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BOR - Papers in Press, published online ahead of print July 14, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.027987
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BIOLOGY OF REPRODUCTION 71, 1671–1676 (2004)
DOI: 10.1095/biolreprod.104.027987
© 2004 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Bovine Blastocyst Development In Vitro: Timing, Sex, and Viability Following Vitrification1

Tshimangadzo L. Nedambale3, András Dinnyés4,5, Xiangzhong Yang3, and X. Cindy Tian2,3

Center for Regenerative Biology,3 University of Connecticut, Storrs, Connecticut 06269-4243 Research Group for Applied Animal Genetics and Biotechnology,4 Hungarian Academy of Sciences and Szent Istvan University, Godollo 2103, Hungary Department of Animal Biology,5 Agricultural Biotechnology Center, Godollo 2100, Hungary


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Selection of blastocysts based on their morphological characteristics and rate of development in vitro can skew the sex ratios. The aim of this study was to determine whether an embryo's developmental rate affects its survival after vitrification, and whether male and female embryos survive vitrification differently. In vitro fertilized bovine oocytes were cultured in potassium simplex optimized medium (KSOM) + 0.1% BSA for 96 h, and then into KSOM + 1% BSA (KSOM) or in sequential KSOM + 0.1% BSA for 96 h, and then into synthetic oviduct fluid (SOF) + 5% FBS (KSOM-SOF). In part 1 of this study, embryos cultured in each medium that had developed into blastocysts at approximately 144, 156, or 180 h were recovered from culture, graded, and then vitrified. After warming, blastocyst survival rates were immediately evaluated by reexpansion of the blastocoels. In the second part of the study, all blastocysts (n = 191) were sexed by polymerase chain reaction 48 h after warming. When cultured in KSOM medium, more 144-h blastocysts survived vitrification (68%) than blastocysts vitrified at 180 h (49%). Blastocysts derived at 156 h in KSOM-SOF survived vitrification better (87%) than blastocysts vitrified at either 144 h or 180 h, and subsequently hatched at a greater rate than those vitrified at 180 h. The overall blastocyst survival rates did not differ significantly whether embryos were cultured in KSOM or sequential KSOM-SOF. Blastocysts derived at 144 and 156 h in KSOM or KSOM-SOF were predominately male, and significantly more of them survived vitrification 48 h after warming. However, blastocysts cultured in KSOM-SOF, and then vitrified at 180 h were predominately female. Overall, blastocysts that survived vitrification, and subsequently hatched 48 h after warming, were male. In summary, embryos that reached the blastocyst stage earlier were predominantly males; these males had better morphology, endured vitrification, and subsequently hatched at a greater rate than did female blastocysts.

assisted reproductive technology, early development, embryo, fertilization, in vitro fertilization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The end point conventionally used to measure the efficacy of a culture system for in vitro embryo studies is the percentage of blastocyst formation, and it has become routine to select blastocysts, following a standard culture period, for cryopreservation or embryo transfer [1]. Furthermore, the faster-developing blastocysts in in vitro culture systems are generally considered more viable, and better able to survive following cryopreservation or embryo transfer than those that develop more slowly [2]. However, there is evidence that a female embryo might take longer than a male embryo to reach the developmental transition stage and form a blastocoel, and to advance in development from an early to an expanded blastocyst [3–5]. Several other studies indicated that in vitro-produced bovine blastocysts were predominantly male [4, 6, 7]; this was contrary to the report by Larson et al. [8] claiming that in vitro-produced male bovine embryos did not have an inherently faster rate growth than females in synthetic oviduct fluid (SOF) medium.

In vitro culture systems, especially a coculture system, plays a role in increasing the ratio of male over female embryos reaching the most advanced blastocyst stage [3, 5]. Glucose metabolism has, interestingly, been shown to be greater in male blastocysts than in female blastocysts [9], which may explain the rapid development of male embryos to the blastocyst stage. Additionally, in vitro culture systems containing serum have been associated with high blastocyst yields by inducing earlier blastocyst formation, which again might result in more male blastocysts [5]. Even so, it is not trivial to replace serum in in vitro studies, and results obtained with serum substitutes have been either inconsistent or inferior to those obtained with serum/BSA [10, 11]. Eliminating serum during the early stages of embryo development, however, has been reported to be beneficial [12] and could yield embryos with high cryoresistance and developmental potential, provided that serum is present near the time of compaction [13, 14]. We have previously tested sequential embryo culture media in which serum was added only in later stages of embryo development, and in doing so, obtained improved embryo development rates [14].

Embryos produced in vitro are more sensitive to low temperature and have less cryotolerance than those produced in vivo [15–17], due to deficiencies in the in vitro culture conditions [16]. Therefore, advances in embryo survival following vitrification could be achieved by improving their culture conditions, or by selecting embryos for vitrification based on the kinetics of their development. However, no studies have been conducted to compare the survivability of male and female embryos following cryopreservation. In this study, we aimed to determine 1) whether the rate of embryo development in defined or sequential media affect the embryos' survival after vitrification, and 2) whether male and female embryos survive vitrification differently.


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

Oocyte Collection and In Vitro Maturation

Bovine oocytes were collected from ovaries obtained from a slaughterhouse (Laceyville, PA), and matured in Tissue Culture Medium-199 (TCM-199) plus 10% FBS (Hyclone, Logan, UT), 1% antibiotic/antimycotic (Gibco BRL, Grand Island, NY), and 10 ng/ml epidermal growth factor [14]. Briefly, cumulus-oocyte complexes (COCs) were placed in sterile cryovials containing pregassed maturation medium and shipped to the laboratory overnight in a portable incubator (Minitube of America, Verona, WI) at 38.5°C. On arrival, oocytes were cultured to 24 h from the onset of maturation in 5% CO2 in air at 39°C.

In Vitro Fertilization and Culture

In vitro fertilization (IVF) was conducted in BO medium [18]. Briefly, matured oocytes with multiple layers of expanded cumulus cells were washed in TL-HEPES (Bio-Whittaker, Walkersville, MD) [14] and then in BO fertilization medium supplemented with 6 mg/ml essentially fatty acid-free (FAF)-BSA and 10 µg/ml heparin. For fertilization, 15–20 COCs were placed in 50 µl of BO medium, under mineral oil, containing frozen-thawed sperm (10 x 106 sperm/ml) from a single bull for 6 h in 5% CO2 in air at 39°C. The start of IVF = 0 h.

Presumptive zygotes (n = 796) were randomly allocated between the two culture media; potassium simplex optimized medium (KSOM) or KSOM-SOF) as described previously [14]: 1) KSOM + 0.1% FAF-BSA for 96 h, and then into KSOM + 1% FAF-BSA until 180 h (KSOM, n = 398); 2) KSOM + 0.1% FAF-BSA for 96 h, and then into SOF + 5% FBS until 180 h (KSOM-SOF, n = 398). Cleavage and blastocyst rates were recorded at 48 h postinsemination; and at 144, 156, or 180 h of culture. In this study, we deliberately chose 180 h as our end point stage of development because by this time, the majority of embryos are expected to have reached the blastocyst stage in vitro [19]. Blastocysts were graded according to the International Embryo Transfer Society (IETS) manual standard [20] as excellent (C1) or fair (C2).

Blastocyst Vitrification, Thawing, and Postwarming Culture

The vitrification protocol used in this experiment was adopted from that described by Rall [21] and has been described previously [14]. Briefly, 0.25-ml plastic straws were prepared prior to vitrification by pushing a cotton plug 2 cm into the straw, and placing a 7.5-cm column of 1.0 M sucrose in Dulbecco phosphate-buffered saline (DPBS) into the straw using a 1-ml syringe with a 27-gauge needle (Vetpharm, Sioux Center, IA). Then, a 1-cm column of vitrification solution (VS3a) was placed adjacent to the sucrose column but separated by a 0.5-cm air space.

For vitrification, blastocysts at 144, 156, or 180 h were equilibrated in three steps at room temperature. First, blastocysts were placed for 3 min in DPBS with 6% BSA (PB1) and then equilibrated in 25% VS3a (1.6 M glycerol with 6% BSA in DPBS) for 20 min. Blastocysts were then rinsed for 30 sec in 65% VS3a (4.2 M glycerol and 6% BSA in DPBS), and transferred into a column of 100% VS3a in a 0.25-ml straw. The straw was heat-sealed and allowed to remain at room temperature for 1 min, then held in liquid nitrogen (LN2) vapor (–150°C to –180°C) for 3 min and then placed directly into LN2. Straws were stored for several months in LN2 at –196°C.

Warming was accomplished by holding the frozen straw for 10 sec in air, 10 sec in a 22°C water bath, and then shaking it vigorously to mix the contents. The sealed end of the straw was cut and its contents emptied into a Petri dish. Warmed blastocysts were recovered and transferred into 1.0 M sucrose and then 0.5 M sucrose for 2 min each. Finally, blastocysts were rehydrated in modified PBS (mPBS; 3 mg/ml BSA in DPBS) for 4 to 5 min, washed, and transferred into their respective culture media: 1) KSOM + 1% BSA; or 2) SOF + 5% FBS. Reexpansion and development of blastocysts and hatching of blastocysts were recorded at 6, 24, or 48 h postthawing.

Determination of Sex of Blastocysts Following Postwarming Culture

Sexing was conducted as described by Park et al. [22]. Embryos that reached blastocyst at 144, 156, or 180 h were sexed after being vitrified/ warmed to determine whether the rate of blastocyst formation results in an imbalance in the sex ratio of in vitro produced bovine embryos.

Blastocysts at 144, 156, or 180 h were washed three times in DPBS without magnesium and calcium. One microliter of DPBS containing a single blastocyst was placed into a sterile, 0.6-ml polymerase chain reaction (PCR) tube. Then, 5 µl of K-buffer (10 x PCR buffer without MgCl2, Qiagen Proteinase K, and 5% Tween-20) was added into the PCR tube containing the blastocyst. The tubes were placed in a PTC-200 Petter Thermal Cycle (MJ Research, Inc, Watertown, MA) and lysed at 56°C for 1 h, followed by 10 min at 95°C to denature or inactivate the proteinase K [23]. The tubes were stored at –20°C until PCR analysis. Genomic DNA isolated from skin tissues of male and female cattle (50 ng/µl) was used as positive controls for embryo sexing.

The bovine DNA sequences, BOV97M and bovine 1.715 satellites, were used for sex determination. Amplification of BOV97M, located on the Y chromosome, produces a PCR product of 141 base pairs (bp). Amplification of the bovine 1.715 satellite, located on an autosome, produces a PCR product of 216 bp, indicating the success of the PCR procedure. The presence of both PCR products indicates a male embryo, and the presence of only the 216-bp product indicates a female embryo (Fig. 1). The BOV97M primers were forward, 5'-GAT CAC TAT ACA TAC ACC ACT-3'; and reverse, 5'-GCT ATG ACA CAA ATT CTG-3'. The sequence of the bovine-specific primers were forward, 5'-TGG AAG CAA AGA ACC CCG CT-3'; and reverse, 5'-TCG TCA GAA ACC GCA CAC TG-3'. One-third of a blastocyst lysate (2 µl of the total 6 µl of blastocyst lysate) was used in a final volume of 25 µl. The reaction mixture contained 1x PCR buffer (Invitrogen-Life Technology, Carlsbad, CA) without MgCl2, 2 mM MgCl2 (Invitrogen-Life Technology), 10 mM total dNTP, 1 U of Tag DNA polymerase (Invitrogen-Life technology) and 3 ng/µl of each BOV97M primer, 1.5 ng/µl of each bovine-specific primer. Amplification was performed for a total of 40 cycles. Each consisted of template denaturation at 95°C for 30 sec, primer annealing at 50°C for 30 sec, and primer extension at 72°C for 45 sec. After 40 cycles, the samples were incubated at 72°C for 5 min and cooled to 4°C. The amplified products were then electrophoresed on a 1.5% agarose gel (Invitrogen-Life technology), stained with ethidium bromide, and evaluated using UV light (Fig. 1).



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  		FIG. 1. Sex determination of blastocysts (144 h) cultured in KSOM alone or sequential KSOM-SOF: ethidium bromide-stained agarose gel electrophoresis (1.5%) of Y/bovine satellite duplex PCR. Arrows indicate the position of the specific bands: in PCR, males exhibit two bands, whereas females reveal only one band. Mr represents the molecular weight marker, positive (+) controls (lanes 2 and 3) represent the presence of female (F) and male (M) DNA, and negative (–) control (N) represents the absence of DNA

Experimental Design

Part one: to determine whether the rate at which the blastocyst stage is reached influences survival following vitrification This experiment was designed to compare vitrified blastocysts that had developed in vitro by 144, 156, or 180 h in KSOM alone or in sequential KSOM-SOF. Blastocysts were examined under a stereomicroscope and graded as C1 (excellent or good) or C2 (fair) according to the IETS standard [20] before vitrification.

Part two: determination of blastocyst sex following vitrification Embryos that reached the blastocyst stage at 144, 156, or 180 h were sexed after being vitrified/warmed to determine whether the rate of blastocyst formation correlated with a skewed sex ratio of in vitro-produced bovine embryos (KSOM alone vs. sequential KSOM-SOF).

Statistical Analysis

The data were subjected to a one-way analysis of variance. Differences between rates of blastocyst development by the number of hours in culture media were determined using a Bonferroni test for pair-wise comparison of means. The sex ratio of blastocysts that developed in KSOM or KSOM-SOF was analyzed by chi-square tests. P values < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Part One

Blastocysts that were cultured for 144, 156, or 180 h in KSOM alone or in sequential KSOM-SOF were vitrified, and their viability was evaluated postwarming at 6, 24, or 48 h. Additionally, we recorded the percentage of all hatched blastocysts cultured in each medium (Fig 2 and Table 1).



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  		FIG. 2. Postwarming development of all vitrified blastocysts cultured in KSOM alone or in sequential KSOM-SOF (solid bars = KSOM, open bars = KSOM-SOF)


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  		TABLE 1. Survival rate and grade of vitrified bovine blastocysts derived at different times (hours) in culture media (KSOM vs. KSOM-SOF)

In KSOM alone, of the blastocysts vitrified at 144 or 156 h, the survival percentage was not statistically different at 6 h postwarming. However, more 144-h blastocysts developed 48 h postwarming, compared with 156-h blastocysts. Furthermore, blastocysts formed at 144 h survived vitrification and subsequently hatched better (P < 0.05) than those from 180 h of culture (Table 1).

In sequential KSOM-SOF, a significantly (P < 0.05) higher percentage of the blastocysts vitrified at 156 h (P < 0.05) reexpanded and developed at 6, 24, or 48 h postwarming than did those from 144 h or 180 h. Furthermore, a greater percentage (P < 0.05) of the blastocysts that were vitrified at 156 h hatched, compared with those vitrified at 144 h or 180 h (Table 1).

As shown in Table 1 (KSOM alone vs. sequential KSOM-SOF), a significantly (P < 0.05) greater percentage of 156-h blastocysts from sequential KSOM-SOF reexpanded and developed at 6, 24, or 48 h postwarming compared with all hours of culture in KSOM alone. Subsequently, embryos reaching the blastocyst stage at 144 h in the sequential media hatched at a rate equal to those from 144 h in KSOM, but better (P < 0.05) than those from 180 h of culture in either media (Table 1). Furthermore, a greater (P < 0.05) percentage of the blastocysts that appeared at 144 or 156 h, in either KSOM or KSOM-SOF, were graded as excellent (C1), while more of the blastocysts that appeared at 180 h were graded as fair (C2, Table 1).

There were no statistical differences in the overall percentage of blastocysts that reexpanded, developed, and subsequently hatched (39% vs. 50%) from either KSOM or KSOM-SOF at 6, 24, or 48 h postwarming (Fig. 2).

Part Two

When embryos that had been cultured in KSOM alone for 144 or 156 h were vitrified, a greater percentage of the survivors that hatched after 48 h in culture were determined to be males rather than females (i.e., 44% vs. 14%, and 29% vs. 5%). A similar pattern was found for embryos cultured in KSOM-SOF for 144 h (i.e., 43% vs. 3%); for embryos cultured in KSOM-SOF for 156 h, the difference between the sexes of the survivors was not as large (i.e., 49% vs. 22%), yet the difference was still significant. However, of the blastocysts formed at 180 h of culture, a significantly greater (P < 0.05) percentage of females survived vitrification, and subsequently hatched (Table 2).


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  		TABLE 2. Postwarming survival rates of vitrified bovine blastocysts derived from KSOM or sequential KSOM-SOF media

Overall, a significantly (P < 0.05) greater percentage of the blastocysts that survived vitrification at 48 h postwarming and that subsequently hatched, were males. This was true when male and female blastocysts were compared, regardless of culture in either KSOM or KSOM-SOF (Fig. 3).



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  		FIG. 3. The overall rate of male vs. female blastocysts produced in KSOM alone or in sequential KSOM-SOF that survived vitrification 48 h postwarming, and hatched. Forty-eight hour-male or 48 h-female = 48 h postwarming male/female blastocysts, H-male or H-female = hatched male/female blastocysts. Solid bars = KSOM, open bars = KSOM-SOF

The overall pooled percentage of male (T-male) or female (T-female) blastocysts that developed throughout the culture period, from 144 to 180 h, did not, interestingly, differ significantly, in either KSOM or KSOM-SOF (Fig. 4). Furthermore, embryos cultured in KSOM and that reached the blastocyst stage early by 144 or 156 h did not show a skewed sex ratio between male and female (58% vs. 42%, and 51% vs. 49% for 144 and 156 h, respectively). However, there was a significant culture effect on the sex ratio of embryos that reached the blastocyst stage later, by 180 h (31% male vs. 69% female), in the KSOM group. In contrast, there was a culture effect on the sex ratio of embryos that reached the blastocyst stage in sequential KSOM-SOF by 144 h (61% male vs. 39% female).



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  		FIG. 4. Overall pooled percentage of male and female blastocyst development in KSOM vs. KSOM-SOF. Solid bars = T-male (total number of male blastocysts), open bars = T-female (total number of female blastocysts)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The most significant finding of this study is that male blastocysts cultured in KSOM or sequential KSOM-SOF survived vitrification better, had better morphology, and subsequently hatched at a greater rate than did female blastocysts 48 h postwarming. The results of this study also indicated that in vitro-produced embryos reaching the blastocyst stage earlier were more likely to be males than females, as previously reported in bovine [3, 4, 24] mouse [25, 26], ovine [27, 28], and human [29].

Embryos that formed blastocysts at 156 h in sequential KSOM-SOF survived vitrification better than all other embryos in this study. This finding demonstrated that embryos that cleave and reach the blastocyst stage earlier survive cryopreservation better than those that take a longer time in development before reaching the blastocyst stage. It is interesting that a preimplantation embryo development gene (Ped) has been identified that provides important information for the timing differences observed in in vitro embryos. Early cleaved embryos have been reported to express a Ped fast gene, which confers a developmental advantage over late-cleaved embryos that express a Ped slow gene [30, 31]. In addition, the quality of our blastocysts correlated with rapid development, because more of the blastocysts that appeared at 144 and 156 h were graded as excellent (C1), whereas more blastocysts appearing at 180 h were graded as fair (C2). This is consistent with the frequent practice of using the timing of development as an indicator of bovine embryo quality [32].

In part 2 of this study, our findings indicate that male embryos develop more rapidly and reach the blastocyst stage earlier than females, in either KSOM or KSOM-SOF media. The higher rates of postwarming survival of blastocysts that formed at 144 or 156 h indicated that predominantly males, rather than females, will tolerate vitrification. This suggests that a greater number of male fetuses will result from successful vitrification and transfer of in vitro-produced bovine embryos selected for quality. This observation provides further evidence that more male calves will be born following embryo transfer of in vitro-produced embryos, as reported previously by Hasler [33]. These data were also consistent with our recent report [14] in which all of the fetuses, from a 30% pregnancy rate, were determined to be male after Caesarean delivery at Day 75 of pregnancy. However, our sample size was small in that study due to a limited number of recipients. It is interesting that blastocysts that formed at 180 h showed no sex bias in KSOM alone, although a large proportion of male embryos had already been removed from the culture at 144 or 156 h. However, a significant increase was found in the percentage of female blastocysts appearing at 180 h, indicating that female embryos took longer to develop to the blastocyst stage (Table 2). The higher survival rate of male blastocysts 48 h postwarming (Fig. 3) from both culture systems was probably due to female blastocysts developing slower, reaching the blastocyst stage later, and more often being graded as fair or poor, under in vitro conditions, as reported previously [3, 4, 6]. Perhaps the enhanced survival of male blastocysts is related to the fact that male blastocysts have been shown to have a greater number of cells than female blastocysts [4, 27]. By contrast, in the study by Beyhan et al. [34], male and female embryos had the same total cell numbers at Day 8. Total cell numbers have long been regarded as a valuable and reliable indicator of blastocyst quality [32].

One possible explanation for enhanced cryosurvival of male blastocysts in this study is that they developed faster; it is reasonable to assume that they also formed more cells. This assumption is based on the rate of development, as no direct comparison of cell numbers between male and female blastocysts were performed in our study. Our female embryos lagged behind the males in their development, and it may be that female embryos under in vitro culture conditions are affected by suboptimum conditions of nutrition, or environment, to a greater extent than male embryos [3– 5]. Another indication that the female embryos were not under the most favorable conditions for culture in our study, was that most of them were graded as being only of fair quality (C2). Significantly more (P < 0.05) of those embryos that reached the blastocyst stage earlier (144 or 156 h) were graded as excellent. Moreover, they survived vitrification better than those that reached the blastocyst stage at 180 h, which supports an earlier finding by Van Soom et al. [32] that embryo morphology is correlated to blastocyst formation and hatching ability. Not surprisingly, in vitro-produced embryos graded as excellent have been reported to yield higher pregnancy rates following transfer [35].

Although male embryos predominately developed faster and reached the blastocyst stage earlier when all embryos were included, the overall sex ratio did not deviate significantly from 50:50; this held true whether culture was in KSOM or KSOM-SOF (Fig. 4). Thus, the male to female ratio might not be altered if blastocysts were not selected based on their morphological quality and rate of development. Selection of blastocysts based on their developmental stage or general morphological qualities (or both) will likely alter their sex ratio, although survival is enhanced by such selection [3, 4, 35]. Due to such practice, the sex ratio of embryos used for cryopreservation or embryo transfer becomes skewed to favor the males.

Clearly, there is a sex ratio discrepancy in in vitro-produced bovine embryos that does not occur in vivo. The mechanism or mechanisms by which male and female embryos differ in their rates of development to the blastocyst stage is not clearly understood. It has been suggested that a deleterious effect of a double dose of the X chromosome might be a factor for the differences in growth rate between male and female embryos [36]. Female embryos carrying two X chromosomes potentially produce twice the amount of X-linked enzymes compared with male embryos [37], and probably more importantly, in vitro culture systems seemed to delay X chromosome inactivation in female mammalian embryos [36, 37]. Some researchers have suggested that the X-linked enzymes (G6PDH, HPRT) related to glucose metabolism might also play a role in the growth rate differences observed in vitro between male and female embryos. The fact that female embryos express more of these enzymes than do male embryos might put them at a disadvantage with respect to the need to adapt metabolically to the subphysiological in vitro conditions [25]. Additionally, glucose and oxygen concentrations have been shown to control sex-related growth rate differences in bovine in vitro-produced embryos [38]. This would suggest a possible role for levels of oxygen radicals that would normally be lower in female embryos, due to the double expression of G6PDH and HPRT, and would occur prior to X chromosome inactivation [39]. Oxygen radical levels might then be too low to activate growth-promoting genes. The presence of glucose in medium has also been reported to favor the growth of male over female bovine embryos. Perhaps, due to X-linked gene expression, or overexpression, glucose metabolism is more efficient in male embryos [9].

In conclusion, although the overall sex ratio was maintained close to 50:50, in both KSOM alone and sequential KSOM-SOF, embryos that reached the blastocyst stage earlier were predominantly male, and they survived vitrification and subsequently hatched better than did female blastocysts. This raises the possibility that cryobiologists and embryologists might be biasing their selection of embryos toward males by using conventional embryo grading techniques based on morphology and the rate of in vitro development. This potential skewing of sex ought to be taken into account when preserving beef or dairy blastocysts, in which the sex of the progeny is of major economic importance. Furthermore, gene banking efforts must develop a strategy for addressing this effect when preserving various breeds and species. Embryo sexing prior to cryopreservation or transfer might provide one solution by offering selection based on sex.


   ACKNOWLEDGMENTS
 
We thank Marina Julian for critically reading the manuscript, Drs. Jie Xu and Thomas A Hoagland for help with statistical analyses, and Yuquin (Linda) Zhang for helpful suggestions regarding PCR.


   FOOTNOTES
 
1 This research was supported by a joint grant (FY2001) from the Internship Program for Early Career South African Scientists (U.S. Department of Agriculture, South African National Department of Agriculture, Agricultural Research Council, and Colorado State University). Back

2 Correspondence: X. Cindy Tian, Connecticut Center for Regenerative Biology/Department of Animal Science, University of Connecticut, 1392 Storrs Road, U-4243, Storrs, CT 06269-4243. FAX: 860 486 8809; xtian{at}canr.uconn.edu Back

Received: 29 January 2004.

First decision: 18 February 2004.

Accepted: 9 July 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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