THE JOURNAL OF EXPERIMENTAL ZOOLOGY 280:189–196 (1998)

© 1998 WILEY-LISS, INC.

Birth of Live Mice Derived by In Vitro FertilizationWith Spermatozoa Retrieved Up to Twenty-FourHours After Death

N. SONGSASEN, J. TONG, AND S.P. LEIBO*Department of Biomedical Sciences, University of Guelph, Guelph, Ontario,Canada, N1G 2W1

ABSTRACT The objective of this study was to determine the viability and fertility of mousespermatozoa obtained at various postmortem intervals. Male mice were euthanized, and the bod-ies were kept at ~22°C for up to 24 hr. The epididymides were removed and spermatozoa wereallowed to swim out into Minimum Essential Medium supplemented with bovine serum albumin.Motility of spermatozoa retrieved within 12 hr postmortem was ~60%, whereas motility of thoseobtained 6 to 12 hr later decreased significantly (P < 0.05). There appeared to be no differences inthe percentages of spermatozoa with intact plasma and acrosomal membranes regardless of thetime after death. After in vitro fertilization of oocytes with spermatozoa collected immediatelyafter death or at 6, 12, 18, or 24 hr postmortem, the cleavage rates were 81%, 70%, 64%, 34%, and19%, respectively. Once oocytes were fertilized, more than 65% developed into morulae/blasto-cysts. Transfer of a total of 166 embryos produced in vitro with postmortem spermatozoa resultedin the birth of 44 live pups (26.5%). Of these 44, 3 live mice were derived by transfer of 11 embryos(27.3%) produced with 24-hr postmortem spermatozoa. Histological examination of the testes andepididymides obtained at various postmortem intervals revealed that degenerative changes of thetestes occurred within 6 hr, whereas those of the epididymides were less obvious until 6 hr later.These changes included pyknosis, release of intracellular contents, and disruption of intercellularbridges of the germ cells. This study has demonstrated that spermatozoa recovered from a deadanimal as long as 24 hr after death can be used to fertilize oocytes, and that the resulting zygotescan develop into live young. J. Exp. Zool. 280:189�196, 1998. © 1998 Wiley-Liss, Inc.

Male gametogenesis involves a sequence of cy-tological events that occur within the seminif-erous tubules of the testes, resulting in theformation of spermatozoa (Eddy and O’Brien,’94). Gametogenesis begins at puberty and con-tinues until old age. Fully differentiated sper-matozoa leave the testis and pass through theepididymis where they mature and acquire theability to fertilize oocytes (for review, seeYanagimachi, ’94). Spermatozoa from differentspecies gain their fertilizing ability at differ-ent parts of the epididymis. In all mammalianspecies, however, spermatozoa are capable offertilizing oocytes by the time they reach thecaudal part of the epididymis.

Schroeder et al. (’91) have demonstrated withfemale gametes that postmortem degeneration isreversible, reporting that oocytes recovered frommice 3 to 9 hr postmortem were capable of com-pleting maturation in vitro. In vitro maturation(IVM) and fertilization (IVF) of oocytes obtainedfrom mice 6 hr after they had been killed resultedin 36% of the oocytes developing into two-cell em-

bryos. When such embryos were transferred, livepups were obtained. The results of that study ledseveral other investigators to conduct analogousobservations with female and male gametes fromseveral species (Johnston et al., ’91; Christian etal., ’93; Hay and Goodrowe, ’93). For male ga-metes, Hay and Goodrowe (’93) recovered motilespermatozoa from the epididymides of domesticcats that had been stored at 5°C overnight. Al-though those authors showed that such sperma-tozoa were able to attach to the zona pellucida ofcat oocytes, functional survival of spermatozoa,that is, the ability to fertilize oocytes resulting inembryonic development, was not determined. Pre-viously, we reported that motile spermatozoa couldbe recovered from the epididymides of mice thatwere euthanized 24 hr earlier (Christian et al.,

Contract grant sponsor: Natural Science and Engineering ResearchCouncil of Canada.

*Correspondence to: S.P. Leibo, Building 165, Department of Bio-medical Sciences, Ontario Veterinary College, University of Guelph,Guelph, Ontario, Canada N1G 2W1. E-mail: sleibo@uoguelph.ca

Received 31 July 1997; accepted 23 September 1997.

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190 N. SONGSASEN ET AL.

’93). We also demonstrated that blastocysts couldbe obtained after IVF of oocytes with spermatozoaobtained at 15 hr postmortem at a rate comparableto that using fresh spermatozoa (46% vs. 62%).

In the present study, we investigated the effectsof postmortem changes on characteristics of mousespermatozoa in more detail. Fertilizing ability andother characteristics (e.g., viability, motility, andacrosome integrity) of spermatozoa collected frommale mice at various times after death were deter-mined. Transfer of embryos into recipients wasused as the ultimate criterion to test spermfunction and quality of the embryos producedin vitro with postmortem spermatozoa. Histo-logical changes of the testes and epididymidesobtained at various postmortem intervals werealso examined.

MATERIALS AND METHODSChemicals were obtained from Sigma Chemical

Company (St. Louis, MO) and media from Cana-dian Life Technologies, Inc. (Burlington, ON,Canada), unless otherwise stated. Animals werepurchased from Charles River, Canada (St.-Con-stant, QC, Canada). Their care and use at the Uni-versity were reviewed in advance and performedaccording to standards of the Canadian Councilon Animal Care.

Experiment 1: Characteristics of mousespermatozoa retrieved at various

postmortem intervalsMale B6D2F1 mice of proven fertility (2 to 3

months old) were killed by cervical dislocation. Af-ter death, the mice were kept at room tempera-ture (22°C) for 0, 6, 12, 18, or 24 hr before theircaudae epididymides were removed and placedinto 0.9 ml Minimum Essential Medium (MEM)supplemented with 3 mg/ml BSA covered with sili-cone oil (Corning Medical Fluid 200; Paisley Prod-ucts, Scarborough, ON, Canada). At each interval,epididymides were removed from the mice andminced into small pieces, and spermatozoa wereallowed to swim out into the solution for 10 minat 37°C. Each sperm suspension was transferredinto a plastic tube (12 × 75 mm, Cat. T405-3;Simport, Beloeil, QC, Canada), and the concen-tration of each sample was determined using aNeubauer haemocytometer (Fisher Scientific,Pittsburgh, PA). The integrity of the plasma andacrosomal membranes, as well as motility and fer-tilizing ability, of spermatozoa were assessed us-ing the methods described below. This entireexperiment was repeated twice; thus, there were

three replicates (i.e., 3 males/postmortem inter-val) in this experiment.

Membrane integrity of spermplasma membrane

Integrity of the sperm plasma membrane was as-sessed by a method modified from that describedby Garner and Johnson (’94) using the fluorescentdouble stain FertiLight® (Molecular Probes, Inc.,Eugene, OR). This vital stain contains two nucleicacids dyes: (1) SYBR-14, which permeates intactplasma membranes causing viable sperm to fluo-resce green; and (2) propidium iodide (PI), whichpermeates only damaged plasma membranes, caus-ing nonviable sperm to fluoresce red. The SYBR-14was diluted 1:10 in dimethyl sulfoxide and kept asa stock solution at –20°C. For staining, the dye wasfurther diluted 1:5 in modified Tyrode’s buffer solu-tion (Parrish et al., ’86) and 2.5 µl of this solutionwere added to 100 µl of the sperm sample and in-cubated at 37°C for 10 min. Then 5 µl of PI wereadded to the sperm sample, and the sample wasincubated for another 10 min. After staining, 10 µlof the sperm suspension were placed on a micro-scope slide, covered with a coverslip, and observedunder a fluorescent microscope (Zeiss IM35, filterset # 487709, excitation filter = 450 to 490 nm). Ineach replicate, two slides were observed, and ap-proximately 200 spermatozoa were counted for eachslide (a total of 400 sperm/replicate). In sampleswith highly motile sperm, 5 µl of 0.037% formalde-hyde (Fisher Scientific Co., Fairlawn, NJ) in PBSwere added to immobilize them for observation.

Sperm motilitySperm motility was determined by microscopy

using the criteria described by Jequier and Crich(’86), by which both percentages of motile sper-matozoa and the rate of forward movement areassessed. The rate of forward progression was as-sessed on a scale of 0 to 4: 0 = absent, 1 = weakor sluggish, 2 = definite, 3 = good, 4 = vigorous.

Acrosome integrityAcrosome integrity was determined using a

method modified from that described by Cross etal. (’86). Spermatozoa were stained with Pisumsativum Agglutinin (PSA) that was conjugated tofluorescein isothiocyanate (FITC) after fixationand permeabilization. Lectin from Pisum sativumbinds to acrosomal contents resulting in sperma-tozoa with intact acrosomes exhibiting a band ofgreen fluorescence of FITC at their anterioracrosomal region. Briefly, 20 µl of sperm sample

FERTILITY OF MOUSE SPERM RETRIEVED AFTER DEATH 191

were spread on a microscopic slide and allowedto dry. The spermatozoa were then fixed andpermeabilized with 100% methanol at 0°C for 1min. A 20-µl volume of FITC/PSA (100 mg/ml inD-PBS) was dropped onto the slide, which wasthen covered with a plastic sheet and left for 20min. Then, the slide was rinsed, left in distilledwater for 15 min, and allowed to dry in air. Thespecimen was covered with antifade mounting me-dium (Johnson and Nogueira Araujo, ’81) and ob-served under a fluorescence microscope. In eachreplicate, two slides were observed, and approxi-mately 200 spermatozoa were counted for each slide.

In vitro fertilizationIn vitro fertilization was performed using a

method modified from that described by O’Brienet al. (’93). Ovulated oocytes were obtained fromsuperovulated B6C3F1 mice (age 4 to 6 weeks) at13 to 14 hr after injection with 5 IU hCG. Theoocytes were collected into Waymouth’s MB 752/1medium + 5% fetal bovine serum, and thenwashed in the same medium three times. A fewminutes before fertilization, the oocytes werewashed in MEM + BSA, and placed into IVF drops(50 to 60 oocytes/drop) containing 0.5 ml MEM +BSA and covered by sterile silicone oil. Sperma-tozoa were added to the IVF drops (final totalsperm concentration of 2 × 105 sperm/ml) for in-cubation at 37.0 ± 0.5°C in a humidified atmo-sphere of 5% O2: 5% CO2: 90% N2 for 4.5 hr. Atthat time, oocytes that showed signs of degenera-tion, such as contraction of cytoplasm, were dis-carded. The remaining presumptive zygotes werewashed in MEM + BSA, and cultured in 2.5 mlMEM + BSA under the same conditions. At 24 hrpost-insemination, the numbers of two-cell em-bryos were counted. The embryos were thenwashed in KSOM medium (Lawitts and Biggers,’91), and 10 to 15 embryos were cultured in a 10-µl drop of KSOM covered by sterile silicone oil.The embryos that developed into compact moru-lae or blastocysts were counted at 72 hr post-in-semination. Morulae and blastocysts were thencryopreserved in 1.5 M ethylene glycol using themethod described by Songsasen et al. (’95). As re-cipients became available, the embryos werethawed rapidly, washed in M2 medium (Hogan etal., ’86) and transferred into recipients.

Embryo transferAfter being thawed, cryopreserved embryos pro-

duced by IVF were transferred into pseudopreg-nant CD-1 females (age 6 to 8 weeks old) using

the method described by Hogan et al. (’86). Briefly,female mice were placed in cages of vasectomizedmales (CD-1) that had been shown to be sterileand observed for the presence of vaginal plugseach morning. The day on which a vaginal plugwas observed was considered as day 0.5 of pseudo-pregnancy. Ten to 12 blastocysts were transferredinto each uterus of a day-1.5 to day-2.5 pseudo-pregnant recipient (5 to 6 per horn). Pregnancieswere allowed to proceed to term, and the num-bers of live young were recorded.

Experiment 2: Histological changes of thetestes and epididymides obtained at various

postmortem intervalsFive B6D2F1 males were killed by cervical dis-

location. Their testes and epididymides were re-moved immediately after death or 6, 12, 18, or 24hr later, and were fixed in Bouin’s solution for 4hr. The fixed tissues were dehydrated in a seriesof increasing concentrations of ethanol (70%, 90%,and 100%), and cleared in xylene. The processedtissues were then embedded in a paraffin block,sectioned, and stained with haematoxylin-eosin(Russell et al., ’90).

The proportional data of sperm viability, motil-ity, acrosome integrity, and embryonic develop-ment were transformed using the equation y´ =arcsin y. Analysis of transformed data were thenperformed using a General Linear Proceduremodel (SuperANOVA programme, Abacus Con-cepts, 1989). A one-way Anova was performed tocompare differences among groups (various post-mortem times). Comparisons of means amonggroups were performed using the Duncan NewMultiple Range test. Differences were consideredsignificant when P < 0.05.

RESULTSExperiment 1: Characteristics of mouse

spermatozoa retrieved at variouspostmortem intervals

Viability, motility and acrosome integrity ofsperm collected at various times postmortem areshown in Figure 1. The viability and acrosome in-tegrity of spermatozoa retrieved at various post-mortem intervals up to 24 hr were similar to thosecollected immediately after death (Figs. 1A, C).In contrast, compared to those collected at 0 to12 hr after death, spermatozoa suffered substan-tially decreased motility when they were collectedat 18 and 24 hr (P < 0.05; Fig. 1B). At 0 to 12 hrpostmortem, the percentage of motile spermato-

192 N. SONGSASEN ET AL.

Figure 1.

FERTILITY OF MOUSE SPERM RETRIEVED AFTER DEATH 193

zoa was ~60% with a progressive movement scoreof 3 (good movement), whereas that of spermato-zoa collected 6 to 12 hr later was less than 30%with a progressive movement score of 2 (definitemovement).

Table 1 shows development of oocytes into two-cell embryos and morulae/blastocysts after theywere exposed to spermatozoa collected at varioustimes after the male mice had been killed. Therewere no significant differences in development ofoocytes into two-cell embryos and morulae/blas-tocysts when spermatozoa collected at 0 to 12 hrpostmortem were used for IVF. However, percent-ages of cleavage and blastocyst formation weresignificantly lower when oocytes were fertilizedwith spermatozoa retrieved at 18 and 24 hr post-mortem (P < 0.05). Once the oocytes were fertil-ized, more than 85% of them developed intomorulae or blastocysts at 72 hr post-insemination,except for those resulting from spermatozoa re-trieved at 24 hr postmortem.

The results of embryo transfer are shown inTable 2. Pregnancies were established aftertransferring cryopreserved embryos produced invitro with spermatozoa collected at all postmor-tem intervals. Eleven embryos produced in vitrowith spermatozoa retrieved at 24 hr postmor-tem were transferred into one recipient that be-came pregnant and delivered three live pups(27.3%). Similar proportions of transferred em-bryos from the other groups developed into liveyoung, except for the somewhat lower rateachieved with the 12-hr group.

Experiment 2: Histological changes of thetestes and epididymides obtained at various

postmortem intervals

TestisChanges within the seminiferous tubules and

interstitial compartments were observed as earlyas 6 hr after death (Fig. 2). Disorganization re-sulting in sloughing of the germ cells into the lu-men of the seminiferous tubules was the mostprominent change observed in this group. Al-though the testes retrieved from one mouse im-mediately after death contained some germ cellsundergoing pyknosis, degenerating germ cells

were more abundant in the testes obtained at 6hr postmortem. At 12 and 18 hr postmortem, de-generative changes had become more obvious,compared to the control at 0 hr, as disorganiza-tion and degeneration of germ cells occurred. By24 after death, the seminiferous tubules becamequite diffuse as the basal lamina surrounding eachtubule disintegrated.

EpididymisPostmortem changes of the epididymis occurred

more slowly than those within the testis. At 6 hrafter death, the epithelial cells of epididymis weregenerally normal compared to the control (Fig. 3).However, degenerative changes became more ap-parent at 18 and 24 hr as the epithelial cells be-came pyknotic and released their intracellularcontents into the lumen of the epididymides. By24 hr postmortem, the structure of the epididy-mal tubule appeared to be breaking down.

DISCUSSIONWe have demonstrated that viable and fer-

tile epididymal spermatozoa can be retrievedfrom a dead mouse as long as 24 hr after death.In vitro fertilization of oocytes with postmor-tem spermatozoa resulted in development of oo-cytes into two-cell embryos and blastocysts. Theembryos produced in vitro with postmortemspermatozoa were fully capable of developinginto live pups after they were transferred intorecipients.

Attempts to rescue genetic material from ani-mals that die have been reported previously forboth female and male gametes. Schroeder et al.(’91) demonstrated that mouse oocytes collectedup to 9 hr postmortem retained meiotic compe-tence and developmental capacity after IVF. De-velopmental capacity of the oocytes retrieved atlonger postmortem intervals decreased substan-tially; however, none of the oocytes retrieved at12 hr after death developed into blastocysts afterIVF. Importantly, these investigators also demon-strated that live mice could be obtained aftertransferring embryos produced in vitro with post-mortem oocytes. For wild animals, it has beenshown with felid species that viable oocytes canbe retrieved several hours after death. Such oo-cytes can be matured and fertilized in vitro(Johnston et al., ’91), although live young producedthis way were not reported.

Hay and Goodrowe (’93) demonstrated that mo-tile spermatozoa can be recovered from cold-storedepididymides of the domestic cat. The spermato-

Fig. 1. Characteristics of mouse spermatozoa retrieved atvarious postmortem intervals; (A) viability, (B) motility, and(C) acrosome integrity. Each point represents the mean ± SEof data from three replicates. Different letters indicate sig-nificant differences (P < 0.05).

194 N. SONGSASEN ET AL.

zoa were capable of binding to the zona pellucidaof cat oocytes, but embryonic development afterIVF was not assessed in that study. Christian etal. (’93) reported that motile and fertile sperma-tozoa could be recovered as long as 15 hr afterthe death of a mouse. In that study, 22 of 110oocytes developed into hatched blastocysts afterbeing exposed to spermatozoa retrieved at 15 hrpostmortem. In the present study, motility of sper-matozoa obtained at 24 hr after death was simi-lar to that in the previous study (23% vs. 15%).Moreover, we confirmed the results of the previ-ous study that the plasma membranes of epididy-mal spermatozoa remain intact as long as 24 hrafter the mouse had been killed.

In contrast to oocytes, spermatozoa obtainedat 12 hr postmortem were able to fertilize oo-cytes in vitro at a rate similar to that with sper-matozoa collected immediately after death(Table 1). Some spermatozoa obtained at 24 hrpostmortem retained their fertilizing capacity,although the percentages of two-cell embryo andblastocyst development after IVF were signifi-cantly reduced. These differences between oo-cytes and spermatozoa may be attributable tothe fact that epididymal sperm have completedmeiosis and differentiation and are ready forfertilization. Oocytes recovered from the ovarymust undergo further processes of maturation.

TABLE 1. Development of oocytes after in vitro fertilization with spermatozoa retrieved from miceafter various postmortem intervals1,2

Oocytes Morulae/Blastocysts at 72 hrPostmortem % Cleaved As % of total As % of 2-cellinterval (hr) Total (mean ± SE) (mean ± SE) (mean ± SE)

0 169 81.0 ± 7.1a 78.3 ± 8.8a 96.3 ± 2.56 194 70.1 ± 18.0ab 68.0 ± 20.1a 93.9 ± 6.1

12 167 63.5 ± 12.6ab 57.9 ± 11.5ab 91.4 ± 4.918 182 34.2 ± 1.3bc 29.1 ± 4.0b 84.7 ± 9.724 166 19.1 ± 6.1c 13.8 ± 6.0c 64.4 ± 19.41Each group consisted of three replicates of IVF.2Different letters within a column indicate significant differences (P < 0.05).

TABLE 2. Pregnancies and births of live young after transferring embryos produced withsperm collected at various postmortem intervals1

TotalPostmortem Total Pregnant embryos Live pups Pup sexesinterval (hr) recipients recipients transferred (% of total) (female/male)

0 4 2 42 13 (31.0) 4/86 6 4 68 24 (35.3) 10/12

12 4 3 44 5 (11.4) 1/018 4 4 43 12 (27.9) 3/324 1 1 11 3 (27.3) 3/01Some pups were born alive, but were cannibalized by their mother within first 24 hr postpartum before their sexes could be determined.

Meiotic competence and developmental capac-ity of oocytes depend greatly on interactions be-tween the oocytes and somatic cells surroundingthem. Thus, changes occurring within a follicleor oocyte itself that upset this balance may besufficient to compromise oocyte maturation re-sulting in failure of fertilization and embryonicdevelopment.

The fertilizing capability of spermatozoa col-lected after death appears to be correlated withtheir motility. The cleavage rates obtained withspermatozoa at various postmortem intervals(Table 2) have been plotted as a function of themotility at the same intervals (Fig. 1B). Theresults in Figure 4 show that there is a highcorrelation between motility and fertilizing abil-ity of sperm. In contrast, there were no signifi-cant differences in viability and acrosomeintegrity of sperm collected at various postmor-tem intervals (Figs. 1A, C). The finding thatsperm motility is more susceptible to deleteri-ous conditions than other characteristics, suchas integrity of the plasma and acrosomal mem-branes, agrees with those reported by others(Gao et al., ’93; Curry and Watson, ’94; Wi-lloughby et al., ’96). For example, Willoughbyet al. (’96) reported that, compared to the lossof integrity of plasma membranes, the loss ofmotility of mouse spermatozoa after exposure

FERTILITY OF MOUSE SPERM RETRIEVED AFTER DEATH 195

Fig. 2. Light micrographs of mouse testes obtained at various postmortem intervals: 0,6, 18, and 24 hr. The bar represents 0.1 mm.

Fig. 3. Light micrographs of mouse epididymides obtained at 0 and 24 hr postmortem.The bar represents 0.1 mm.

196 N. SONGSASEN ET AL.

to anisotonic solutions followed by dilution into iso-tonic medium occurred over a narrower range.

Normal histological and postmortem changes ob-served in this study were similar to those reportedelsewhere (Seaman, ’87; Feldman and Seely, ’88;Russell et al., ’90). These changes included pycnoticnuclei, release of intracellular components, and dis-ruption of intercellular bridges between germ cells.Histological examination of the epididymides re-vealed that distinct degenerative changes did notoccur until 12 hr postmortem. Nevertheless, degen-erative changes occurring within the testis did notaffect the fertilizing ability of epididymal sperma-tozoa. Although changes within the testis occurredas early as 6 hr postmortem, there were no signifi-cant differences in development into two-cell em-bryos of the oocytes fertilized with sperm retrievedat 0, 6, and 12 hr postmortem.

To our knowledge, this is the first report dem-onstrating that mouse spermatozoa retrieved aslong as 24 hr after death are able to fertilize oo-cytes in vitro. The results reported here togetherwith a recently developed cryopreservation methodfor mouse spermatozoa (Songsasen et al., ’97) of-fer practical ways to preserve spermatozoa fromgenetically valuable mice that die prematurely. Al-though caution is necessary to extrapolate fromthese results to other species, the present studysuggests the feasibility of using epididymal sper-matozoa retrieved many hours after death to res-cue the genetics of endangered animals.

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Fig. 4. Fertilization of oocytes obtained at various post-mortem intervals as a function of sperm motility of speci-mens retrieved at the same intervals. The fertilization dataare from Table 1 and motility data from Figure 1B.