Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print August 18, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.032839
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/6/1974    most recent
biolreprod.104.032839v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, J.-Y.
Right arrow Articles by Tsang, B. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, J.-Y.
Right arrow Articles by Tsang, B. K.
Agricola
Right arrow Articles by Jiang, J.-Y.
Right arrow Articles by Tsang, B. K.
BIOLOGY OF REPRODUCTION 71, 1974–1979 (2004)
DOI: 10.1095/biolreprod.104.032839
© 2004 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Optimal Conditions for Successful In Vitro Fertilization and Subsequent Embryonic Development in Sprague-Dawley Rats1

Jin-Yi Jiang, and Benjamin K. Tsang2

Reproductive Biology Unit and Division of Reproductive Medicine, Departments of Obstetrics & Gynecology and Cellular & Molecular Medicine, University of Ottawa; Hormones, Growth and Development Program, Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), Ottawa, Ontario, Canada K1Y 4E9


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was conducted to determine the optimal conditions for successful in vitro fertilization (IVF) in Sprague-Dawley (SD) rats. The IVF of oocytes from SD and Wistar rats was compared in different fertilization media (mR1ECM, IVF-20, and modified Krebs-Ringer bicarbonate solution [mKRB]), and IVF conditions were then optimized for oocytes of the SD strain. Results showed that in mR1ECM medium, fertilization rates were markedly lower in SD rats (15%) than in the Wistar strain (73%), although this response was significantly improved by increasing the NaCl concentration. In addition, fertilization rates in SD rats were higher in modified IVF-20 (73%) than in IVF-20 (18%) and mKRB (53%). In contrast, fertilization rates in Wistar rats were higher in IVF-20 and modified IVF-20 than in mKRB (78%, 74%, and 36%, respectively). Further investigation concerning the effects of the NaCl supplementation (10– 40 mM) in IVF-20 on the fertilization of oocytes in the SD strain indicated that significantly higher percentages of oocytes were fertilized in IVF-20 supplemented with 30 mM NaCl (66%) and developed to the blastocyst stage (47%) in vitro. After transfer, embryos derived from this IVF system developed to term at a percentage comparable to that of in vivo-fertilized controls. In conclusion, differences exist in optimal IVF conditions between rat strains, and a modified culture medium has been successfully developed for assessment of the developmental competence of oocytes in SD rats.

assisted reproductive technology, embryo, fertilization, gamete biology, in vitro fertilization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In vitro fertilization (IVF) is an important approach for the study of basic physiological, morphological, and molecular events in gametes and during their interaction, and of propagation of animals that have been rendered infertile by mutations [1]. It is widely used in human infertility treatment [2, 3] and for the production of large numbers of embryos in the domestic animal industry [4, 5].

Capacitated sperm and mature, unaged oocytes are two basic requirements for successful fertilization in vitro [6]. It has been demonstrated that genetic background is an important determinant in the in vitro response of oocytes [7– 9] and spermatozoa [10–13], as observed in different species, including mice and rats, as well as in different strains of the same species. These factors may be responsible for the different environmental requirements for successful IVF in different strains and species. In the mouse, extensive studies have indicated that IVF rates vary greatly in mouse strains under the same IVF conditions, especially when cryopreserved sperm are used [10, 14–16]. However, in the rat, no study, to our knowledge, has reported the comparison of IVF conditions for different strains.

Studies of folliculogenesis in Sprague-Dawley (SD) rats have produced increased interest in assessment of the developmental competence of oocytes in this strain by IVF [17–19]. Although successful IVF in Wistar rats has been reported extensively [20–24], information concerning IVF in SD rats is limited, which may, in part, be responsible for the difficulty in successfully applying this technique to the assessment of reproductive function in this species. Indeed, to our knowledge, only one such study has been reported [25]. Moreover, in that study, penetration and pronuclear formation with corresponding sperm tail were not assessed to determine fertilization. In contrast, the fertilization was determined by the number of 2-cell embryos at 36 h after gamete coculture, and the possibility that the low percentage of 2-cell embryos observed might have been caused by pathenogenetic activation could not be excluded. Furthermore, the developmental competence of the 2-cell embryos in vitro and pregnancy outcome following embryo transfer were not assessed.

Recent investigations have indicated that oocytes from Wistar and SD rats respond differently to pathenogenetic activation [8, 9], suggesting that different strains of rats may have different optimal conditions for successful IVF. Therefore, in the present study, we compared the IVF requirements for two rat strains (SD and Wistar), and we defined the optimal conditions for successful fertilization in vitro of oocytes from the SD rat.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All reagents were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise stated.

Animal Preparation

Female Wistar and SD rats (age, 21 days) were purchased from Charles River Canada (Montreal, PQ, Canada). Animals were then placed in polycarbonated cages with wood shavings on the floor in a room at a controlled temperature of 21°C and humidity of 50%, with lights-on at 0700 h and lights-off at 1900 h, and were given bullet-type commercial rat feed and tap water ad libitum. The present study was carried out in accordance with the Guide to Care and Use of Experimental Animals of the Canadian Council on Animal Care and was approved by the Animal Care Committee of the Ottawa Health Research Institute.

Medium

The mR1ECM used in the present study [26, 27] contained 76.7 mM NaCl, 3.2 mM KCl, 0.5 mM MgCl2·6H2O, 2.0 mM CaCl2·2H2O, 25.0 mM NaHCO3, 7.5 mM D-glucose, 0.5 mM sodium pyruvate, 10.0 mM sodium lactate, 0.1 mM glutamine, 2% (v/v) minimal essential medium (MEM) essential amino acid solution (50x; Gibco BRL, Grand Island, NY), 1% (v/v) MEM nonessential amino acid solution (100x; Gibco BRL), and 1.0 mg/ml polyvinylalcohol. Three fertilization media were used: 1) mR1ECM containing different NaCl (110–150 mM) and 4 mg/ ml of BSA (Fraction V) [21], 2) IVF-20 (Vitrolife AB, Göteborg, Sweden) with different amounts of NaCl supplementation, and 3) modified Krebs-Ringer bicarbonate solution (mKRB) [21]. The IVF-20 contained calcium chloride, EDTA, glucose, human serum albumin, magnesium sulfate, penicillin G, potassium chloride, potassium dihydrogen phosphate, sodium bicarbonate, sodium chloride, sodium lactate, and sodium pyruvate (precise information on each component not available because of proprietary restriction). Drops of fertilization and culture media (400 µl each) were covered with mineral oil (M-8410) in polystyrene culture dishes (60 x 15 mm; Falcon 353002; Becton Dickinson, NJ) and equilibrated overnight at 37°C in 5% CO2 in air.

Preparation of Sperm Suspension and Capacitation

Spermatozoa were obtained from adult males with a protocol described previously [22, 23, 28]. Briefly, one drop of dense mass of spermatozoa was introduced into the preequilibrated insemination media (400 µl) described above. After warming in an incubator at 37°C in 5% CO2 in air (~5 min), 10–30 µl of the sperm suspension were transferred into drops of insemination medium to attain a final sperm concentration of 1 x 106 cells/ml. The diluted sperm suspensions were preincubated for 5–7 h at 37°C in 5% CO2 in air for capacitation.

Hormone Administration and Collection of Cumulus-Oocyte Complexes

Immature rats (age, 25–27 days) were injected with eCG (10 IU s.c.; G4877) and hCG (10 IU i.p.; CG-5) 48 h later. Fourteen hours after hCG injection, animals were killed by cervical dislocation under anesthesia by isoflurane USP (Abbott Laboratories, Montreal, PQ, Canada) [20, 22, 23]. Oviducts were isolated, removed of the surface liquid and blood with a piece of sterilized filter paper, and placed into dishes containing diluted sperm suspensions. The cumulus-oocyte complexes in the oviducts were carefully released into the sperm suspensions and kept at 37°C under 5% CO2 in air for 12 h.

In Vitro Fertilization

Penetration, polyspermy, and pronuclear formation were examined as described previously [20, 23, 28]. Briefly, after incubation with sperm suspensions at 37°C under 5% CO2 in air for 12 h, eggs were transferred into 100 µl of culture medium and freed from surrounding cumulus cells by being drawn up repeatedly into a fine pipette. The denuded eggs were placed in the center of four vaseline spots on a glass slide, compressed gently with a cover-slip, fixed briefly with 2.5% glutaraldehyde in phosphate buffer solution and 10% neutral formalin at room temperature for 2–6 h, stained with 0.1% orcein in 45% acetic acid, and examined by phase-contrast microscopy. Eggs were considered to be penetrated if a spermatozoon tail could be visualized in the perivitelline space or if the eggs had pronuclei with sperm tail(s) in the vitellus [20]. Eggs containing two or more enlarged sperm heads or male pronuclei with corresponding tail in the vitellus were considered to be polyspermic [29].

To compare IVF of oocytes between Wistar and SD rats using mR1ECM supplemented with varied concentrations of NaCl, cumulus-oocyte complexes from the two strains were cocultured with sperm in the mR1ECM with 110, 140, and 150 mM NaCl. The osmolarity of media was measured by a VAPRO Vapor Pressure Osmometer 5520 (Mandel Scientific Company, Inc., Guelph, ON, Canada). In addition, based on the results of the above experiment, IVF outcomes of oocytes between the two rat strains were also compared in IVF-20, modified IVF-20 with 33 mM NaCl, and mKRB. Furthermore, cumulus-oocyte complexes of SD rats were also cocultured with sperm in IVF-20 supplemented with 0, 10, 20, 30, or 40 mM of NaCl for 12 h to determine the optimal NaCl concentration for IVF.

Embryo Culture

The culture of embryos and examination of their development were conducted as described previously [22, 23, 26]. Briefly, eggs were freed from cumulus cells 12 h after insemination, washed 15 times with culture medium, and observed under a phase-contrast microscope for evidence of fertilization. Ten to 20 eggs with female and male pronuclei and corresponding tail were transferred into 400 µl of culture medium and cultured at 37°C under 5% CO2 in air for 5 days. Blastocyst cell numbers were counted following Hoechst 33342 epifluorescein staining as described previously [30].

To assess the developmental competence in vitro, zygotes fertilized in IVF-20 supplemented with 0, 10, 20, 30, or 40 mM of NaCl were cultured in mR1ECM. The rates of embryos that had developed to the 2-cell, 4-cell, and blastocyst stages were determined at 24, 72, and 120 h of culture, respectively.

Embryo Transfer

To determine the developmental competence in vivo of embryos fertilized in vitro in IVF-20 supplemented with optimal NaCl, 1-cell embryos from IVF in IVF-20 with optimal NaCl (30 mM) were transferred into the oviducts of recipients after induction of pseudopregnancy as described previously [22, 23]. As controls, embryos from in vivo fertilization were also transferred. These embryos were collected from eCG-primed, immature female rats the day after hCG injection and were paired with adult males. Briefly, female rats (age, 72–82 days; weight, 230–300 g) with at least two consecutive, regular, 4-day estrous cycles immediately before study were stimulated vaginally three times (on and off for 3 sec, respectively) in the afternoon of proestrus and on the morning of estrus (Day 1) with a plastic rod connected to two electrodes (24 V AC). Nine to 10 embryos at the 1-cell stage were transferred to the oviducts of each recipient at Day 1. Vaginal smears of recipients were examined on Days 1, 4, and 12–14 after transfer to confirm successful induction of pseudopregnancy and signs of pregnancy, respectively. Recipients that showed estrus on the fourth day were considered to be nonpseudopregnant and were excluded from the present study. Females were killed if they were pregnant but did not deliver an offspring by Day 24 of pregnancy, and their uterine horns were examined for implantation sites. Offspring were counted and the body weight measured on the day of parturition.

To determine the fertility of rats derived from IVF, these animals were mated at 3 mo of age with SD males or females. Offspring were counted and weighed.

Statistical Analysis

Data in Tables 1–5 were analyzed by the chi-square test; data in Table 6 were analyzed by the Student t-test. Differences at P < 0.05 were considered to be statistically significant. Values are mean ± SEM throughout.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Effects of NaCl concentrations in mR1ECM on IVF in Wistar and SD rats


View this table:
[in this window]
[in a new window]
  		TABLE 5. Pregnancies and offspring derived from oocytes of SD females fertilized in vitro in modified IVF-20 medium followed by transfer to pseu dopregnant recipients


View this table:
[in this window]
[in a new window]
  		TABLE 6. Summary of F1 pups with IVF and in vivo-fertilized embryo mothers


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of NaCl Concentrations in mR1ECM on IVF of Oocytes from Wistar and SD Rats

In mR1ECM medium with 110 mM NaCl, higher percentages of oocytes (79% vs. 15%) were penetrated by either one or more sperm (polyspermy) and developed to pronuclei in Wistar rats than in SD rats (P < 0.05). The increase of NaCl concentrations to 140 and 150 mM in the fertilization medium markedly decreased the rates of oocyte penetration and development to pronuclei in Wistar rats (P < 0.05). However, in SD rats, 140 mM NaCl significantly increased these responses (P < 0.05), although the rate of polyspermy was also higher (Table 1).

Effects of Different Fertilization Media on IVF of Oocytes in Both Wistar and SD Rats

The IVF of oocytes in Wistar and SD rats was assessed in IVF media of IVF-20, modified IVF-20, and mKRB (Table 2). In Wistar rats, the percentages of oocytes penetrated and those developed to pronuclei were markedly higher in IVF-20 and modified IVF-20 (IVF-20 + 33 mM NaCl) than in mKRB (P < 0.05), although no significant difference in the rates of polyspermy was observed among the three media. In SD rats, however, the percentages of oocytes penetrated were markedly higher in modified IVF-20 and mKRB than in IVF-20 (84%, 73%, and 55%, respectively; P < 0.05). In addition, the percentages of oocytes with normal pronuclear formation were significantly higher in modified IVF-20 than in IVF-20 and mKRB (73%, 53%, and 18%, respectively; P < 0.05). In contrast, the percentage of oocytes with polyspermy was markedly lower in modified IVF-20 than in IVF-20 and in mKRB (5%, 18%, and 13%, respectively; P < 0.05) (Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Effects of different media on IVF in Wistar and SD rats

Effects of Different Concentrations of Supplemented NaCl in IVF-20 on IVF of Oocytes in SD Rats

The IVF of oocytes in SD rats was determined in IVF-20 supplemented with 10–40 mM NaCl (Table 3). The percentages of oocytes penetrated and of those with normal pronuclear formation were markedly higher in IVF-20 supplemented with 30 mM NaCl (75% and 66%, respectively) than with other concentrations (19–48% and 14–34%, respectively; P < 0.05), although no significant difference was noted in the rates of polyspermy between the groups (Table 3).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Effects of added NaCl concentrations in IVF-20 on IVF in SD rats

Effects of Varied NaCl Concentrations in IVF-20 Medium on In Vitro Development of Oocytes in SD Rats

Higher percentages of oocytes developed to the 2- and 4-cell stages when fertilized in IVF-20 supplemented with 20 mM NaCl (97% and 77%, respectively) or 30 mM NaCl (100% and 72%, respectively) than in other NaCl concentrations (76–88% and 23–36%, respectively; P < 0.05) (Table 4). Furthermore, the percentage of embryos derived from IVF in IVF-20 supplemented with 30 mM NaCl developed to blastocysts was higher than those in other groups (P < 0.05). No significant differences were noted in the rates of the 2-cell stage embryos and of blastocysts between IVF in IVF-20 supplemented with 30 mM NaCl and in vivo-fertilized embryos. In addition, no significant differences were noted in the cell numbers of blastocysts developed from oocytes fertilized in IVF-20 supplemented with 30 mM NaCl and those fertilized in vivo (P > 0.05) (Table 4).


View this table:
[in this window]
[in a new window]
  		TABLE 4. In vitro development of oocytes fertilized in IVF-20 supple mented with NaCl in SD rats

In Vivo Development of IVF Embryos

All recipients transferred with embryos from either IVF or in vivo fertilization established pregnancy. Surprisingly, the percentage of pregnant recipients delivering offspring was significantly higher (100%) when IVF embryos were transferred than when embryos from in vivo fertilization were transferred (75%, P < 0.001). Two pregnant recipients receiving embryos from in vivo fertilization did not deliver an offspring by Day 24 after transfer and had four and seven implantation sites in their uterine horns, respectively. No significant differences were observed in the percentages of transferred embryos developing to term, sex ratios of offspring, and body weight at birth between the two groups (Table 5).

Fertility of Rats Derived from IVF

Of the 37 rats (21 males and 16 females) born from IVF embryos, 31 (19 males and 12 females) were paired with females or males at 3 mo of age and then killed at 4 mo of age after delivery of F1 offspring. No obvious abnormalities were noted in any of these animals, which had a body weight of 342.3 ± 12.0 g at 4 mo of age, comparable to that of controls (332.9 ± 10.5 g), in females. After being paired with males, all IVF embryo-derived females became pregnant after mating and delivered normal offspring, with an average litter size (14.8 ± 0.7) comparable to that of controls (15.1 ± 1.0). No differences were noted in body weight and sex ratio of F1 offspring between IVF-derived females and controls (Table 6).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that differences in IVF conditions exist between rat strains and that successful IVF can be achieved in the SD rat using a currently available medium (IVF-20) with a simple modification. Although the detailed mechanisms for the different requirements of successful IVF between rat strains remains unknown, genetic background and components in the medium may be major factors. It has been demonstrated that a genetic influence exists on the variability of water permeability and its activation energy of oocytes in mice and hamsters [7]. Recent studies in rats have shown that oocytes from Wistar and SD strains response differently to pathenogenetic activation [8]. Oocytes from the SD strain were more resistant to the strontium treatment than were those from the Wistar strain, and the overall activation efficiency was higher for SD oocytes [8]. However, when oocytes were electrically stimulated and then treated with 6-dimethylaminopurine, more activated oocytes from Wistar-Imamichi strain developed to the 2-cell and blastocyst stages than did those from the SD rats [9]. In addition, fertilized rat eggs of the SD outbred strain had a high capability of developing from the 1-cell to the blastocyst stage, whereas those of the SD inbred strains performed much more poorly [31]. Extensive studies have indicated that IVF rates vary greatly in mouse strains under the same IVF conditions, especially when cryopreserved sperm are used [10, 14–16]. Differences in osmotic sensitivity of spermatozoa from different genetic backgrounds have been observed in mice [10], cattle [11], humans [12], and pigs [13]. In addition, differences exist between mitochondrial function of spermatozoa from the two mouse strains [32]. Therefore, differences in genetic background between two rat strains may lead to differences in characteristics of spermatozoa (i.e., integrity of plasma membranes and acrosome and mitochondrial function) and, thus, in their osmotic tolerance, as reported previously [10], although this possibility needs to be investigated further.

It has also been reported that an increase of NaCl concentration in the medium is important for sperm penetration in Wistar rat IVF. High NaCl concentrations (100–130 mM) in mR1ECM medium significantly increased the percentages of oocyte penetration compared with those of other NaCl concentrations [21]. In the present study, we confirmed that oocytes from Wistar rats have high fertilization rates in mR1ECM with 110 mM NaCl, indicating that our IVF system is optimal for the Wistar strain, as reported previously [22, 23]. However, in SD rats, few oocytes were fertilized in this condition. Higher NaCl concentrations significantly improved the fertilization, although penetration and pronuclei formation rates were still low. These findings suggested that IVF of oocytes in SD rats requires a medium not only with a high NaCl concentration but also with some essential components that are absent in mR1ECM. Therefore, we compared the effects of two other media (mKRB and IVF-20) on the fertilization of oocytes in SD rats and Wistar rats. The mKRB has been widely used in rat IVF with Wistar and CD strains [20, 21, 28, 29, 33]. Currently, IVF-20 is used extensively in human IVF and embryo cultures [34–38]. In the present study, significantly high polyspermic rates were evident with both mKRB and IVF-20 in the SD group, as has previously been reported in the Wistar strain [21, 28]. Further investigations indicated that when 30 mM NaCl was added in IVF-20, penetration and pronuclei formation rates were high and comparable to those reported in the Wistar strain [21]. Oocytes fertilized in this optimal condition developed to blastocysts at a markedly higher percentage (47%) than in other groups and at a rate comparable to that (51%) in other studies [28]. These embryos also developed to term after embryo transfer at a percentage similar to that of embryos fertilized in vivo, which was comparable to the finding (44%) of Jiang et al. [22], and higher than that (21%) reported by Toyoda and Chang [20]. These findings suggest that one or more components in IVF-20 and a high NaCl concentration are necessary for successful IVF in this rat strain, although further investigations are needed to define the identity of these essential components and how they are crucial in this process.

The SD strain is a popular rat model used in studies of folliculogenesis, oocyte maturation, and reproductive toxicology [17–19, 39–44]. These studies usually require use of IVF to assess quality of oocytes or sperm as well as overall fertility and reproduction [45–47]. Unlike IVF in mice, however, IVF is difficult to conduct in the SD strain [25]. This difficulty led many research groups to use zona pellucida-free oocytes to assess the fertility of oocytes [39, 48] or sperm [45, 49]. The limited availability of media that can support rat IVF is, in part, responsible for the relative lack of progress in this area for this species. To our knowledge, only mKRB and mR1ECM have been used for rat IVF [20–22, 28, 29, 33]. The present study demonstrated, to our knowledge for the first time, that IVF-20, a commercial medium widely used in human IVF, can be successfully applied to IVF in Wistar rats as well as in the SD strain if supplemented with NaCl. This may result from the glucose [50, 51], human serum albumin [28, 52], phosphate [28, 53], and NaCl [21] contained in this modified medium (or from its osmolarity [54, 55]), because they are crucial for the sperm acrosome reaction and successful fertilization. The IVF-20 is formulated according to the "low-glucose and low-phosphate" principle of culture media for IVF, embryo culture, and embryo transfer in humans. Its successful application makes SD rat IVF easier, simpler, and more repeatable. Indeed, this system is now used to assess the fertilization and subsequent developmental competence of oocytes from SD rat follicles following different treatments to define potential biomarkers of oocyte quality (data not shown).

Studies of human IVF have shown that infants conceived in this way have twice the risk of a major birth defect as naturally conceived infants. These defects include low birth weight and preterm delivery [56]. However, in the present study, none of these defects were found in offspring derived from IVF embryos, and no difference was observed in the birth weight of pups between IVF and in vivo-fertilized controls. These IVF-derived pups did not show any obvious abnormalities until 4 mo of age, when killed. They showed normal fertility with an average litter size comparable to that of controls.

In conclusion, differences exist in IVF conditions between rat strains. Oocytes of Wistar rats respond well to IVF in varied conditions, including mR1ECM with 110 mM NaCl and IVF-20 with or without a supplement of 33 mM NaCl. However, in SD rats, successful IVF of oocytes was achieved only in IVF-20 supplemented with 30 mM NaCl. Oocytes fertilized in vitro in this optimal condition developed to blastocysts in vitro and to normal fertile offspring in vivo. These findings suggest that the optimal conditions for rat IVF may provide a valuable experimental tool that will allow the assessment of developmental competence of oocytes in both Wistar and SD rats.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Yifang Wang at the Fertility Center of the Ottawa Hospital for suggesting the use of IVF-20 in the present studies and staff of Animal Care Services at Ottawa Health Research Institute for the maintenance and care of the animals used.


   FOOTNOTES
 
1 Supported in part by a grant from the Canadian Institutes of Health Research (MOP-15691). In addition, the studies described were part of the Program on Oocyte Health (http://www.ohri.ca/oocyte) funded under the Healthy Gametes and Great Embryos Strategic Initiative of the Canadian Institutes of Health Research (CIHR) Institute of Human Development, Child and Youth Health (IHDCYH), grant number HGG62293. J.Y.J. is a recipient of a CIHR-STIRRHS Postdoctoral Fellowship. Back

2 Correspondence: Benjamin K Tsang, Hormones, Growth, and Development Program, Ottawa Health Research Institute, The Ottawa Hospital (Civic Campus), 725 Parkdale Avenue, Ottawa, ON K1Y 4E9, Canada. FAX: 613 761 4403; btsang{at}ohri.ca Back

Received: 4 June 2004.

First decision: 1 July 2004.

Accepted: 3 August 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bleil JD. In vitro fertilization. In: Wassarman PM, DePamphilis ML (eds.), Methods in Enzymology: Guide to Techniques in Mouse Development, vol. 225. San Diego, CA: Academic Press; 1993:253–263
  2. Menezo YJ, Veiga A, Pouly JL. Assisted reproductive technology (ART) in humans: facts and uncertainties. Theriogenology 2000 53:599-610[CrossRef][Medline]
  3. Menezo YJ, Chouteau J, Torello J, Girard A, Veiga A. Birth weight and sex ratio after transfer at the blastocyst stage in humans. Fertil Steril 1999 72:221-224[CrossRef][Medline]
  4. Wheeler MB, Clark SG, Beebe DJ. Developments in in vitro technologies for swine embryo production. Reprod Fertil Dev 2004 16:15-25[CrossRef][Medline]
  5. Taneja M, Bols PE, Van de Velde A, Ju JC, Schreiber D, Tripp MW, Levine H, Echelard Y, Riesen J, Yang X. Developmental competence of juvenile calf oocytes in vitro and in vivo: influence of donor animal variation and repeated gonadotropin stimulation. Biol Reprod 2000 62:206-213[Abstract/Free Full Text]
  6. Fraser LR. In vitro capacitation and fertilization. In: Wassarman PM, DePamphilis ML (eds.), Methods in Enzymology: Guide to Techniques in Mouse Development, vol. 225. San Diego, CA: Academic Press; 1993:239–252
  7. Benson CT, Critser JK. Variation of water permeability (Lp) and its activation energy (Ea) among unfertilized golden hamster and ICR murine oocytes. Cryobiology 1994 31:215-223[CrossRef][Medline]
  8. Kato M, Hirabayashi M, Aoto T, Kazumi I, Ueda M, Hochi S. Strontium-induced activation regimen for rat oocytes in somatic cell nuclear transplantation. J Reprod Dev 2001 47:407-413[CrossRef]
  9. Mizutani E, Jiang JY, Mizuno S, Tomioka I, Shinozawa T, Kobayashi J, Sasada H, Sato E. Determination of optimal conditions for parthenogenetic activation and subsequent development of rat oocytes in vitro. J Reprod Dev 2004 50:139-146[CrossRef][Medline]
  10. Songsasen N, Leibo SP. Cryopreservation of mouse spermatozoa. II. Relationship between survival after cryopreservation and osmotic tolerance of spermatozoa from three strains of mice. Cryobiology 1997 35:255-269[CrossRef][Medline]
  11. Beatty RA, Stewart DL, Spooner RL, Hancock JL. Evaluation by the heterospermic insemination technique of the differential effect of freezing at–196 degrees C on fertility of individual bull semen. J Reprod Fertil 1976 47:377-379
  12. Cohen J, Felten P, Zeilmaker GH. In vitro fertilizing capacity of fresh and cryopreserved human spermatozoa: a comparative study of freezing and thawing procedures. Fertil Steril 1981 36:356-362[Medline]
  13. Gilmore JA, Du J, Tao J, Peter AT, Critser JK. Osmotic properties of boar spermatozoa and their relevance to cryopreservation. J Reprod Fertil 1996 107:87-95
  14. Nakagata N, Takeshima T. Cryopreservation of mouse spermatozoa from inbred and F1 hybrid strains. Jikken Dobutsu 1993 42:317-320[Medline]
  15. Kaleta E. Influence of genetic factors on the fertilization of mouse ova in vitro. J Reprod Fertil 1977 51:375-381
  16. Sztein JM, Farley JS, Mobraaten LE. In vitro fertilization with cryopreserved inbred mouse sperm. Biol Reprod 2000 63:1774-1780[Abstract/Free Full Text]
  17. Wang Y, Chan S, Tsang BK. Involvement of inhibitory nuclear factor-{kappa}B (NF{kappa}B)-independent NF{kappa}B activation in the gonadotropic regulation of X-linked inhibitor of apoptosis expression during ovarian follicular development in vitro. Endocrinology 2002 143:2732-2740[Abstract/Free Full Text]
  18. Wang Y, Asselin E, Tsang BK. Involvement of transforming growth factor alpha in the regulation of rat ovarian X-linked inhibitor of apoptosis protein expression and follicular growth by follicle-stimulating hormone. Biol Reprod 2002 66:1672-1680[Abstract/Free Full Text]
  19. Wang Y, Rippstein PU, Tsang BK. Role and gonadotrophic regulation of X-linked inhibitor of apoptosis protein expression during rat ovarian follicular development in vitro. Biol Reprod 2003 68:610-619[Abstract/Free Full Text]
  20. Toyoda Y, Chang MC. Fertilization of rat eggs in vitro by epididymal spermatozoa and the development of eggs following transfer. J Reprod Fertil 1974 36:9-22
  21. Oh SH, Miyoshi K, Funahashi H. Rat oocytes fertilized in modified rat 1-cell embryo culture medium containing a high sodium chloride concentration and bovine serum albumin maintain developmental ability to the blastocyst stage. Biol Reprod 1998 59:884-889[Abstract/Free Full Text]
  22. Jiang JY, Umezu M, Sato E. Characteristics of infertility and the improvement of fertility by thyroxine treatment in adult male hypothyroid rdw rats. Biol Reprod 2000 63:1637-1641[Abstract/Free Full Text]
  23. Jiang JY, Miyoshi K, Umezu M, Sato E. Superovulation of immature hypothyroid rdw rats by thyroxine therapy and the development of eggs after in vitro fertilization. J Reprod Fertil 1999 116:19-24
  24. Jiang JY, Umezu M, Sato E. Vitrification of two-cell rat embryos derived from immature hypothyroid rdw rats by in vitro fertilization in ethylene glycol-based solutions. Cryobiology 1999 38:160-164[CrossRef][Medline]
  25. Bone W, Jones NG, Kamp G, Yeung CH, Cooper TG. Effect of ornidazole on fertility of male rats: inhibition of a glycolysis-related motility pattern and zona binding required for fertilization in vitro. J Reprod Fertil 2000 118:127-135
  26. Miyoshi K, Abeydeera LR, Okuda K, Niwa K. Effects of osmolarity and amino acids in a chemically defined medium on development of rat one-cell embryos. J Reprod Fertil 1995 103:27-32
  27. Miyoshi K, Funahashi H, Okuda K, Niwa K. Development of rat one-cell embryos in a chemically defined medium: effects of glucose, phosphate, and osmolarity. J Reprod Fertil 1994 100:21-26
  28. Miyoshi K, Kono T, Niwa K. Stage-dependent development of rat 1-cell embryos in a chemically defined medium after fertilization in vivo and in vitro. Biol Reprod 1997 56:180-185[Abstract]
  29. Miyamoto H, Chang MC. Fertilization of rat eggs in vitro. Biol Reprod 1973 9:384-393[Abstract]
  30. Jiang JY, Mizuno S, Mizutani E, Sasada H, Sato E. Parthenogenetic activation and subsequent development of rat oocytes in vitro. Mol Reprod Dev 2002 61:120-125[CrossRef][Medline]
  31. Iannaccone P, Taborn G, Garton R. Preimplantation and postimplantation development of rat embryos cloned with cumulus cells and fibroblasts. Zygote 2001 9:135-143[CrossRef][Medline]
  32. Willoughby CE, Mazur P, Peter AT, Critser JK. Osmotic tolerance limits and properties of murine spermatozoa. Biol Reprod 1996 55:715-727[Abstract]
  33. Miyamoto H, Chang MC. In vitro fertilization of rat eggs. Nature 1973 241:50-52[CrossRef][Medline]
  34. Vendrell J, Bonada M, Marqueta J, Torres M, Roses A, Idrobo J. Relationship between the culture of spare embryos and implantation rate: a reflection of intrinsic quality of the embryos. Revista Iberoamericana de Fertilidad 2003 20:89-98
  35. Pedrero S, Gutierrez-Corchado L, Guarnizo MC, Bachiller J, Estrade A, Gasca L, Salido E, Bebek H. Microdrops embryo culture cover by mineral oil—comparison of two types of mineral oil. Revista Iberoamericana de Fertilidad 2003 20:103-107
  36. Martinez F, Rienzi L, Iacobelli M, Ubaldi F, Mendoza C, Greco E, Tesarik J. Caspase activity in preimplantation human embryos is not associated with apoptosis. Hum Reprod 2002 17:1584-1590[Abstract/Free Full Text]
  37. Sakkas D, Leppens-Luisier G, Lucas H, Chardonnens D, Campana A, Franken DR, Urner F. Localization of tyrosine phosphorylated proteins in human sperm and relation to capacitation and zona pellucida binding. Biol Reprod 2003 68:1463-1469[Abstract/Free Full Text]
  38. Reynier P, May-Panloup P, Chretien MF, Morgan CJ, Jean M, Savagner F, Barriere P, Malthiery Y. Mitochondrial DNA content affects the fertilizability of human oocytes. Mol Hum Reprod 2001 7:425-429[Abstract/Free Full Text]
  39. Lu Q, Smith GD, Chen DY, Han ZM, Sun QY. Activation of protein kinase C induces mitogen-activated protein kinase dephosphorylation and pronucleus formation in rat oocytes. Biol Reprod 2002 67:64-69[Abstract/Free Full Text]
  40. Xiao CW, Asselin E, Tsang BK. Nuclear factor kB-mediated induction of flice-like inhibitory protein prevents tumor necrosis factor {alpha}-induced apoptosis in rat granulosa cells. Biol Reprod 2002 67:436-441[Abstract/Free Full Text]
  41. Xiao CW, Ash K, Tsang BK. Nuclear factor-{kappa}B-mediated X-linked inhibitor of apoptosis protein expression prevents rat granulosa cells from tumor necrosis factor {alpha}-induced apoptosis. Endocrinology 2001 142:557-563[Abstract/Free Full Text]
  42. Wang Y, WS O, Rippstein PU, Tsang BK. Role and gonadotrophic regulation of X-linked inhibitor of apoptosis protein expression during rat ovarian follicular development in vitro. Biol Reprod 2000 62:suppl 1108[Abstract/Free Full Text]
  43. Davis TE. Interstitial cell adenoma of the rat testis: why is it such a problem in toxicology studies using Sprague-Dawley rats?. Toxicol Pathol 1995 23:83-86[Free Full Text]
  44. Barton TS, Wyrobek AJ, Hill FS, Robaire B, Hales BF. Numerical chromosomal abnormalities in rat epididymal spermatozoa following chronic cyclophosphamide exposure. Biol Reprod 2003 69:1150-1157[Abstract/Free Full Text]
  45. Berger T, Miller MG, Horner CM. In vitro fertilization after in vivo treatment of rats with three reproductive toxicants. Reprod Toxicol 2000 14:45-53[CrossRef][Medline]
  46. Holloway AJ, Moore HD, Foster PM. The use of in vitro fertilization to detect reductions in the fertility of male rats exposed to 1,3-dinitrobenzene. Fundam Appl Toxicol 1990 14:113-122[CrossRef][Medline]
  47. Holloway AJ, Moore HD, Foster PM. The use of rat in vitro fertilization to detect reductions in the fertility of spermatozoa from males exposed to ethylene glycol monomethyl ether. Reprod Toxicol 1990 4:21-27[CrossRef][Medline]
  48. Berger T, Horner CM. In vivo exposure of female rats to toxicants may affect oocyte quality. Reprod Toxicol 2003 17:273-281[CrossRef][Medline]
  49. Sokol RZ, Okuda H, Nagler HM, Berman N. Lead exposure in vivo alters the fertility potential of sperm in vitro. Toxicol Appl Pharmacol 1994 124:310-316[CrossRef][Medline]
  50. Tsunoda Y, Chang MC. In vitro fertilization of rat and mouse eggs by ejaculated sperm and the effect of energy sources on in vitro fertilization of rat eggs. J Exp Zool 1975 193:79-86[CrossRef][Medline]
  51. Niwa K, Iritani A. Effect of various hexoses on sperm capacitation and penetration of rat eggs in vitro. J Reprod Fertil 1978 53:267-271
  52. Davis BK. Influence of serum albumin on the fertilizing ability in vitro of rat spermatozoa. Proc Soc Exp Biol Med 1976 151:240-243[CrossRef][Medline]
  53. Wu R. Control mechanisms of glycolysis in ehrlich ascites tumor cells. J Biol Chem 1965 240:2827-2832[Free Full Text]
  54. Miyamoto H, Chang MC. Effect of osmolality on fertilization of mouse and golden hamster eggs in vitro. J Reprod Fertil 1973 33:481-487
  55. Niwa K, Chang MC. Effect of pretreatment of rat spermatozoa with high concentrations of NaCl on sperm capacitation and fertilization of eggs in vitro. Biol Reprod 1975 13:187-189[Abstract]
  56. Hansen M, Kurinczuk JJ, Bower C, Webb S. The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med 2002 346:725-730[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
Y. Seita, S. Sugio, J. Ito, and N. Kashiwazaki
Generation of Live Rats Produced by In Vitro Fertilization Using Cryopreserved Spermatozoa
Biol Reprod, March 1, 2009; 80(3): 503 - 510.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/6/1974    most recent
biolreprod.104.032839v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, J.-Y.
Right arrow Articles by Tsang, B. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, J.-Y.
Right arrow Articles by Tsang, B. K.
Agricola
Right arrow Articles by Jiang, J.-Y.
Right arrow Articles by Tsang, B. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS