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Biology of Reproduction 67, 256-262 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

In Vitro Fertilization of In Vitro-Matured Equine Oocytes: Effect of Maturation Medium, Duration of Maturation, and Sperm Calcium Ionophore Treatment, and Comparison with Rates of Fertilization In Vivo after Oviductal Transfer1

K. Hinrichs2,a, C.C. Lovea, S.P. Brinskob, Y.H. Choia, and D.D. Varnerb

a Departments of Veterinary Physiology and Pharmacology and b Large Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843-4466


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 µM vs. 7.14 µM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 µM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40–44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.

embryo, fertilization, in vitro fertilization, oviduct, sperm capacitation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In vitro fertilization (IVF) has had limited investigation in the horse. Although the first paper reporting IVF of equine oocytes was published more than 12 yr ago [1], only 11 additional studies involving standard (nonassisted) IVF in the horse have subsequently been reported [2–12], many of which use IVF as a control for more effective methods of fertilization such as zona removal or intracytoplasmic sperm injection (ICSI) [6, 9–11]. Two foals have been born from IVF, both of these from oocytes matured in vivo [4, 5]. Reported fertilization rates (penetration with pronucleus formation) after standard IVF have ranged from 4% [6] to 33% [2, 8], but only one laboratory reporting a high fertilization rate has published subsequent studies utilizing the same IVF technique, and the rates were not repeatable [9, 10]. The major barrier to IVF in the horse appears to be penetration of the zona pellucida, as fertilization rates are greatly increased by partial or total zona removal or zona drilling, and in fact, polyspermy results if this is done [6, 13].

It is possible that the failure of sperm penetration of horse oocytes is related to changes in the zona pellucida in vitro, which render it resistant to penetration. Zona hardening may occur when cortical granules are prematurely released from the oocyte in culture due to aging of the oocyte or to inappropriate culture conditions [14, 15]. Dell'Aquila et al. [11] evaluated zona hardening in horse oocytes after in vitro maturation (IVM). They reported that culture with fetuin, a protein that prevents zona hardening in mouse oocytes [16], reduced zona hardening of horse oocytes in culture but did not increase IVF rates.

The duration of oocyte maturation associated with optimum IVF rates in the horse is not known. Horse oocytes reach metaphase II at 24–36 h, and oocytes initially having compact (Cp) cumuli take longer to mature than do oocytes having expanded (Ex) cumuli [17, 18]. Culture beyond the time needed for oocytes to reach MII may be beneficial to IVF results. The first successful IVF was reported in oocytes recovered from the follicle 35 h after gonadotropin stimulation and cultured an additional 6–12 h before IVF; a live foal was produced from this protocol [4]. Predicted rates of IVF (penetration with pronucleus formation) were higher in Ex than in Cp oocytes (30% and 14%, respectively) and tended to be higher in oocytes matured 26–40 h than in oocytes matured 18–24 h for both cumulus types [12].

Male pronucleus formation after ICSI was also higher in Ex than in Cp oocytes [9]; when oocytes were matured with 20% follicular fluid from preovulatory follicles, pronucleus formation rates for Cp oocytes after ICSI were improved [10]. Similar supplementation did not increase rates of standard IVF. Supplementation of maturation medium with epidermal growth factor (EGF) has been shown to increase fertilization rate in pigs [19] and rate of pronucleus formation after ICSI in humans [20]. The effect of EGF on oocyte IVM has been investigated in horses [21, 22], but no work has been done on the effect of EGF on rates of IVF in the horse.

Pregnancy rates after transfer of in vivo-matured oocytes to the oviduct of inseminated recipient mares are excellent (75% to 82%) [23, 24]. However, similar transfer of IVM oocytes resulted in low blastocyst recovery (5/29, 17% [17]) and a low pregnancy rate at Day 16 (4/40, 10% [25]). It is unknown whether the poor embryo development associated with transfer of IVM oocytes in these studies was due to failure of fertilization or failure of development after fertilization. Research in IVF in the horse cannot readily progress until the competence of IVM oocytes for fertilization is ascertained.

Most studies reporting penetration and pronucleus formation after IVF in the horse have used calcium ionophore (CaI) treatment to capacitate sperm [1–4, 7, 12]; fertilization has also been achieved using heparin treatment of frozen-thawed sperm [8–12]. The optimal concentration and duration of exposure to ionophore treatment is not known; however, relatively good fertilization rates have been reported when using 3 µM CaI for 5 min (15% to 26% penetration [7]) and 7.14 µM CaI for 10 min (33% decondensation/pronucleus formation [2]; 21% pronucleus formation/cleavage [12]). Fertilization rates of equine oocytes exposed to untreated sperm are essentially 0 [2, 4, 13].

The objective of this study was to evaluate the effects of duration of oocyte maturation (to the time of polar body extrusion, 24 h, or "aged," 42 h), maturation medium, calcium ionophore concentration, and sperm preparation technique on IVF rates and to compare these with the rate of fertilization seen after transfer of IVM oocytes to the oviducts of mares for in vivo fertilization. The extent of embryo development 40–44 h after fertilization of IVM oocytes in vivo was also determined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
General Methods

Horse ovaries were obtained from 2 horse slaughterhouses and were transported 3–4 h in PBS at room temperature (22–25°C) to the laboratory. All visible follicles were opened with a scalpel blade, the granulosa layer of each follicle was scraped using a 0.5-cm bone curette, and the cells were washed from the curette into individual Petri dishes using holding medium (M199 with Hanks salts and 12.5 mM Hepes [Gibco Life Technologies, Inc., Grand Island, NY] with added ticarcillin [0.1 mg/ml; SmithKline Beecham Pharmaceuticals, Philadelphia, PA]).

The contents of the Petri dishes were examined using a dissection microscope at 10–20x magnification. Oocytes were classified as being compact (Cp) or expanded (Ex) on the basis of both cumulus and mural granulosa cell morphology. If any aspect of the recovered granulosa cells showed signs of expansion (ranging from cells protruding from the surface layer to full expansion with copious intercellular matrix), the oocyte was classified as Ex even if the cumulus appeared to be compact. For this study, only Ex oocytes were used. Oocytes were maintained in holding medium at 22–25°C until all oocytes had been collected (0–3 h).

Semen from 1 fertile Quarter Horse stallion was used for all experiments. The basic sperm processing media used in experiment 1 were modified Tyrodes media for sperm culture (Sp-TALP) and in vitro fertilization (Fert-TALP), as described by Parrish et al. [26]. The sperm processing media used in experiment 2 consisted of those used in experiment 1 as well as capacitating medium used by Alm et al. [12], based on that described by Lonergan et al. [27], which we termed CAP. All media were prepared without bovine serum albumin (BSA), sterilized by filtering through a 0.22-µm filter, then frozen in 50-ml aliquots at -80°C until used. On the day of use, aliquots of media were thawed, BSA was added, medium osmolality was measured, and then the pH of the media was adjusted to 7.4 using NaOH and HCl. All media were equilibrated for 6–12 h at 38.2°C in a humidified atmosphere of 5% CO2 in air prior to use.

Experiment 1: Effects of Duration of Culture, Maturation Medium, and Calcium Ionophore Exposure on Fertilization Rates

Oocyte maturation After collection, oocytes were placed randomly into 1 of 4 media for culture 1) matM, M199 with Earle salts (Gibco), with 25 µg/ml gentamycin (Gibco), 10% FBS, and 5 µU/ml FSH (Sioux Biochemical, Sioux Center, IA); 2) EGF, matM with 50 ng/ml EGF (Gibco); 3) 20% FF, M199 with Earle salts (Gibco), and 25 µg/ml gentamycin (Gibco) with 20% follicular fluid recovered from a preovulatory follicle in vivo 24 h after administration of hCG; or 4) 100% FF, follicular fluid as described above, with 25 µg/ml gentamycin.

Oocytes were distributed to all four media on each collection day. Oocyte-cumulus complexes were incubated in droplets of medium at a ratio of 10 µl medium per oocyte at 38.2°C in a humidified atmosphere of 5% CO2 in air for either 24 or 42 h. At the end of the culture period, oocytes were transferred to 100-µl microdroplets of Fert-TALP under light white mineral oil. Ten to 15 oocytes were transferred per droplet.

Sperm preparation On each day fertilization was performed, a sperm-rich ejaculate (first 3 jets of semen) was collected from the stallion by use of a Missouri-model artificial vagina (Nasco, Ft. Atkinson, WI). The gel-free semen was mixed 1:2 (vol:vol) with Sp-TALP, then transferred to 15-ml capacity centrifuge tubes and centrifuged at 500 x g for 6 min. The supernatant was aspirated, and the sperm pellet was resuspended in fresh Sp-TALP to obtain a final concentration of approximately 80 x 106 sperm/ml. Within 15 min following collection, the Sp-TALP-extended semen was aliquoted into 1.5-ml aliquots and dimethylsulfoxide (DMSO) or DMSO containing calcium ionophore (A23187, Calbiochem, La Jolla, CA; stock concentration of 10 mM in DMSO) was added and the sperm was further incubated to result in 3 treatments 1) CaI-3, 3.0 µM calcium ionophore for 3 min; 2) CaI-7.14, 7.14 µM calcium ionophore for 10 min; or 3) DMSO, DMSO without calcium ionophore (0.06% [vol:vol] or 0.07% [vol:vol] for controls for CaI-3 and CaI-7.14 treatments, respectively) for 3 or 10 min. All incubations were performed at 38.2°C in a humidified atmosphere of 5% CO2 in air. Following incubation, the aliquots were centrifuged at 320 x g for 2 min, the supernatant was removed, and the sperm pellet was resuspended to approximately 60–100 x 106 sperm/ml in Fert-TALP.

Motion characteristics of sperm suspensions were assessed after exposure to Sp-TALP alone and after exposure to the 3 DMSO/ionophore treatments (DMSO only, CaI-3, or CaI-7.14, all followed by resuspension of sperm in Fert-TALP media) using a computerized spermatozoal-motility analyzer equipped with a heated stage (HTM IVOS, Version 10.8; Hamilton Thorne Research, Beverly, MA). Four motility variables were evaluated 1) percentage of motile spermatozoa, 2) percentage of progressively motile spermatozoa, 3) mean curvilinear velocity of spermatozoa (VCL; µm/sec), and 4) amplitude of lateral head displacement (ALH, calculated by average sperm track width; µm). Data were analyzed using a general linear model procedure (SAS Institute, Inc., Cary, NC). Variables measured in percentages were transformed to angles corresponding to arcsine square root of percentage prior to variance analyses. The Student-Newman-Keuls statistic was used to separate main effect means when treatment F-ratios were significant ( P < 0.05).

Sperm treatments CaI-3 or CaI-7.14 were used for fertilization on separate days. Sperm concentrations were determined using the motility analyzer and an aliquot of the final sperm suspension containing 1 x 106 sperm (volume of 10–18 µl) was added to the 100-µl fertilization droplet containing the oocytes. The oocytes and sperm were incubated at 38.2°C in a humidified atmosphere of 5% CO2 in air for 24 h, then oocytes were removed and processed for assessment as described below. Four replicates of each sperm treatment were performed for each maturation time.

Experiment 2: Effect of Capacitating Medium and Sperm Incubation on In Vitro Fertilization

Oocytes were obtained as described for experiment 1. Based on the results of experiment 1, oocytes were matured in 100% FF for 42 h before fertilization. At the end of the culture period, oocyte-cumulus complexes were transferred to 100-µl droplets of Fert-TALP under oil (10–15 oocytes per drop). Oocytes were inseminated with sperm prepared as in the CaI-7.14 treatment in experiment 1 or with supernatant from that sperm treatment or with sperm prepared as described by Alm et al. [12] with or without incubation for 45 min after capacitation treatment, as detailed below.

Sperm preparation Sperm-rich ejaculates were collected as described in experiment 1. Aliquots of semen were mixed 1:2 (vol:vol) with Sp-TALP (treatment 1) or nonfat dry milk extender (E-Z Mixin CST; Animal Reproduction Systems, Chino, CA; treatment 2), then centrifuged at 500 x g for 6 min. The sperm pellet was then resuspended in the same medium to a concentration of approximately 80 x 106 sperm/ml. Aliquots (1.5 ml) of extended semen were recentrifuged at 320 x g for 2 min, then sperm pellets were resuspended to 1.5 ml in Sp-TALP containing 7.14 µM A23187 (treatment 1) or in CAP containing 7.14 µM A23187 (treatment 2) and incubated at 38.2°C in a humidified atmosphere of 5% CO2 in air for 10 min. Subsequently, the diluted semen was recentrifuged at 320 x g for 2 min, and the sperm pellet was resuspended in Fert-TALP (treatment 1) or CAP (treatment 2) to obtain a final sperm concentration of 60–100 x 106/ml. One aliquot of treatment 2 was used immediately, and 1 aliquot was incubated an additional 45 min at 38.2°C in 5% CO2 in air prior to addition to fertilization droplets (treatment 3). Sperm processed as above but diluted only with Sp-TALP at each step were evaluated as a control. Motility variables were assessed and analyzed for treatments 1–3 and the Sp-TALP control as described in experiment 1. An aliquot of treatment 1 was centrifuged and the supernatant passed through a 3-mm diameter 0.22-µm pore-size filter to obtain the medium for addition to fertilization drops in the supernatant treatment. For treatments 1, 2, and 3, an aliquot of the final suspensions containing 1 x 106 sperm (volume of 12–18 µl) was added to the 100-µl fertilization droplet containing the oocytes. For the supernatant treatment, a volume of supernatant equal to the volume used for treatment 1 was added to the oocyte droplet. The oocytes and sperm or supernatant were incubated at 38.2°C in a humidified atmosphere of 5% CO2 in air for 24 h, then oocytes were removed and processed for assessment as described below. Five replicates were performed; all sperm treatments were evaluated concurrently in each replicate.

Experiment 3: In Vivo Fertilization of IVM Oocytes

Oocytes were recovered from slaughterhouse-derived ovaries and were placed into matM or into 100% FF, as described for experiment 1. The oocyte-cumulus complexes were incubated for 24 h in droplets of 10 µl medium per oocyte under light white mineral oil at 38.2°C in a humidified atmosphere of 5% CO2 in air.

Four mares scheduled for euthanasia due to nonreproductive abnormalities were used as oocyte recipients. The mares' reproductive tracts were assessed via transrectal ultrasonography to determine the stage of cycle. Mares with corpora lutea visible on ultrasonography were treated with prostaglandin F2{alpha}, 10 mg i.m., 6–7 days before transfer. Mares were treated with a long-acting preparation of estradiol-17ß (estradiol in Saber, Thorn Bioscience, Lexington, KY), 100 mg i.m., 5–13 days before transfer.

On the morning of the day of transfer, recipient mares were artificially inseminated with a minimum of 500 x 106 sperm from the same stallion whose semen was used for the in vitro fertilization studies. Transfer of IVM oocytes to the recipient oviduct was performed via standing flank laparotomy, as previously described [24]. Oocytes matured in follicular fluid were transferred to one oviduct and oocytes matured in matM were transferred to the contralateral oviduct of the same mare. On the first transfer, 25 oocytes were transferred to each oviduct. On subsequent transfers, 12 oocytes were transferred to each oviduct. Mares were treated with antibiotics (ampicillin, 9 g i.v., immediately prior to surgery and b.i.d. thereafter until euthanasia) and flunixin meglumine (500 mg i.v., immediately prior to surgery and 12 and 24 h afterward).

Forty to 44 h after transfer, the mares were killed and the oviducts and ovaries removed. The oviducts were dissected free of excess connective tissue and the uterine end of the oviduct was cannulated using a blunt 25-ga needle. Each oviduct was flushed with at least 50 ml of Dulbecco phosphate-buffered saline. The flush medium was collected into Petri dishes, which were searched with the aid of a dissecting microscope to locate the oocytes/embryos. Oocytes that were flattened and misshapen or had pale or scant cytoplasm were considered to be the recipient mare's own oocytes retained from previous cycles. All collected oocytes and embryos were stained as described below and the chromatin configurations assessed.

Assessment of fertilization After fertilization, oocytes were denuded of cumulus by pipetting. Denuded oocytes and embryos were fixed briefly in buffered formal saline at 22–25°C. Fixed oocytes/embryos were labeled for chromatin evaluation by placing them on a glass slide with 6.5 µl of mounting medium (3:1 glycerol:PBS containing 2.5 µg/ml Hoechst 33258) and covering them with a coverslip. The oocytes were evaluated using a fluorescence microscope with a 365-nm excitation filter. Chromatin configurations were classified as metaphase, having a linear or circular metaphase plate with or without visible polar body chromatin; pronuclear, having one or more pronuclei present within the oocyte cytoplasm; cleaved, having two or more cells with nuclei present; abnormal, chromatin appearing in one to several dense to hairlike masses within the oocyte; and degenerating, no chromatin or chromatin spread throughout the cell. The method of assessment (Hoechst staining) did not allow visualization of the sperm tail. Oocytes exhibiting pronucleus formation or cleavage were considered to be fertilized; oocytes having 2 pronuclei (PN) or cleavage with nuclei present in each blastomere were considered to be normally fertilized.

Fertilization rates were expressed as a percentage of total viable oocytes/embryos (disregarding degenerating oocytes). Differences in fertilization rate and embryo development among treatments were evaluated using chi-square analysis, with the Fisher exact test used when a value of less than five was expected for any given parameter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1

Sperm motility Fifteen ejaculates were processed. The percentages of motile sperm in the Sp-TALP, DMSO, CaI-3, and CaI-7.14 treatments ranged from 83 ± 0.9% (mean ± SEM) to 87 ± 1.0%; progressively motile sperm in these treatments ranged from 60 ± 2.5% to 63 ± 1.1%. These parameters were unaffected by treatment (P > 0.05). Sperm VCL was significantly lower in the Sp-TALP treatment (202 ± 2.3 µm/sec) than in the DMSO, CaI-3, and CaI-7.14 treatments (246 ± 3.8, 243 ± 5.5, and 236 ± 8.6, respectively; P < 0.001). Similarly, ALH was significantly lower in sperm in the Sp-TALP treatment (7.1 ± 0.1 µm) than in the DMSO, CaI-3, or CaI-7.14 treatments (9.3 ± 0.2, 9.2 ± 0.3, and 9.0 ± 0.3, respectively; P < 0.001).

Fertilization The volume of added sperm suspension was 13.6 ± 2.23 µl and 13.7 ± 2.71 µl (mean ± SD) for the CaI-3 and CaI-7.14 treatments, respectively. The final concentration of sperm within the droplet was therefore a mean of 8.85 x 106 sperm per ml for both sperm treatments.

The fertilization rates for the different oocyte maturation and sperm treatments are given in Table 1. There was no significant difference between sperm treatments in percentages of either normal fertilization (12/207 [6%] for CaI-3 and 12/177 [7%] for CaI-7.14; P > 0.1) or total fertilization (23/207 [11%] for CaI-3 and 22/177 [12%] for CaI-7.14; P > 0.1). Similarly, there was no significant difference in normal or total fertilization rates for oocytes matured 24 or 42 h (normal fertilization: 10/201 [5%] and 14/183 [8%], respectively, P > 0.1; total fertilization: 19/201 [9%] and 26/183 [14%], respectively, P > 0.1); therefore, sperm treatments and maturation times were combined.


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  		TABLE 1. Fertilization (pronucleus formation) rates for horse oocytes subjected to different maturation media and maturation times

There was an overall effect of maturation medium on rates of normal fertilization (P < 0.05) and total fertilization (P < 0.01). The rate of normal fertilization for oocytes matured in 100% FF (11/94 [12%]) was significantly higher than that for oocytes matured in 20% FF (1/94 [1%]; P < 0.01) and tended to be higher than that for matM (3/90 [3%]; P = 0.06). There was no significant difference in normal fertilization between the 100% FF and the EGF treatment (9/106 [8%]; P > 0.1). Similarly, the total fertilization rates for oocytes matured in 100% FF (20/94 [21%]) were significantly higher than those for oocytes matured in 20% FF (5/94 [5%]; P < 0.01) or matM (8/90 [9%]; P < 0.05) and tended to be higher than those for the EGF treatment (12/106 [9%]; P = 0.08). The highest normal fertilization rates (16%) were achieved in oocytes matured 24 h in 100% FF and fertilized with CaI-3 sperm and in oocytes matured 42 h in 100% FF fertilized with CaI-7.14 sperm. The latter treatment was used in experiment 2. Photomicrographs of horse oocytes 24 h after in vitro fertilization, stained with Hoechst and visualized under fluorescence, showing two pronuclei and 2 polar bodies and a 2-cell embryo are presented in Figure 1.



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  		FIG. 1. Fluorescent photomicrographs of Hoechst-stained oocytes/embryos fertilized in vitro (A, B) or in vivo (C, D) showing pronuclei or nuclei (open arrows), polar bodies (arrowheads), and accessory sperm (solid arrows). Bar = 50 µm. A) Oocyte with 2 pronuclei and 1 polar body, with accessory sperm in the zona pellucida. B) Two-cell embryo with 2 nuclei, polar body, and accessory sperm. C) Two-cell embryo with 2 nuclei and 2 polar bodies. D) Four-cell embryo with 4 nuclei and 2 polar bodies

Experiment 2

Sperm motility Motility data were obtained from 4 ejaculates. The percentages of motile sperm in the Sp-TALP control and treatments 1, 2, and 3 ranged from 87 ± 2.0% to 91 ± 2.5%; progressively motile sperm ranged from 67 ± 2.0% to 75 ± 1.4%. These parameters were unaffected by treatment (P > 0.1). Sperm VCL tended to be lower (P < 0.1) in the control (Sp-TALP only; 203 ± 4.8 µm/sec) compared with treatment 3 (CAP/CaI/45-min incubation; 239 ± 6.8). Mean amplitude of ALH was significantly lower in the control (6.9 ± 0.3 µm) compared with treatment 3 (9.3 ± 0.4; P < 0.05).

Fertilization

The volume of sperm suspension added to the 100-µl fertilization droplets for treatments 1, 2, and 3 were 14.18 ± 0.78, 15.54 ± 1.46, and 15.14 ± 1.31 µl (mean ± SD), respectively. Thus, the mean final sperm concentrations in the fertilization droplets for the three treatments were 8.76, 8.66, and 8.69 x 106 sperm/ml, respectively. Volume of supernatant added in the supernatant group was the same as the sperm suspension volume for treatment 1.

The fertilization rates for the different oocyte maturation and sperm treatments are given in Table 2. There were no significant differences in fertilization rates among treatments 1, 2, and 3, although the rate of normal fertilization in treatment 1 tended to be higher than that for treatment 3 (P = 0.09). In the supernatant treatment, only 1 of 71 oocytes (1 of 50 nondegenerating oocytes) was seen to have a pronucleus-like structure after culture. There was a significantly lower prevalence of pronucleus formation in the supernatant treatment than in treatments 1 and 2 when compared in both normal fertilization (P < 0.05) and total fertilization (P < 0.01) classifications.


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  		TABLE 2. Fertilization rates of oocytes matured 42 h in 100% follicular fluid and inseminated with 1 of 4 sperm treatments

Experiment 3

Of 122 oocytes intended for transfer, 4 were found in the pipette after the transfer procedure; therefore, 118 oocytes were transferred. One hundred and two oocytes were recovered upon flushing the oviducts, of which 15 were considered to originate from the recipient mare; thus, 87 of the 118 transferred oocytes (74%) were recovered. The recovery rate for the first transfer, in which 25 oocytes were transferred to each oviduct, was 28/48 (58%); the recovery rate for the subsequent 3 transfers (12 oocytes per side) was 59/70 (84%). One replicate of 9 recovered oocytes, in the matM group, was not used for calculation of fertilization rate because of apparent inflammation and infection of the oviduct. The oocytes recovered from this oviduct were found to have bacteria and white blood cells present on the oocytes and in the surrounding fluid when evaluated. The proportions of oocytes recovered and the fertilization rates for oocytes for each maturation treatment are presented in Table 3.


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  		TABLE 3. Fertilization rate of oocytes matured in vitro in either 100% follicular fluid or in maturation medium (matM) and transferred to the oviducts of mares in vivo

All fertilized oocytes recovered from the oviducts appeared to be normally fertilized, i.e., had 2 PN or had normal cleavage. The fertilization rates expressed in relation to the proportion of viable oocytes (those fertilized or in metaphase, disregarding degenerating oocytes) were not significantly different between maturation treatments (27/35 [77%] for 100% FF and 16/21 [76%] for matM; P > 0.1).

Of the 43 fertilized oocytes recovered, 16 (37%) had cleaved to 2 or more cells. The cleavage rate was 6/27 (22%) in the 100% FF group and 10/16 (63%) in the matM group; this difference was significant (P < 0.05). Four of 10 cleaved embryos (40%) had reached 3–4 cells in the matM group, whereas 1 of 6 cleaved embryos (17%) reached this stage in the 100% FF group; this difference was not significant (P > 0.1). Photomicrographs of Hoechst-stained embryos recovered from the oviduct 96 h after oocyte transfer, visualized under fluorescence, showing a 2-cell embryo and a 4-cell embryo are presented in Figure 1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study show that, while the rate of normal fertilization, i.e., 2 PN to cleavage, of IVM oocytes in vitro is low under many different oocyte and sperm treatments (0–16%), the in vivo fertilization rate for IVM oocytes is high (77%). This indicates that IVM horse oocytes are capable of normal sperm penetration and activation and that irreversible zona hardening, as would be seen with premature release of cortical granules, probably does not play a part in the failure of sperm to fertilize horse oocytes in vitro. The high rate of fertilization in vivo may be related either to effective sperm capacitation within the oviduct or to oviduct-mediated changes in the permeability of the zona pellucida [28–32].

The rates of in vitro fertilization achieved in the present study are lower than those in the reports on which our procedures were based but were repeatable between experiments 1 and 2. Only 1 stallion, of known high fertility, was used in this study. While the efficiency of IVF may have been influenced by the stallion used, the rates achieved in this study are within the range generally reported for IVF in the horse, and the in vivo fertilization rates using this stallion were high. Comparison of fertilization rates among published reports is complicated by differences in the method of fertilization assessment; however, even within laboratories, high rates of fertilization have not been repeatable in later studies (e.g., rates of 33%, 4% to 17%, and 6% to 8% in 3 studies using the same IVF technique [8, 9, 11]).

The capacitating medium used in experiment 1 was that reported by Zhang et al. [2]. In experiment 2, we compared this medium with that reported by Alm et al. [12], who also used 7.14 µM CaI, 10 min, for capacitation. The major procedural differences in the latter report included initial extension of semen in skim milk-glucose extender, use of CAP medium for capacitation and subsequent resuspension, and incubation of sperm for 45 min at 38.5°C after CaI exposure. CAP is a TALP-based medium, but unlike Sp-TALP, contains glucose (5 mM) and does not contain calcium [27]. However, use of this procedure (with or without the 45-min incubation) in experiment 2 did not result in significantly different rates of fertilization from those for the Sp-TALP procedure.

Treatment of stallion sperm with concentrations of CaI over 3 µM has been reported to greatly reduce stallion sperm motility [13]; however, progressive motility was maintained well in both 3 µM and 7.14 µM CaI groups in our study. We analyzed VCL and ALH to detect motility patterns suggesting hyperactivation that might be accompanied by high fertilization rates. However, there was no significant difference in VCL or ALH between CaI treatments nor between sperm treated with CaI and sperm treated with DMSO alone. Sperm in the DMSO and CaI treatments, which were evaluated after transfer to Fert-TALP, had significantly greater ALH and VCL than did sperm maintained in Sp-TALP alone. This may be related to the presence of sperm motility stimulants, penicillamine, hypotaurine, and epinephrine, in the Fert-TALP but may also be related to the glucose in this medium. Glucose or other glycolytic sugars are reported to support hyperactivated motility in human sperm [33]. However, exposure of sperm to CaI in the presence of glucose (in CAP medium in experiment 2) did not affect initial motility patterns (treatment 2). It is clear that detailed work on the effect of medium components on sperm capacitation, as has been done in other species [26, 34, 35], needs to be performed with stallion spermatozoa. Unfortunately, until a basic method for repeatable fertilization is achieved, study of factors affecting functional capacitation of horse sperm will continue to be problematic.

Maturation of oocytes in the presence of 100% FF significantly increased the rate of IVF over that obtained with standard maturation medium; however, it had no effect on fertilization in vivo. In fact, the cleavage rate after in vivo fertilization was significantly lower in oocytes matured in 100% FF than in those matured in matM. Variability between oviducts in the recipient mares should have been minimal because there was no preovulatory follicle present. We incubated 100% FF-matured oocytes with sperm supernatant in experiment 2 to determine the rate of parthenogenetic activation in these oocytes. Inclusion of treated sperm supernatant rather than medium alone is important as a control because treatment with CaI alone can cause parthenogenetic activation of horse oocytes [36, 37]. The low rate of pronucleus formation seen in the supernatant treatment agrees with previous studies in the horse, in which parthenogenetic activation rates of 4% to 5% [36] and 0% [11, 38] were reported.

Efficient fertilization and embryo development were achieved after transfer of IVM oocytes to the oviduct. The recipient mares used in this study were mares without active corpora lutea, treated with a long-acting estrogen [39] before transfer. Use of nonovulating, estrogen- and progesterone-treated mares as recipients for in vivo-matured oocytes has been previously reported [40–42]. In the present study, because embryo recovery was to be done within 2 days of transfer, no progesterone was administered to the recipient mares. The high fertilization rate achieved under these conditions is notable in relation to current understanding of the oviductal sperm reservoir. It has been held in many species, including the mare [43], that sperm are stored in the caudal oviductal isthmus until release is signaled around the time of ovulation (review, [44]). It has been postulated that the final stages of sperm capacitation necessary for fertilization are programmed by ovarian hormonal signals acting via the endosalpinx [45]. However, in the present study, high rates of fertilization were achieved under constant estrogen stimulation and in the absence of a periovulatory follicle, exogenous progesterone administration, or exposure to follicular fluid. It is possible that procedures associated with the transfer may have provided a signal for sperm release and capacitation; alternatively, these results suggest that, in the mare at least, capacitated sperm are released from the isthmus and travel through the oviduct constantly, without an ovulation-specific release of capacitated sperm. Transfer of oocytes to the oviducts of hormone-treated, nonovulating recipients in other species may help answer questions regarding ovulation-induced sperm release.

The development of the recovered oocytes may have been delayed compared with those reported for normally ovulated ova recovered from the oviducts. Betteridge et al. [46] found that in vivo-derived embryos recovered at 24–36 h after ovulation were in the 3- to 4-cell stage, and Ball et al. [47] reported the recovery of 4- to 8-cell embryos 35–58 h after ovulation. The apparent developmental delay in the present study may explain the poor embryonic development noted in the two previous studies on in vivo fertilization of IVM horse oocytes [17, 25]. It is possible that embryos derived from IVM oocytes either fail to develop to the blastocyst stage in vivo or fail to signal normal oviductal transport to the uterus on Day 5 [48]. Further work is needed to determine the fate of embryos resulting from transfer of IVM oocytes in vivo.

In conclusion, in this study, we found that oocyte maturation medium significantly affected the rate of fertilization in vitro whereas duration of maturation and sperm treatment did not. The rate of normal fertilization in vitro was low (maximum 16%). Results of our in vivo transfers showed that large numbers of oocytes may be transferred to the oviducts of mares in vivo and may be efficiently recovered, that horse oocytes matured in vitro have high rates of fertilization after transfer to the oviduct in vivo, and that cleavage of these fertilized oocytes to the 4-cell stage will occur by 44 h in vivo but at an apparently delayed rate compared with that of normally ovulated oocytes.


   ACKNOWLEDGMENTS
 
We thank Ms. Linda Love for excellent technical assistance.


   FOOTNOTES
 
First decision: 13 December 2001.

1 Supported by the Link Equine Research Endowment Fund, Texas A&M University. Back

2 Correspondence: Katrin Hinrichs, Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Room 300A, VMA Building, TAMU 4466, College Station, TX 77845-4466. FAX: 979 845 6544; khinrichs{at}cvm.tamu.edu Back

Accepted: February 6, 2002.

Received: November 27, 2001.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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