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548 Theriogenology

rate and low in vitro development of transferable embryos have continued to impede thistechnology. This is partly due to unsuccessful superovulation (19,30,30), difficulty in obtainingslaughterhouse material and thus limited availability of mature oocytes for IVF (29,1) and the lackof an efficient in vitro capacitation system for stallion spermatozoa (5,43,21,11). In the equinespecies, the only offspring born after IVF was originated from an in vivo matured oocyte, (37)and, to our knowledge, no foals have been obtained from any in vitro matured oocytes to date.

The in vitro maturation (IVM) of horse oocytes was first performed by Fulka and Okolski in1981 (23). They reported that compact-cumulus equine oocytes needed more than 24 h to bematured in vitro (68%). Subsequent studies confirmed this observation (43.29). In addition, it hasbeen shown that a comparable maturation rate could be reached within 24 h of culture if theoocytes had an expanded cumulus investment. It seems that neither follicular atresia nor abnormalsteroid levels in the follicular fluid of slaughtered mares ovaries had any influence on IVM (36).Ho\\*ever, oocytes from pregnant mares or from mares in the luteal phase, those collected fromhighly vascularized follicles as well as those showing a heterogeneous cytoplasm need to bediscarded (14). Recent studies on equine IVM have been focused on improving oocyte recoveryand on investigating the effects of different kinds of media, sera, hormones and different cultureperiods. Oocyte recovery methods have not shown any influence on maturation (10). However,techniques such as slicing the ovaries, flushing the follicles with PBS or rupturing the isolatedfollicles may increase the number of recovered oocytes compared with that of aspiration offollicular fluid (10,40,1). Tissue culture medium 199 seems to be more effective than MCnCzo B2or Hams F-IO for equine IVM (41,40). Studies investigating serum-hormone balance to supportmaturation are currently in progress. Willis et al. (42) obtained the highest polar body formationrate (67%) after 32 h of culture in TCM 199 supplemented with serum collected on the first day ofestrus. They also observed that the Metaphase II (MII) formation rate was similar, whatever theculture time, if using mare serum from the day of ovulation (48% after 15 h vs 52% after 32 h),with various concenlrations of either bovine or equine LH, with or without FSH. Moreover, intheir study, culture with PMSG increased not only the number ofoocytes that reached MI1 but alsothat of the degenerating oocytes. Grondahl et al. (25), using equine pituitary FSH and LH,obtained 64% MI1 oocytes with 5% ECS, and 53 and 79% in two trials with 10% fetal equineserum. Alm and Torner (I) reported 73% oocyte maturation 30 h after culture in TCM 199supplemented with horse serum with additional granulosa cells. Shabpareh et al. (40) obtained86% maturation by culturing oocytes for 30 h in TCM 199 with 10% EMS in association withFSH and estradiol.

The in vitro capacitation of stallion semen is not yet an efficient procedure in equines(5,43,31 ,I I). Ellington et al. (21) demonstrated that the co-culture of stallion sperm cells onmares oviductal epithelial monolayer increased the zona binding ability. Arns and Sheperd (2)observed increased rates of sperm/oocyte binding and sperm penetration in non-viable equineoocytes after separation of equine spermatozoa by Percoll discontinuous gradient in Hams F-IO.In vitro fertilization technology in the equine is quite new, having started in 1989. The very firststudies have used in vivo matured oocytes and semen treated with A23187 calcium ionophore, butlow fertilization as well as low cleavage rates were obtained (4,5,37). To date, the best resultsobtained in the equine species in terms of fertilizalion and clea\.age of in vitro matured oocytesfertilized with conventional IVF procedures have been achieved after the treatment of sperm cellswith calcium ionophore (2 to 33% and from 3 to 24%, respectively; 13,44,25). However, theseresults are quite low compared with those obtained in other animal species, and efforts to improvethe in vitro capacitation system continue.

The aim of our study \vas to establish an efficienl IVM/IVF procedure for compact-cumulusoocytes recovered from the ovaries of marts slaughtered during the breeding season, using frozen-thawed semen prepared with swim-up and treated with heparin. The influence of EMS and ECS inthe IVM medium on the maturation rate was compared and the effects of increasing semen


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Theriogenology 549

concentrations, homologous serum supplementation in IVM medium and partial cumulus massremoval on the fertilization rate were investigated.MATERIALS AND METHODS

Oocytes CollectionOvaries from mares of unknown reproductive history were obtained during the breeding season(April to July) at local slaughterhouses in Southern Italy (41 North latitude). The ovaries were putin physiological saline (9 g NaCI/L) containing 40 mg/L gentamycin sulphate and were transported(at 25 to 3OC) to the laboratory within 1 to 3 h in a thermos container. Follicular fluid wasaspirated from follicles less than 3 cm in diameter through an 18-gauge x 1 l/3- (I .3-x a-mm)Terumo needle and collected into sterile M-ml Falcon tubes which were mantained in waterbath at38C. The aspiration pressure of water vacuum pump was set at 100 to 150 mm Hg. Duringaspiration, a scraping motion within the follicle dislodged the cocyte from the follicular wail. Afterbrief sedimentation (2 to 3 min), the pellet (5 to 10 ml) was collected and esamined to locate theoocytes under a stereomicroscope equipped with an heating stage set at 38C. We didnt search forthe oocytes in the remaining supematant because previously we had noted a very low oocyterecovery rate (3 to 5%) and we wished to shorten the interval from collection to IVM culture.Oocytes were collected in TCM 199 supplemented with 30% ECS or EMS, checked for theirmorphology, and those selected for culture were washed 4 times in the same medium. Eventhough the oocytes were kept at 38C during all the procedures, the overall time from collection toculture was never longer than 30 min.

Oocyte Classification and SelectionCumulus-oocyte complexes were classified, based on their cumulus morphology, into 7categories according to the criteria established by Liebfried and First (32) for bovine oocytes, andwere further subdivided as suggested by Hinrichs et al. (18) for equine oocytes: 1) compactcumulus - a sheet of compact cells completely surrounding the oocyte like a hood; 7) partial

cumulus - a compact cumulus only partially covering the oocytc; 3) corona radiata - oocyte withonly the corona radiata without a cumulus; 4) denuded - cumulus and corona cells completelyabsent; 5) partially expanded - an expanding cumulus but still appearing cellular; 6) totallyexpanded - a completely expanded cumulus with a sparsely cellular gelatinous cloud aroundoocyte, and 7) degenerated - clumped, dark cumulus cells. We selected only those oocytes with acompact cumulus and showing a homogeneously appearing cytoplasm, as suggested by Leibfriedand First (32) in the bovine.Sera Preparation

Blood samples were collected from echographically monitored estrus mares on the day beforeovulation and from cows on the day of estrus. The blood was centrifuged twice at 500 s g for 10min and the serum was heat inactivated at 56C for 30 min, aliquoted and frozen at -30C until use.In Vitro Maturation

The in vitro maturation method was the same as previously used for bovine oocytes in ourlaboratory (15). The basic medium was TCM 199 (Sigma M-0148, Milan, Italy) with Earles saltsbuffered with 4.43 mM Hepes (Sigma H-9136) and 33.9 mM sodium bicarbonate (Sigma S-5761)and supplemented with 0.1 g/l L-glutamine (Sigma G-7513) 1 mM sodium pyruvate (Sigma P-X56), 3.91 mM calcium lactate (Serva Feinbiochem GmbH & Co Heidelberg, Germany No.29760) and 50 ug/ml gentamycine (Sigma G-1772), as described by Berg and Brem (3). Afterpreparation, pH was adjusted to 7.18 and medium was filtered through 0.X? pm filters (LidaManifacturing Corp., Kcnosha WI, USA No. 5003-o). Then 30% of either EMS or ECS,


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gonadotrophins (10 &ml ovine FSH and 20 pg/mI ovine LH, NIDDK, NHPP, Baltimore, MD,USA) and 176 estradiol (1 pglml, Sigma E- 3357) were added. Medium was filtered again andallowed to equilibrate for 1 h under 5% CO? in air before being used. The oocytes were culturedfor 24 to 36 h at 38SC under 5% CO? in air. The four-well multidishes (Nunc IntermedRoskilde, Denmark No. 176740) mere used as culture containers into which 10 to 20 oocytes wereplaced in 400 ~1 of medium per well and covered with pre-equilibrated lightlveight paraffin oil(Sigma M-3516).Sperm In Vitro Capacitation and Insemination

Semen samples (0.4 ml/straw) frozen at concentration of 1 s 108 sperm cells/ml were rapidlythawed in a water bath at 37C. Total motility after thawing was 70%. with 50 to 60% progressivemotility. Sperm cells were prepared using the swim-up procedure described by Parrish et al. (38)in Tyrode-Lactate Medium modified for sperm treatment (Sp-Talp, for details of chemicalcomposition, refer to Reference 39). Semen was layered (0.2. ml/tube) in conical Falcon tubes(17~170 mm) under 1 ml Sp-Talp and incubated in a tilted position at 38SC for 1 h under 5%COzin air. The top (0.4 to 0.5 ml of medium) from each tube containing motile spermatozoa wasremoved, pooled and centrifuged (300 s g) for 10 min. The sperm pellet was resuspended in thesame medium up to 1 ml and was equilibrated for 5 min at room lemperature. Additional medium(4 ml) was added and the whole was centrifuged (300 s g) for 10 min. The supernatant wasdiscarded and the pellet l!as resuspended for a total of 100 ~1, then the concentration and motilitywere calculated. The total sperm concentration recovered after swim-up, per thawed straw, was1~10 of sperm cells/ml. The total percentage of motile spermatozoa was 80 to 90%, with 30 to40% progressively motile sperm cells. The final sperm concentration of 1x106, 5~10~ or 1x10sperm cells/ml \vas added to 400 ~1 of culture medium containing cxxytes for insemination in four-well multidishes. The IVF medium (Fert-Talp, whose chemical composition is described inReference 39) was modified in our procedure by eliminating glucose, pcnicillamine, hypotaurineand epinephrine whereas heparin (Sigma H-3393 ) was added at final concentration of 1 &ml.The sperm cells and oocytes (10 to 3-Ooocytes/well) were kepl together for 34 h at 38SC under5% CO2 in air.Maturation and Fertilization Assessment

Methods used to categorize maturation and fertilization were the same as we previously used inthe bovine (16). Oocytes submitted to IVM trials (Esperiment 1) were first assessed for thecumulus mass expansion under the stereomicroscope (30 to 60 magnification) and then denudedby incubating 3 to 3 min in 0.3% hyaluronidase (Sigma H-3506) followed by the mechanicalremoval of cumulus cells. Oocytes were examined for the first polar body extrusion and then fixedin ethanokacetic acid (3: 1) for 24 h, stained with 1% Lacmoid (Sigma L-7512) m 45% acetic acidand examined under a phase-contrast microscope (s 400) to visualize the nuclear structures.Nuclear chromatin status was classified as follows: germinal vesicle; meiosis resumption,comprised of stages from germinal vesicle breakdown to telophase I; and complete maturation atmetaphase II with the first polar body extruded. Oocytes showing either muliipolar meiotic spindleor irregular chromatin clumps were considered to be abnormal. In Experiment 3 fertilization wasconsidered to be normal when the second polar body extrusion or cleavage under thestereomicroscope or 3 pronuclei with sperm tail remnants in the ooplasm or a normal nucleus ineach blastomere were observed after fisation and staining. The frequency of sperm headdecondensation, a single pronucleus with sperm head in the cytoplasm and polyspermy were alsoevaluated.Esperiment 1

The objective of this experiment was to compare the effects of IVM medium supplementationwith EMS or ECS on maturation. Oocytes were randomly allocated to IVM medium containing


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Theriogenology 551

either 20% EMS or 20% ECS and cultured for 24 to 26 h. Each group in the experiment werereplicated 4 times.Experiment 2

Based on the results of Experiment I, IVF was conducted to determine if 1) increasing spermconcentrations (1x1@, 5~10~ and lx107 sperm cells/ml); 3) IVM medium supplementation with30% EMS or ECS; or 3) partial mechanical dispersion of cumulus mass, carried out before IVFincubation, would intluence the fertilization rates of equine oocytes. Group- 1 oocytes which hadbeen matured in IVM medium supplemented with ECS were randomly divided into 3 subgroupsand fertilized with spermatozoa at different concentrations: lxl@(shown in Table 2 as la); 5x106(Table 2, 1b) and 1~10 (Table 1, lc) sperm cells/ml. Group-? oocytes were matured in vitro inmedium containing EMS and fertilized with lxl@sperm cells/ml. Group-3 oocytes were maturedand fertilized in vitro as in Group 2, except that the cumuli were partially removed with a finelydravvn Pasteur pipette before fertilization. Each experimental condition was replicated 3 times. Inone replicate of Group 3, the oocytes were allon,ed to develop further after IVF for 72 h at 385Cunder 5% COZ in air in 20% EMS supplemented IVM medium vvithout hormones but in presenceof granulosa cells from the cumulus (3,15). Embryonic development was evaluated at successive14-h interv.als. The uncleaved ova vvere removed from culture and fixed as described previously.Fertilization rates in this replicate included the oocytes that developed further into embryos as wellas those that were found uncleaved but with evident signs of fertilization after staining.Statistical Analysis

Continuity-adjusted Chi-square anaIysis was used, and results with PcO.05 were considered tobe statistically significant. In Erperiment 1 the results obtained with EMS and ECS werecompared. In Experiment 1 comparison was made with a 3x2 contingency table between Groupsla and lb, la and lc, and 1b and lc (Table 2) for different sperm concentrations; between Groupsla and 2 for effects of EMS as the IVM medium supplement; and between Groups 3 and 2 toevaluate the effects of partial cumulus mass removal b&ore fertilization.RESULTS

During the period of April to July 1994, ovaries from 151 mares were collected and processed.From the follicular fluid, 683 COCs (2.3 oocyteslovary) were recovered, 335 of which (49%)showed an intact cumulus (Category 1); 160 (27%) showed incomplete cumulus investment(Categories 2 to 4); 55 (8%) had a partially expanded cumulus (Category 5); 20 (3%) had acompletely espanded cumulus (Category 6); and 86 (13%) had degenerative signs on the cumuluscells (Category 7). All 335 Category-l oocytes were cultured (103 in Experiment 1 and 332 inEsperiment 2) and could be analyzed. Figure la shows a group of these oocytes.Espenment 1

The 3 different sources of serum (EMS and ECS) added to the IVM medium did notsignificantly influence the results (Table 1). After 14 to 36 h of culture, both groups showed highrates of cumulus mass expansion (87% vvith EMS vs 91% with ECS; Figure lb). After denudingmost of the oocytes showed an evenly granulated and distributed ooplasm, and only 19% (8/39) ofthe oocytes cultured with EMS and 12% (8/56) with ECS had uneven distribution of lipidicgranulae. These oocytes were also included in the results. High rates of nuclear maturation at themetaphase II stage with the first polar body were obtained with both sera. Figure 2. shows anoocyte that remained immature at the germinal vesicle stage, observed under a stereomicroscopeafter denuding (Figure ?_a), and under a phase-contrast microscope after staining (Figure 2b).Figure 3 shows an in vitro matured oocyte. In Table I, continuity-adjusted Chi-square values


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552 Theriogenology

Table 1. Effects of serum type on in vitro maturation of equine oocytes

Sem

No.(%) ofculturedoocytes

germinalvesicle

No.(%) of oocytes showing

meiosisa metaphase IIresumption with polar body abnormali ties

EMS 39 (loo) 3 (7.7) 3 (7.7) 32 (82.0) 1 (2.6)ECS 64(lOO) 2 (3.1) 6 (9.4) 56 (87.5) 0 (0)

EMS=Estrous mare serum; ECS=Ektrous cow serum. aComprised of stages from germinal vesiclebreakdown to telophase 1.In each column Chi-square analysis resulted in non significance.

between the 2 treatment groups were calculated by comparing frequences of each maturation stageagainst the remainder (e.g., Metaphase II with polar body against that of the overall of germinalvesicle, meiosis resumption and abnormalities: Chi-square = 0. 22, NS). All the comparisons werenot significant.Experiment 2

These experiments were conducted as successive sets of trials based on the results obtained inExperiment 1. First the effects of increasing sperm concentrations were investigated. Then inlanes la,lb and lc of table 2 the results of IVF experiments conducted with lx106, 5x106 and1x107 sperm cells/ml, respectively, were described. No statistically significant differences wereobserved between the sperm concentrations used in terms of either monospermic or polyspermicpenetration rates. An example of a normal in vitro fertilized oocyte is shown in Figure 4. Once weobserved these results, any effects of EMS supplementation in the IVM medium on the fertilizationrates were investigated. The comparison between Groups 2 and la of the same table indicates thatthe use of homologous serum in the maturation medium does not influence subsequentfertilization. A statistically significant difference (PcO.05) was observed in the last set of trials, inwhich a significantly higher normal fertilization rate was obtained after partial cumulus massremoval than that obtained in oocytes fertilized with a whole cumulus. A low incidence ofpolyspermy as well as delayed pronuclear development (oocytes with a decondensing sperm heador with only a single pronucleus and sperm tail) were observed in all examined parameters (Table2). In a replicate of Group 3 in which 12 occytes were fertilized after cumulus removal and furtherculture for 72 h, two embryos (1 at the l-cell stage and the other at the 4-cell stage) were obtained(17% cleavage), with a normal nucleus in each blastomere being observed after staining (Figures 5and 6). Continuity adjusted Chi-square values were calculated by comparing the frequency of eachstage against the total number of frequencies of the remaining stages in the same line.

DISCUSSIONIn the mare seasonal polyestrous reproductive activity related to latitude and photoperiod is wellknown, and consequent variations in follicular development may influence the number of femalegametes available for use in reproductive technologies. Oocyte yield per follicle does not seem tobe conditioned by seasonal variations. In fact, Palmer et al.(37) observed no different oocyteyields per follicle between spring and autumn in pony mares. On the other hand, our group (34)


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Theriogenology

Figure 1.

Figure 2.

Figure 3.

Equine cumulus-owyte complexes of wt. I. a) At collection. b) Note the cumulus massexpansion after 3-4 to 26 hours of IVM culture (I 60).

Equine oocyte remained immature 24 to 25 hours after IVM culture. a) The peri\,ltelllnspace obser\.ed in \ivo re\.ealed to be minimum (x 200). b) Oocyte at germinal \xsiclestage (GV) obsened after fixing and staining lvith Lacmoid (I 400).

Equine oocyte matured in vitro for 14 to 26 hours. a) The arro\\ indicates the first polarbody estruded (z 300). b) Set of chromosomes in Metaphase II (MII) and the first polarbody (PB) after staining nith Lacmoid (s 400).


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Theriogenology

Figure 4. Fertilization of in vitro-matured equine oocyte. a) Two polar bodies observed after 24hours of IVF incubation (s 300). b) Two pronuclei (PN), sperm tail (arrow) in theooplasm and 2 polar bodies (PB) after staining with Lacmoid (s 400).Figure 5. Two-cell stage equine embryo issuing from in vitro-matured and fertilized oocyte. a)Two regular blastomeres 24 hours after in vitro embryo culture (s 300). b) Bothblastomeres contain 1 nucleus (N) stained with Lacmoid (x 400).Figure 6. Four-cell stage equine embryo 48 hours after in vitro culture obtained from in vitromatured and fertilized oocyte. a) Three blastomeres are well shown and the fourth is notin focus (s 200). b) Even though only 3 nuclei (N) could be photographed (x 400) all 4\sere observed after staining with Lacmoid.


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556studying follicle size distribution over the course of a year, observed a high frequency of 5 to 29-mm follicles during spring and a high frequency of >30-mm follicles during summer. Thedistribution of different COC categories, based on cumulus and cytoplasm appearance, alsodiffered among seasons; however, oocytes with a compact cumulus and a homogeneous cytoplasmcould be harvested all year long. Thus we chose the spring and early summer periods in which toincrease the number of cumulus-intact oocytes available for experiments. Moreover, oocytesduring this time may also be more sensitive to hormonal conditions in culture media

The oocyte recovery rate by aspiration (3.3 oocytes/ovary) in our study was higher than the3.08 oocyteslmare reported by Okolski et al. (35) and 1.75 oocytes/ovary by Choi et al. (10). Thiscould be related to the season in which our study had been conducted as well as to details in theretrieval methodology such as needle size (18 gauge), low aspiration pressure (100 to 150 mmHg), and scraping motion to better dislodge the oocyte from the follicular wall. The study of Choiet al. (10) was conducted during the same period as our study, but this information was notavailable for the study of Okolsky et al. (35). In the 2 studies above, the follicles were aspiratedwith a syringe fitted to a thinner needle than the one we used (11 gauge) and without control ofaspiration pressure ky water pump. With our aspiration method, we were able to obtain a highnumber of oocytes with compact cumulus investment (49% from follicles -20 mm). Our recoveryresults were higher than those of Choi et al. (34%; 10) and Okolski et al. (29.3%; 35) of compact-cumulus oocytes following aspiration. In the last study (35), a large number of remaining oocyteshad no cumulus cells. Recently, Alm and Torner (1) reported 42.9% cumulus-compact oocytesfrom follicles cl.5 mm and 43.7% from those up to 30 mm with aspiration using an l&gaugeneedle, these are comparable to our results. Others have reported high compact oocyte recoveryrates using retrieval methods other than aspiration: 89% by rupturing isolated follicles (35) and55% by slicing the follicles (10). However, these methods are labor intensive and time-consuming, and since the time between collection and culture is a very important factor in equinematuration and fertilization, aspiration was our preferred method. Few oocytes (3%) with acompletely espanded cumulus and with cells in a gelatinous matrix were found. This was probablyrelated to the size of the punctured follicles.In our present study only cumulus-compact cocytes were selected for in vitro maturation since anumber of observations in the bovine (reviewed by Brackett and Zuelke; 6) reported the presenceof an intact complement of cumulus cells surrounding the oocyte and a homogeneously appearingooplasm as the best indicators of an immature oocytes ability to undergo maturation andembryonic development. In the equine, however, some studies have shown no difference inmaturation to the MI1 stage of oocyles with compact and expanded cumulus masses (43,29), thussuggesting the possibility that oocytes can be recovered with an expanded cumulus followingIVM-IVF. In our study, espanded oocytes represented only a limited percentage of the collectedoocytes (8%, 55/683 with a partially expanded and 3%. 201683 with a completely espandcdcumulus mass), which is probably related to the season as well as to the size of the aspiratedfollicles, and these were excluded from the study.The high maturation rates obtained in our study after 24 to 26 h of culture are in contrast withdata from some authors (43,41,40,10,29,1), who reported comparable rates only after 30 h, butare in agreement with the data of Willis et al. (43_),who observed no significant differences in MI1formation, after 15 and 32 h of culture, in TCM supplemented with serum on the day of ovulation,and with that reported by Del Campo ct al. (14), who observed that the maturation rate did notchange between 24 and 48 h of culture in TCM 199 supplemented with gonadotrophins. In aprevious experience (12). we observed that 48 h of IVM incubation resulted in a marked increase(38%) of oocytes showing uneven distribution of dark lipid granulae in the cytoplasm. Since thesesigns are indicative of oocyle degeneration in the bovine (32), prolonged culture time was avoidedin our study, thus \ve obtained low rates of cytoplasmic degeneration following IVM (13% withECS and 19% with EMS).


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Theriogenology 557

The results of Experiment 1 show that both ECS collected on the day of estrus and EMScollected on the day before ovulation induce high nuclear maturation of compact-cumulus equineoccytes recovered during the breeding season. No other study has reported data on the comparisonbetween these 2 sera on the maturation rate of equine oocytes. Our results were higher than thosereported previously either for EMS or other kinds of sera. Zhang et al. (43) reported a 63%maturation rate with 30% late estrous mare serum and 64% with 10% fetal calf serum, Willis et al.(41) obtained their best results (67%) using 15% serum from the first day of estrus; Grondahl etal. (35) observed a 64% maturation rate with 5% ECS and 53 to 79% with 10% fetal equineserum; Alm and Torner (1) obtained a 73% MI1 formation rate with 10% horse serum. The onlycomparable maturation rate to ours (86%) was reported by Shabpareh et al. (40), which wasobtained with 10% EMS, but this maturation rate had been reached after 30 h of IVM culture.Perhaps the high serum concentration (20%) in our study allowed a high number of oocytes tomature in a short (14-h) period of culture. High MI1 rates observed in our study could have beendue also to the addition of exogenous gonadotrophins in the IVM medium. The same IVM mediumcomposition allowed us to obtain 16% blastocyst formation in the bovine after IVF and IVC (15).Rapid aspiration as well as controlled temperature during all procedures could probably have beenvalid contributions to improving the results. In our experience, IVM medium supplementation withEMS did not improve the fertilization rate compared with ECS (12.2 vs 11.9%, respectively).Grondahl et al. (35) observed higher fertilization rates vvhen ECS was used in the IVM medium(15 to 26%) in comparison with fetal equine serum (0 to 19%). Estrus mare serum, with thepeculiar estrous and preovulatory LH peak duration in this species, could contain variableconcentrations of factors supporting the in vitro maturation of the cumulus-oocyte complex, inboth somatic and nuclear compartments, with consequent influence on the oocytes ability toundergo pronuclear development.

The use of frozen-thawed semen ensures high repeatability of IVF systems and gives theadvantage of long-term storage of viable sperm cells with the same characteristics. Only a smallnumber of studies have used frozen-thawed semen in the equine (37,25,11); fresh semen has beenthe choice of most of authors. Swim-up separation in Talp-Hepes and heparin treatment tocapacitate in vitro bull semen has been shown to be a highly efficient and repeatable system forbovine IVF (38). Although calcium ionophore has been suggested by many authors as one of themost promising treatments for obtaining in vitro capacitation of stallion semen, data on in vitrofertilization are not yet conclusive and need further investigation. Moreover, the use of calciumionophore may have limited application with stallion semen. Farlin et al. (32) observed anincreased acrosome reaction rate for stallion spermatozoa after 2 h of incubation with increasingionophore concentrations up to 5 FM, which may be of interest for techniques related to oocytemicromanipulation but not for conventional IVF. In addition, increasing the ionophoreconcentration resulted in reduced motility in their study. Research on assisted fertilizationtechniques ha1.e been begun in the equine. Partial zona dissection was not more effective than IVFof zona-intact oocytes. Treatment of partial or total zona removal yielded high penetration mtes (52and 86%, respectively) but was associated with high polyspermy (13 and 29%, respectively; 1 I).Insemination of zona drilled oocytes with calcium ionophore-treated spermatozoa and subsequentco-culture of embryos using bovine oviduct cells along with mouse embryos resulted in a highcleavage rate, reaching 79% (33), but information on the fertilization rate is laking in this study.Our IVF results can be compared with that of other studies that used different experimentalprocedures: Palmer et al. (37) used frozen-thawed semen and Talp medium, but no fertilizationoccurred among any of the 5 in viva matured oocytes; Del Campo et al. (13) and Zhang et al. (44)both using calcium ionophore-treated semen obtained 15 and 33% fertilization rate, respectively,which was comparable to that in our study but obtained with heparin-treated semen. Grondahl etal. (25) also evaluated the effects of ? different semen sources and treatment methods and obtained26% fertilization with frozen-thawed spermatozoa exposed to calcium ionophore, and 3% withfrozen-thawed epididymal spermatozoa treated with heparin in Talp medium. Choi et al. (11)obtained only a 1% monospermic penetration rate when oocytes surrounded by an intact cumuluswere fertilized with semen treated with caffeine and calcium ionophore.


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558 TheriogenologyThe effects of sperm concentration were investigated to increase the number of capacitatedsperm cells for fertilization because of the low recovery rate of motile sperm cells after swim-uppreparation (1x10 sperm cells/ml with SO to 90% motile spermatozoa). In a previous study of thebovine, using straws cryoconditioned at the same sperm concentration (i.e., IxlOsspermcells/ml), we were able to recover 3 to 3x107sperm cells/ml with 80% motility after swim-up (31).

The present study, which to our knowledge is the first to evaluate the effects of semenconcentration on equine IVF, showed no significant differences in monospermic and polyspermicpenetration rates at the sperm concentrations tested. Final sperm concentrations used in previousstudies in the equine species ranged from 05x106 (37) to I to 3x107/sperm cells/ml (1 l), but theseresults are difficult to compare when considering the variables in sperm preparation procedures bythe different authors. However, even with higher sperm concentrations than those used(lxl@/sperm cells/ml) in our study the fertilization rate was still lower than ours (1% penetrationrate of compact-cumulus oocytes; 11).The effects of reducing the amount of cumulus surrounding the oocytes before and aftermaturation on subsequent fertilization and embryo development was examined in the bovine byHawk et al. (16), who demonstrated that detaching most of the cumulus cells from oocytes, either

by pipetting or by hyaluronidase treatment, before insemination could markedly increase thefertilization rate (84 and 74% vs 57%). This procedure is often described in studies aimed toimprove the yield of bovine blastocysts obtained with the IVF technology (17,24,7). Under ourexperimental conditions this technique significantly improved the results, probably enhancing theprogression of equine spermatozoa through the cumulus mass, so favoring sperm-oocyte binding.In the equine, no reports of partial cumulus cell removal by pipetting in conventional IVM-IVFprocedures are available. As far as enzymatic digestion of the whole cumulus is concerned, weobserved that this was detrimental on the fertilization of equine oocytes (48, 3/49 after 0.1%hyaluronidase incubation for 5 min followed by pipetting; unpublished data). This result, which isin agreement with the study of Choi et al. (3% penetration rate for oocytes denuded with 0.35%hyaluronidase; 1 l), may indicate that cumulus cells have an important tunction in normal spermpronucleus formation, as had already been demonstrated in the bovine (8).Although the fertilization mtes in the equine obtained in our present study can be considered to beencouraging, they are still low compared to those obtained for most farm animals under similarexperimental conditions. This may, be due to an incomplete maturation of the cumulus-oocytecomplex, in particular at cytoplasmic level, probably related to the particular requirements of theequine oocyte to be matured in vitro, which has not been well known until recently. This factorcould have implications on the oocytes ability to undergo pronuclear formation. Intracytoplasmicsperm injection of in vitro-matured equine oocytes is under investigation in our unit in order toincrease the fertilization rate and to evaluate cytoplasmic maturity (9,17,18). Preliminary resultsindicate that the oocytes collected with an expanded cumulus investment had a much higherfertilization rate with assisted fertilization technology than micromanipulated compact cumulusoccytes (79% vs 26%, respectively; 18). These results indicate that the ICSI procedure allowsovercoming difficulties in sperm penetration but also that cytoplasmic maturation of oocytes from

the compact cumulus may not be sufficient for successful IVF under the maturation conditionsused in our study as well as in an earlier studies.In conclusion, although it is possible to achieve a high fertilization mte with micromanipulatingtechniques and testing in a large and varied oocyte population, preliminary results of theexperimental protocol we describe in this study may offer a point from which factors involved inIVMlIVF can be further investigated to increase our knowledge of horse reproductive physiology.

REFERENCES1. Alm H, Tomer H. In vitro maturation of horse oocytes. Theriogenology 1994; 42345349.2. Ams M J, Shepherd R E. Percoll gradient selection of equine spermatozoa enhances

ability to bind and penetrate the zona pellucida. Theriogenology 1994;41: 15~ abstr,


13/14


14/14

560 Theriogenology25.

26.

27.28.29.

30.

31.32.33.

34.35.

36.

37.38.

39.40.41.42.43.44.

Grondahl G, Host T, Bruck I, Viuff D, Bezard J, Fair T, Greve T, Hyttel P. In vitroproduction of equine embryos - Preliminary data. Proc VI Int Symp Equine Reprod 1994;75-76.Hawk HW, Nel ND, Waterman RA, Wall RJ. Investigation of means to improve rates offertilization in in vitro matured/in vitro fertilized bovine oocytes. Theriogenology 199238:989-998.Hawk HW, Wall R J. Improved yields of bovine blastocysts from in vitro producedoccytes. I. Selection of oocytes and zygotes.Theriogenology 1994; 41: 1571-1583.Hinnchs K, Kenney DF, Kenney RM. Aspiration of oocytes from mature and immaturepreovulatory follicles in the mare. Theriogenology 1990;34: 107- 112.Hinrichs K, Schmidt AL, Friedman PP, Selgrath JP, Martin MCI. In vitro maturation ofhorse oocytes: characterization of chromatin configuration using fluorescence microscopyBiol Reprod 1993;48:363-370.Hofferer S, Lecompte F, Magallon T, Palmer E, Combamous Y. Induction of ovulationand superovulation in mares using equine LH and FSH separated by hydrophobicinteraction chromatography. J Reprod Fertil 1993;98597-602.Lacalandra G M, Dell Aquila M E, Fusco S, Sciorsci R L, Perrone P, Minoia P. In vitrofertility test of bull semen. Proc 12th Int Congr Anim Reprod 1992;2:656-658.Leibfried L, First N L. Characterization of bovine follicular oocytes and their ability tomature in vitro. J Anim Sci 1979;48:76-86.Li LY, Meinjtes M, Graff KJ, Paul JB, Denniston RS, Godke RA. In vitro fertilization anddevelopment of in vitro matured oocytes aspimted from pregnant mares. Proc VI IntSymp on Equine Reprod 1994;77-78.Minoia P, Cinone M, Sciorsci RL, Perrone P. Attivita follicologenetica nel torso dellannonella cavalla. Proc XLVII Nat Congr Vet Sci 1993;521-534.Okolski A, Babusik P, Tischner M, Lietz W. Evaluation of mare oocyte collection methodsand stallion sperm penetration of zona-free hamster ova. J Reprod Fertil,l987;(Suppl35):191-196.Okolski A, Bezard J, Magistini M, Palmer E. Maturation of oocytes from normal and atreticequine ovarian follicles as affected by steroid concentrations. J Reprod Fertil 1991;(Suppl44):385-392.Palmer E, Bezard J, Magistrini M, Duchamp G. In vitro fertilization in the horse. Aretrospective study. J Reprod Fertil 1991;(Suppl44):375-384.Parrish J J, Susko-Parrish J L, Leibfried-Rutledge ML, Critser ES, Eyestone W H,First NL. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 1986;25:591~600.Parrish J J, Susko-Parrish J L, Winer M A, First N L. Capacitation of bovine sperm byheparin. Biol Reprod 1988;38: 1171-l 180.Shabpareh V, Squires E L, Seidel E Jr, Jasko D J. Methods for collecting and maturingand maturing equine oocyles in vitro. Theriogenology 1993;40: 1161- 1175.Willis P, Fayrer-Ho&en R A, Candle A B. Effect of serum on in vitro maturationof equine oocylcs. Theriogenology 1990;33:345.Willis P, Candle AB, Fayrer-Ho&en RA. Equine oocyte in vitro maturation: influence ofmaturation: influence of sera, time and hormones. Mol Reprod Dev 1991;30:360-368.Zhang JJ, Boyle MS, Allen WR, Galli C. Recent studies on in vivo fertilization of in vitromatured horse oocytes. Equine Vet J 1989;(Suppl8):101-104.Zhang JJ, Muzs LZ, Boyle MS. In vitro fertilization of horse follicular oocytes maturated invitro. Mol Reprod De\, 1990;36:361-365.

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