient, induction of multiple ovulation, embryo recovery rate, method of transfer,method of cooling and transporting embryos and cryopreservation [1]. The cooled storageand transportation of embryos isgainingwider acceptance. Cooling and storingembryos at5  8 C allow shipping to a centralised location for transfer into recipient mares [1]. Equineembryos can be successfully stored for 24 h at 5  8 C in Ham ’ s F-10 media [2]. However,Ham ’ s F-10 contains a bicarbonate-based buffer and requires a speci fi c CO 2  atmosphere tomaintain a correct pH which makes the use of this method less practical for transport of embryos. An alternative to gassed Ham ’ s F-10 is a culture medium with a buffering systemwhich does not depend on CO 2 . This can be achieved using one of the zwitterionic buffers(or  ‘‘ Good ’’  buffers) which have a suitable p K  a  [3]. A zwitterion buffer contains ametabolically inert buffering ion which does not cross cell membranes, thus providinga stable micro-environment at the zona pellucida and avoids the possible inhibition of embryo development by phosphate buffers [4]. McCue et al. [5] reported that the pregnancy rate on Day 16 was similar for transferred embryos stored in Ham ’ s F-10and Emcare Embryo Holding Solution (EHS) for 24 h . We have also demonstrated thatzwitterion buffers are a good alternative for bicarbonate-based buffers [6]. Zwitterionicbuffers have also been shown to improve survival of stored bovine embryos [7,8]. Vigro Holding Plus (VHP) uses HEPES as an inert buffering system, at a concentration that issuited for an air atmosphere [7]. Currently, in the North American bovine ET industry themost widely utilized alternatives to Dulbecco ’ s phosphate-buffered saline (D-PBS) arezwitterion-buffered media [7].Embryo quality is an important determinant for the success of embryo transfer. Theembryonic quality is often evaluated by grading embryo morphology after collection at theblastocyst stage. Morphologic evaluations have been described as method of assessingembryo viability and the probability of establishing pregnancies after transfer [9,10]. However, morphological assessment of an embryo using a light microscope remains asubjectiveparameterwhichmaybeinsuf  fi cientfordecidingwhichembryosaresuitablefortransferring or freezing. Moreover, there are situations when the difference in qualitycannot be distinguished by morphological criteria alone. The ultimate method to evaluateequine embryos is transfer of the embryo to a recipient mare, but this method is costly andlabour intensive. A quick and easy viability test would be desirable to assess embryos invitropriorto transfer and would be useful inthe development ofnew technique, e.g. cooledstorage and cryopreservation.Potts and Wilson [11]  fi rst described the presence of dead cells in the preimplantationmammalian embryo. Light microscope and ultrastructural studies of preimplantationembryos from many species, including mice [12,13], cows [14], baboons [15], rhesus monkeys [16] and humans [14,17,18], have described dead and dying embryonic cells. However, few studies have been conducted on the assessment of the viability of horse embryos stored for longer periods at 5  8 C [19]. Several workers reported the use of  922  M. Moussa et al./Theriogenology 61 (2004) 921–932  vital stain to test the viability of mammalian embryos using non- fl uorescent dyes areneutral red [20] and trypan blue [21,22]. Some  fl uorescent dyes, e.g.  fl uorescein diacetate(FDA), stain live cells [23 – 26] and Rhodamine 123, a mitochondrion-speci fi c  fl uorescentdye, may also be useful for assessing the metabolic integrity of mammalian embryos [27].4 0 ,6 0 -Diamidino-2-phenylindole (DAPI) has been shown to stain the dead cells in bovineandrabbitembryos[28],andcanbeen usedtodeterminethe numberofdead cells inequineembryos. The survival rate after transfer in the recipient mare appears to be related to thenumberofdeadcells[29,30].Alongwiththis,DAPI-staininghasbeenusedtohighlightthe differences in quality of cooled and frozen embryos, and the technique appears to be usefulas a criterion for analysis of frozen-thawed embryos [31].It has become increasingly apparent that embryonic cells die by apoptosis [32], a formofprogrammedcelldeath.Apoptosisplaysa role inmammaliandevelopment as aqualitycontrol mechanism to eliminate cells that are damaged, non-functional, abnormal, ormisplaced [33,34]. The effect of apoptotic activity on the developmental capacity of  embryos, and its potential role in embryonic loss, is not well characterised [35].Assessment of embryonic apoptotic cell death has been carried out using the terminaldeoxynucleotidyl transferase (TdT)-mediated d-UTP nick end-labelling technique(TUNEL), a method which facilitates the analysis of nuclear DNA fragmentation insitu [36,37]. The aim of this study was to compare the effect of cooled storage on viability and theincidence of DNA fragmentation and, to compare the ef  fi cacy of the DAPI and TUNELassay in predicting embryo quality. To this end, embryos were initially stained with DAPIto detect dead cells and subsequently stained with TUNEL to differentiate the earlyapoptotic cells. 2. Materials and methods 2.1. Mare management  Fourty-threeWelshPonymares,agedbetween3and14yearsandweighing200 – 400 kg,were used as donors. Mares had no abnormalities in their reproductivetracts as detected bypalpation and ultrasonography and had a history of being reproductively sound in theprevious year. The genital tract of mares was examined by transrectal palpation andultrasonography at regular intervals and daily during estrus. Ovulation was induced whenthe largest follicle had reached 33 mm in diameter using 20 mg crude equine gonado-trophin (CEG) intravenously injected [38]. Twenty-four hours after injection of CEG, donor mares were inseminated with fresh semen collected from a fertile stallion ( n ¼ 3),using 600  10 6 spermatozoa per insemination. The day of ovulation was designated asDay 0 and embryos were recovered on Days 7 after ovulation. 2.2. Embryo recovery and handling Fifty equine embryos were recovered on Day 7 after ovulation, the embryo recovery ratewas 84%, (ovulation ¼ Day 0). Embryos were recovered by transcervical uterine lavage  M. Moussa et al./Theriogenology 61 (2004) 921  –  932  923  using 3  0 : 5 l Emcare 1 Complete Flush Solution (ICP,Auckland, NZ) at 37  8 C. Embryoswere washed 10 times in warm Emcare Complete Flush Solution at 37  8 C. Embryos weremeasured using an eyepiece micrometer and graded based on morphology as previouslydescribed by McKinnon et al. [9]. 2.3. Preparation and storage of embryos Embryos were assigned at random to  fi ve treatment groups ( n ¼ 9 – 10 per group).Embryos in Group 0 h (control group) were evaluated immediately after collection andwashing. Embryos in Group E-6 h were evaluated after 6 h of storage at 5  8 C in EmcareHolding Solution (EHS, ICP, Auckland, NZ). Embryos stored for 24 h at 5  8 C weremaintained either in Ham ’ s F-10 (Sigma, Chemical Co., St. Louis, MO; Group H-24 h),which needed to be gassed with a mixture of 5% CO 2 , 5% O 2  and 90% N 2 , before using, inEHS (Group E-24 h) or in Vigro Holding Plus (VHP: Group V-24 h; AB Technology,Pullman,WA).Storedembryoswereplacedina5-mltube fi lledwiththeappropriateholdingsolution, which was then placed in a 50-ml centrifuge tube (Corning, NY, USA)  fi lled withcollection medium and stored in an Equitainer I (Hamilton-Thorn, S. Hamilton, MA) aspreviously described [39]. 2.4. DAPI-labelling: viability evaluation After respective treatment, control and stored embryos were washed three times inEHS then placed in EHS containing 1  m g/ml DAPI (Sigma Chemical Co., St. Louis,MO) and incubated for 15 min at room temperature then washed three times in EHS.The number of dead cells (DAPI positive,  fl uorescent cells) per embryo was determinedusing an inverted  fl uorescence microscope (Olympus, IMT-2) with a diachronic mirrorunit (IMT2-DMU: high pressure mercury burner wavelength 334 – 365 nm,  fl uorescentradiation 420 nm and up). After counting, embryos were  fi xed in PBS solutioncontaining 3% paraformaldehyde at room temperature for 1 h and washed three timesin PBS solution. Embryos were kept at 4  8 C in PBS containing 0.05% NaN 3  (Pro-labo,Paris, France) and 1 mM phenylmethylsulfonyl  fl uoride (PMSF) (Serva, Heidelberg,Germany). 2.5. TUNEL-labelling: evaluation of apoptotic cells Each embryo was stained individually throughout the entire experimental procedure.TUNEL-labelling was based on the procedure described by Brison and Schultz [36]. Fixed embryos were washed three times in PBS supplemented with 1 mg/ml polyvinylalcohol(PVA, Sigma) (PBS/PVA, pH 7.4). Embryos were permeabilized by incubation in 0.1%Triton X-100 sodium citrate for 10 min on ice, and then washed twice in PBS/PVA. Eachembryo was incubated in a 20  m l drop of a mixture of TUNEL  fl uorescein-conjugateddUTPandTdT(TUNEL reagents;BoehringerMannheim,RocheDiagnostics,Lewes,EastSussex) under mineral oil for 1 h at 37  8 C in darkness. Positive controls were incubatedin DNase (50 U/ml; Promega, Southampton, UK) for 20 min at 37  8 C and washed inPBS/PVA before TUNEL-staining. Negative controls were incubated in  fl uorescein-dUTP 924  M. Moussa et al./Theriogenology 61 (2004) 921  –  932  in the absence of TdT. Following TUNEL-staining, embryos were washed twice in PBSsolution and then directly mounted on a glass microscope slide and covered with acoverslip using vaseline lines to avoid collapse. 2.6. Fluorescence microscopy Embryos stained with TUNEL were analysed using an epi fl uorescence microscope(BH-2; Olympus, Tokyo, Japan) equipped with a 458  15 nm wavelength band-passexcitation  fi lter, a 470 nm diachronic mirror and a 500 nm long-pass emission  fi lter, whichenable visualization of cells labelled with TUNEL and cells labelled with DAPI simulta-neously by changing  fi lters. The number of cells stained with TUNEL, or DAPI, or bothTUNEL þ DAPI were determined. Fixed embryos were then dismounted and re-stainedwith DAPI to label all nuclei(method described above). Embryos were mounted again on amicroscope slide and all DAPI-labelled cells were counted. 2.7. Cell counting The number of cells stained with both DAPI and TUNEL (DAPI þ  /TUNEL þ ), or onlywith TUNEL (TUNEL þ  /DAPI  ), or only with DAPI (TUNEL   /DAPI þ ) and the totalnumber of cells (DAPI-labelled after  fi xation) were determined using a counting gridmounted on the eyepiece. The percentage of stained cells was calculated by dividing thenumber of stained cells by the total number of cells  100. 2.8. Statistical analysis Analysis of variance was used for comparison of embryo diameter. Chi square test wasused for comparison of morphological grades among treatment groups. Differencesbetween the percentage of dead cells and apoptotic cells for groups of embryos wereanalysed by the Mann-Whitney  U   test using StatXact 5 software (CYTEL, Cambridge,MA) (signi fi cance at  P  <  0 : 05). Correlation between the percent of dead cells and embryosize,andthecorrelationbetweenpercentofcellsstainedwithDAPIandthe percentofcellsstained with TUNEL were analysed using test correlation coef  fi cient. 3. Results We identi fi ed the following cellular staining patterns: TUNEL þ  /DAPI þ¼ dead oradvanced apoptotic cells, TUNEL þ  /DAPI ¼ live, early apoptotic cells, TUNEL   / DAPI þ¼ dead or necrotic cells, TUNEL   /DAPI ¼ live or non-apoptotic cells. 3.1. Percentage of dead cells in fresh and stored embryos The percentage of cells stained with DAPI (dead) for fresh and stored embryosbefore  fi xation are shown in Table 1. The mean percentage of cells labelled with DAPIwas similar in Groups 0 h and E-6 h and was signi fi cantly higher for each of the three  M. Moussa et al./Theriogenology 61 (2004) 921  –  932  925
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