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a Department of Animal Science, University of Connecticut, Storrs, Connecticut 06269
b Genzyme Transgenic Corporation, Framingham, Massachusetts 01701-9322
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The development of embryos produced in vitro from oocytes of juvenile donors has been variable. Armstrong et al. [5] observed no significant differences in the fertilization and development rates of cow and calf oocytes matured either in vivo or in vitro. A 43% pregnancy rate and 33% live births (of embryos transferred) from fresh in vitro-produced (IVP) embryos from calf oocytes were reported in their study. Revel et al. [6] reported pregnancy rates of IVP embryos from calf oocytes that were similar to those for adult cow oocytes; however, higher rates of embryo losses later in pregnancy were observed for the calf oocyte group. Abnormalities in protein synthesis in oocytes from unstimulated calf ovaries were found and interpreted as an indication of cytoplasmic deficiencies responsible for the poor developmental competence of calf oocytes [7]. Similar conclusions were reached by Duby et al. [8] concerning developmental competence of oocytes from calves younger than 6 mo of age. Damiani et al. [9] partly attributed the low developmental competence of calf oocytes to incomplete or delayed ooplasmic maturation. Khatir et al. [10] demonstrated the inability of calf oocytes to respond to serum or follicular fluid supplementation during in vitro maturation for a subsequent improvement in development during culture. They speculated that lack of receptors for gonadotropins or growth factors was the reason for the nonresponsiveness. Significant differences between cow and calf oocytes in terms of their respective sizes, as well as differences in their energy metabolism during in vitro maturation, have also been reported recently [11].
Previously, we have demonstrated [2] that it is not until 11 mo of age that oocytes from unstimulated heifers show an in vitro developmental competence similar to those from adult cows. In other words, juvenile and prepubertal calves do need gonadotropin stimulation prior to oocyte collection and IVP in order to achieve some degree of viability.
In the present study, a group of 10 age-matched juvenile calves of high genetic merit were subjected to gonadotropin stimulation and oocyte retrieval at 2–3 mo of age. This procedure was repeated when the animals reached 4–5 mo of age. The experiment was designed 1) to examine the number of follicles prior to and after gonadotropin stimulation, 2) to correlate the preexisting follicle numbers with the response to gonadotropin stimulation and in vitro embryo production, 3) to compare the animal age effect on oocyte embryogenic competence, and 4) to ascertain the fertility and conception ability of donor calves at maturity. A preliminary trial was also conducted to evaluate the follicular stimulation protocols. Part of this research was presented at the 1998 Society for the Study of Reproduction Annual Meeting [12].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This project was approved by the Institutional Animal Care and Use Committee of the University of Connecticut. Holstein calves used for the research were selected as donors by a local farmer on the basis of their genetic merit. These donor calves were transported to and housed at the University of Connecticut's Kellog Dairy Center during the entire duration of the study and were maintained on a standard balanced ration. Cumulus-oocyte complexes (COCs) were surgically collected twice from these calves—first at 2–3 mo of age (mean 89.9 ± 5.1 days) and then again 8 wk later at 4–5 mo of age (mean 147.3 ± 4.5 days), hereafter referred to as 2-mo cycle and 4-mo cycle, respectively. Calves were stimulated with gonadotropin treatment before the collection of COCs. In order to test whether prior stimulation of calves at 2–3 mo of age had any effect on the subsequent collection and competence of oocytes at 4–5 mo of age, a group of five 4- to 5-mo-old calves with no prior treatment were stimulated and subjected to aspiration for use as controls.
Gonadotropin Stimulation
Calves were stimulated with a total dosage of 130 mg (40, 30, 30, 30 mg) of FSH (Folltropin V; Vetrepharm, Ontario, ON, Canada) over 2 days as described by Kelly et al. [13]. At 2–3 mo of age, calves received progestin treatment for 6 days prior to FSH stimulation, either as a progesterone vaginal sponge (CIDR-G; InterAg, Hamilton, New Zealand) or as a norgestomet s.c. implant (Synchromate-B; Rhone Merieux, Athens, GA). A pilot comparison between Synchromate-B and CIDR-G was made in order to observe their influence on number, quality, and embryogenic competence of calf oocytes on a separate group of 7 age-matched prepubertal Holstein calves (Table 1). COCs were aspirated at 12–14 h after the final FSH injection, and implants or sponges were removed after oocyte collection. For the 4-mo cycle, calves received the progestin support for a similar duration of 6 days, but only in the form of Synchromate-B s.c. implants.
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Laparoscopy and Oocyte Retrieval
In order to observe the ovaries and record the number of visible antral follicles prior to the stimulation, laparoscopy [14,15] was performed 6 days before the start of gonadotropin treatment of the 2-mo cycle. The procedure was done midventrally with calves under ketamine sedation.
Oocytes from prepubertal calves were recovered during midventral laparotomy under general anesthesia with a xylazine-ketamine combination [16]. Antral follicles, 4–7 mm in diameter, were aspirated using an 18-gauge needle attached to a 10-ml syringe. The aspiration medium consisted of Dulbecco's PBS (D-PBS) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin-B (Gibco Laboratories, Grand Island, NY), 3 mg/ml BSA (fraction V), and 60 IU/ml sodium heparin (Leo Pharmaceutical Products, Zaventem, Belgium). Contents of the syringe were then transferred into an Em-Con (Pets Inc., Canton, TX) embryo filter and washed 2–3 times with D-PBS. Filters were then transported to the laboratory, where the COCs were recovered, evaluated under a dissection microscope, washed in D-PBS, and then subjected to in vitro maturation (IVM).
IVM, Fertilization, and Culture (IVM/IVF/IVC)
Successful in vitro fertilization (IVF) procedures with a > 35% routine blastocyst development rate following the insemination of cow oocytes were used for this research [17]. All the chemicals used in this study were from Sigma Chemical Co. (St. Louis, MO) unless stated otherwise. All the cultures were done in 4-well culture plates (Nunclon; VWR Scientific, Bridgeport, NJ) in 500 µl medium under oil and were incubated in a humidified atmosphere of 5% CO2 in air at 39°C, unless otherwise stated. COCs were considered "usable" unless they were damaged or had expanded cumulus. Usable COCs were matured in vitro for 24 h in Tissue Culture Medium 199 with Earle's salts (ICN Biomedicals, Aurora, OH) and 20% fetal bovine serum (Hyclone, Logan, UT), with addition of 5 µg/ml FSH (Vetrepharm), 5 µg/ml LH (NIDDK, Baltimore, MD), and 1 µg/ml estradiol (Fig. 1a).
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Fertilization medium consisted of modified synthetic oviduct fluid (mSOF) [18] supplemented with 6 mg/ml fatty acid-free BSA. Frozen-thawed semen from a proven bull of high merit was used throughout the study. Percoll-selected motile spermatozoa were used for fertilization at a concentration of 1.0 x 106/ml. The Percoll gradient (50, 70, and 90%) was made with sperm preparation medium consisting of Hepes-SOF medium, 5 mg/ml fatty acid-free BSA, 0.5-mg/ml caffeine, 10 µg/ml heparin, and 0.3 mg/ml glutathione. Cumulus cells and excess spermatozoa were removed by repeated pipetting at 24 h after the start of coincubation.
The in vitro culture (IVC) was in mSOF containing 6 mg/ml fatty acid-free BSA, 30 µl/ml essential amino acids, 10 µl/ml nonessential amino acids, 0.5 mg/ml myoinositol, and 30 µg/ml glutamine in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Cleavage development was recorded at 96 h postinsemination (Fig. 1b). Morulae and blastocyst production rates were recorded on Days 6 and 7, and embryos were morphologically assessed using a subjective grading system (1, excellent; 4, poor). Both compact morulae and blastocysts were considered transferable embryos (Fig. 1c).
Embryo Transfer
On Day 7, embryos were transferred nonsurgically into virgin Holstein heifers on a local farm located 25 miles away from the laboratory. A single embryo was transferred into a recipient with the exception of 3 twin transfers. Embryos of grade 1–3 quality were transferred in bovine embryo transfer medium (Life Technologies, Grand Island, NY) into the uterine lumen ipsilateral to the corpus luteum. Recipient synchronization was achieved using the standard double-injection prostaglandin (Lutalyze; Pharmacia & Upjohn, Kalamazoo, MI) treatment [19]. Pregnancies were diagnosed with ultrasonography between Days 50 and 60.
Artificial Insemination (AI) of Donors
All donor heifers were subjected to 2 surgical oocyte collection sessions as described earlier. These heifers then received 4 more (6 in total) hormonal stimulation treatments between 6 and 13 mo of age as reported previously [20]. Additionally, they were subjected to at least 6 more ultrasound-guided TVOR, with or without ovarian stimulation (data not shown). Donor heifers at 14–15 mo of age were bred by AI in order to evaluate the treatment effect on their breeding performances. AI was performed with frozen-thawed semen from the same bull as had been used for IVF.
Statistical Analysis
Statistical analysis was performed using one-way ANOVA for the follicle numbers, oocyte recovery, and embryo development data. The data on number of follicles aspirated and number of oocytes recovered were also tested for correlation with the number of follicles scored before the initial stimulation. Chi-square or Fisher's exact test was used to analyze the effect on pregnancy rates of embryo grade and of age at collection. Percentage data were arcsine transformed [21]. Tadpole III statistics software (Elsevier-Biosoft, Cambridge, UK) was used for the data analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a preliminary trial, two progestin products (Synchromate-B and CIDR-G) were tested in the stimulation regimen of the prepubertal donor calves in order to observe any differences in their ovarian response, as well as the number, quality, and embryogenic competence of recovered oocytes. The results are presented in Table 1. There were no differences (P > 0.05) between the two progestin products in any parameters examined. Therefore, data from the two progestin treatments in the subsequent experiment were pooled.
In the 2-mo cycle, an average of about 23 follicles per donor was observed with laparoscopic examination on the ovarian surface prior to the start of stimulation. These numbers approximately doubled to 55 follicles per donor after stimulation. Follicle sizes were not precisely measured at the time of aspiration; however, they appeared to be in the range of 4–7 mm. After the aspiration of 551 follicles from 10 donor calves, 455 (83%) oocytes were recovered, of which 439 (96%) were usable and were subjected to IVM/IVF/IVC.
At the 4-mo cycle, these same 10 donor calves were again subjected to gonadotropin stimulation; however, this time no laparoscopic observation of ovarian follicles was done prior to stimulation. From 474 follicles aspirated, 398 (84%) oocytes were recovered; 365 (92%) recovered oocytes were deemed usable and were subjected to IVM/IVF/IVC. The number of follicles aspirated, number of oocytes recovered, number of usable oocytes recovered, and the percentage oocyte recovery were not significantly different from those of the 2-mo cycle (Table 2).
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To test whether prior stimulation and follicular aspiration of donor calves at 2–3 mo of age had any negative effect on the subsequent stimulation response and competence of oocytes at 4–5 mo of age, a group of 5 age-matched calves stimulated for the first time at 4–5 mo of age was used as a retrospective control group. The mean number of follicles observed laparoscopically prior to their stimulation was 34.6 ± 4.8 per donor. The corresponding number of follicles aspirated and of oocytes recovered was 47.4 ± 12.4 and 39.8 ± 11.8 per donor. Statistically, these data were not different from those of the age-matched heifers at the 4-mo cycle.
In Vitro Embryo Production
In the first stimulation and aspiration (2-mo cycle) of the 10 donors, out of 439 inseminated oocytes, 182 (41%) cleaved and resulted in 49 (11%) transferable morulae or blastocysts after 6 days of IVC. At the 4-mo cycle, out of 365 inseminated oocytes, 157 (43%) cleaved and resulted in 36 (10%) transferable morulae or blastocysts. The oocyte cleavage and embryo development rates were not different between the two collection ages (Table 2). The rates of cleavage (36.8 ± 5.4 vs. 43.6 ± 11.1) and embryo development (9.3 ± 2.2 vs. 16.2 ± 6.3) were not statistically different between the 4-mo collection cycle and the age-matched stimulated control group (P > 0.05).
Embryo Transfer, Pregnancies, and Offspring Born
Of oocytes recovered from both the 2-mo and 4-mo cycles, 804 oocytes were inseminated, of which 339 (42%) cleaved, resulting in 85 (10%) transferable embryos. Eighty-four IVP embryos were transferred into 81 recipients, resulting in 11 pregnancies (13%) and 9 calves born at full term (278 ± 1.6 days). Of 11 pregnancies, 8 (73%) resulted from embryos produced from oocytes recovered at the 2-mo cycle, although this rate was not significantly different from the 4-mo cycle pregnancy rate. Of 9 calves born, 7 calves (4 males, 3 females, Fig. 1d) are healthy and living, whereas 2 calves (a male and a female) died at birth due to dystocia. These 2 calves were relatively overweight at birth as compared to the 7 living calves (57 vs. 44 kg mean birth weight), and one of them also had an obstetrically difficult presentation. Of the 7 living calves, 2 were born with elective cesarean section and the rest were born vaginally. Of the remaining 2 pregnancies, one was diagnosed as nonviable (fetus estimated to have died at around Day 45 of pregnancy) by ultrasonography; the other was diagnosed as a hydrops pregnancy near term, and this recipient was sold by the farmer.
Individual Donor Variation
A significant variation between the individual donors was seen in terms of number of follicles observed with laparoscopy (range 5–70) before stimulation at 2–3 mo of age. Similarly, a large variation was observed in the number of follicles aspirated (range 5–164) and the number of total and usable oocytes recovered (range 4–152) among the donors at the 2-mo cycle (Fig. 2A). A similar trend of follicular stimulation response was observed at the 4-mo cycle (Fig. 2B). A significant, positive correlation was observed between the initial number of follicles observed laparoscopically before stimulation and the number of follicles observed and aspirated poststimulation, number of oocytes recovered, and number of usable oocytes at the 2-mo cycle (P < 0.001; Table 3). The initial follicle pool observed prior to stimulation at the 2-mo cycle remained highly correlated with the stimulation responses at the 4-mo cycle (P < 0.01; Table 3).
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For the purpose of comparison, the donor calves were arbitrarily divided into two groups: those that had 
20 follicles (n = 4; calves A–D; Fig. 2) and those that had < 20 follicles (n = 6; calves E–J; Fig. 2), on the basis of laparoscopic observation of follicles prior to stimulation of the calves at the 2-mo cycle. The former group ( 20 follicles) demonstrated a significantly greater (P < 0.05) number of follicles poststimulation, at both the 2-mo and 4-mo collection cycles, thereby yielding a greater number of total and usable oocytes (Table 4, Fig. 3). Although the cleavage (41% vs. 37%) and development (11% vs. 9%) rates were not significantly different between the two groups (P > 0.05), significantly more embryos per donor (both cleaved and transferable embryos) were obtained from the 20-follicle group. Of the total embryos produced, 33 of 61 (54%) in the 20-follicle group and 9 of 23 (39%) in the < 20-follicle group were grade 1 or 2 embryos (mean 8.2 ± 2.8 vs. 1.5 ± 0.8; P < 0.05). All 11 pregnancies came from oocytes obtained from 3 of the 4 calves in the 20-follicles group (mean 2.75 ± 1.4) as compared to none from the oocytes of calves in the < 20-follicle group. Altogether, the oocytes recovered from those 3 calves resulted in a greater number (47 vs. 37) and better quality (55% vs. 43% grade 1 and 2 embryos, P > 0.05) of transferable embryos than for the other 5 calves whose oocytes did not result in any pregnancy. Transfer of embryos developed in vitro by utilizing the oocytes recovered from calves belonging to the 20-follicle group, first at 2–3 mo of age and then again at 4–5 mo of age, resulted in 8 of 37 (21%) and 3 of 25 (12%) pregnancies, respectively (P > 0.05). Oocytes recovered from 8 of 10 donors in the 2-mo and 4-mo cycles resulted in embryo production; 3 of 12 oocytes recovered from the remaining 2 donor calves cleaved to 2–4 cells but did not develop further.
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In order to examine the effect of embryo quality on pregnancy rates, the quality grade of the embryos from the 3 donor calves that resulted in pregnancies was matched to the pregnancy results. Grade 1, 2, and 3 embryos resulted in 4 of 10 (40%), 5 of 16 (31%), and 2 of 21 (9%) pregnancies, respectively; however, statistically these data were not significantly different.
Effect of Repeated Oocyte Collection on Donors' Conception Rate
Of 10 heifer donors, 3 (30%), 1 (10%), 4 (40%), and 1 (10%) conceived successfully with AI at the first, second, third, and fourth consecutive estrus, respectively. All 4 heifers that became pregnant at first or second consecutive estrus belonged to the < 20-follicle group. One heifer (Fig. 2, donor A) failed to conceive and has returned to estrus at regular intervals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Influence of donor cow variation on IVP of embryos has been reported by Hasler et al. [23]. Individual animal variations are common in adult cows and sexually mature heifers in terms of superovulatory response, and the reasons are not yet completely understood [22]. The authors of a recent study [24], on the basis of FSH and LH receptor analyses, concluded that the difference in ovulation rates between high- and low-prolific sheep breeds is partly associated with the gonadotropin responsiveness of follicles in the early follicular phase. In the present study, we found that a wide variation also exists among young prepubertal heifers in the number of antral follicles and their subsequent stimulation responses. In this study, oocytes aspirated from 8 of 10 donor calves resulted in in vitro embryo production. Transfer of IVP embryos of 8 donor calves into synchronized recipients resulted in 11 pregnancies, i.e., one pregnancy per donor calf. However, it is noteworthy that all 11 pregnancies were obtained from 3 of the donor calves. Oocytes recovered from these 3 donor calves (donors A–C; Fig. 2) resulted in a greater number of embryos per donor and also better-quality embryos as compared to those of the other 5 calves whose embryos did not result in pregnancies upon transfer. These 3 donor calves all had 20 follicles before gonadotropin stimulation at 2–3 mo of age. On the basis of the significantly different number of follicles available for aspiration and the number of oocytes recovered poststimulation, calves in the 20-follicle group could be classified as "high responders" and those in the < 20-follicle group as "low responders." A highly positive correlation of poststimulation response to the number of antral follicles in the juvenile calves prior to stimulation suggests that high-responding donors may be selected based on the availability of ovarian antral follicles. A similar positive correlation has been recently reported [25] between the superovulatory response and primordial and growing follicles in adult cows.
Several hormone treatment regimens have been used on prepubertal calves with the aim of either obtaining oocytes that have undergone meiotic maturation in vivo [26–28] or obtaining immature oocytes for IVM prior to IVF [5,6,8,13,14,26,29–32]. However, as a general observation, prepubertal calves require lower hormone doses than adults. The high stimulation sensitivity of prepubertal calves may be partly due to lower body mass and perhaps also to greater follicular responsiveness before the intra-ovarian regulatory mechanisms designed to limit ovulation rate become established [5]. The hormonal regimen and aspiration technique used in this study successfully maximized the recovery of immature, healthy oocytes from stimulated calves as evidenced by the fact that less than 10% of the recovered oocytes were found either to be damaged or to have an expanded cumulus.
Oocyte recovery from stimulated juvenile calves has been accomplished by laparoscopy [26], laparotomy [13], and slaughter [6]. In the present study, oocytes from stimulated donor calves were collected by laparotomy. The average recovery rate (83%) in our study was similar to that previously reported (75%; [13]). The satisfactory conception rates observed in the donor calves when bred after 15 mo of age could be due to appropriate care taken during laparotomies and subsequent TVOR. These elite donor calves became pregnant after undergoing successive ovarian stimulations and oocyte collections via laparotomy and TVOR until puberty; therefore these procedures can be considered safe, as they caused little or no damage to the ovaries and reproductive tracts. However, in the present study, heifers having < 20 follicles prior to the gonadotropin treatment at the 2-mo cycle were comparatively easier to breed at maturity in terms of number of inseminations required. Reasons for this observation could be that the ovaries of high responders might have suffered greater injury because of increased aspiration points and/or that the hormone treatment imparted a larger negative effect both systemically and locally on their reproductive tracts, requiring more time to overcome these effects. This hypothesis is further supported by the fact that the best-responding donor (Fig. 2, donor A) failed to conceive after four AI attempts.
Because of the nature of this study, no attempt was made to determine the in vitro oocyte maturation rates. However, previous studies have shown lower maturation rates of calf oocytes as compared to cow oocytes [9,33]. Differences in cow and calf oocytes in terms of size, protein synthesis, and energy metabolism have been reported recently [11]. However, the cleavage rates after IVF of oocytes obtained from stimulated or nonstimulated calves [6,9] have been reported to be similar to those of cow oocytes. Armstrong et al. [14] reported no difference in the cleavage rates between in vitro- or in vivo-matured calf oocytes and cow oocytes. On the other hand, Duby et al. [8] reported a cleavage rate of 22% from IVM and IVF oocytes collected from 75- to 153-day-old calves. In the present study, the cleavage rate observed (40%) was much lower than some previously reported results of > 80% [6]. The reason for this low cleavage rate could possibly be that rather than selecting only the best-quality oocytes for IVM/IVF/IVC, we used the majority (over 90%) of recovered oocytes as they originated from elite donor calves. Only expanded or damaged oocytes were excluded from this study. Quality of the oocytes recovered from prepubertal calves has been previously reported [13] to affect the cleavage and development rates.
In vitro embryo development rates from juvenile calf oocytes in the present study were observed to be much lower (11% in 2-mo cycle and 10% in 4-mo cycle) than our routine development rates from cow oocytes (35–40%). These results agree with previous reports on low developmental competence of calf oocytes [6,9]. However, Armstrong et al. [14] have reported no difference in the in vitro embryo production rates between cow and calf oocytes. The in vitro embryo development of calf oocytes was also observed to be slower than that of cow oocytes. This observation is in agreement with the results reported previously [2].
Direct comparisons were not made between the developmental competence of the calf and cow oocytes in the present study. However, the same IVF and IVC procedures used in this study have routinely resulted in 35–40% embryo development with cow oocytes in our laboratory. Groups recently have attempted to compare various attributes between the cow and calf, such as the characteristics of follicular fluid [10]; kinetics of nuclear maturation and protein profiles of the oocytes [33]; diameter, energy metabolism, and protein synthesis of oocytes [11]; and steroid production by theca cells [34]. With the help of nuclear transfer, the low developmental competence of calf oocytes as compared to cow oocytes has been demonstrated to occur at the cytoplasmic level [35]. However, it would now be desirable to analyze the differences among the calves themselves.
The pregnancy rate observed in the present study with transfer of calf embryos was significantly lower than those reported from IVP cow embryos [23,36]. As has been reported previously for IVP cow embryos [23], we observed a higher pregnancy rate from better-quality (grades 1 and 2) calf embryos than from poor ones. A recent report suggested that pregnancy losses from calf embryos occur before Day 35 [37], which could not be confirmed in the present study since pregnancy checks were carried out after Day 50. However, with ultrasound examination at Day 50–60 of gestation, we could diagnose only a single embryonic loss with an obviously degenerated fetus (small size) and no heartbeat. The average gestation length and birth weight of live calves born through the transfer of calf embryos were not different from those reported after transfer of IVP cow embryos of this breed [38]. Abnormal pregnancies (hydrops or hydroallantois) have been observed earlier from IVP cow embryos [23,36,38], and the single instance of such pregnancy observed in the present study appears to be within the normal range. Hydroallantois is related to renal failure of the fetus, and some cases have been related to 11ß-hydroxysteroid dehydrogenase dysfunction in the human [39].
In the current study, two calves were observed to be relatively overweight, and died at birth. These two calves were not as heavy as some of the calves reported by others [32,38]; however, they died as a result of dystocia and abnormal presentation in the absence of clinical help at the time of birth. Perinatal calf mortality has been previously observed with transfer of IVP cow embryos and has been reported to be 2.4% greater than for calves born through AI [38]. Holm et al. [40] demonstrated that IVM-IVF compromises the subsequent embryonic and fetal development in sheep irrespective of the subsequent in vivo or in vitro culture treatment. Also, there is evidence that birth weights of calves produced from IVM-IVF embryos cultured in vivo are comparable to those of calves produced through AI in contrast to calves produced from IVC embryos [41]. Galli and Lazzari [42] reported that in vitro-derived bovine embryos were successfully cultured in sheep oviducts for commercial purposes and that this improves the freezability of IVP embryos. Some of the other factors known to result in an overweight calf (large calf syndrome) have been reviewed by Walker et al. [43] and include in vitro coculture, fragmentation of the cytoplasm by inclusion of serum during embryo culture, removal of cytoplasm at the enucleation step of nuclear transfer, and asynchronous embryo transfer. On the other hand, in a recent preliminary study it was observed that culturing conditions of bovine IVM-IVF embryos did not affect the birth weights of the calves born [44].
No significant differences were observed between the 2-mo and 4-mo cycles in the parameters examined. The number of follicles aspirated, total and usable oocytes recovered, and in vitro cleavage and development rates of the oocytes inseminated, however, were consistently higher in the 2-mo collection compared to the 4-mo collection. This decline is likely due to the development of refractoriness to repeated administration of gonadotropin [45]. The same trends were observed in slightly greater oocyte recovery, cleavage, and development rates in the control stimulation group (calves stimulated with gonadotropin for the first time at 4–5 mo of age). However, it is important to mention that a confounding variable existed in the control stimulation group, since prior to the gonadotropin stimulation, follicles were observed laparoscopically at 4–5 mo of age rather than 2–3 mo of age as was done in the treatment calves. Interestingly, though, most of the pregnancies in the present study resulted from the 2-mo cycle. The pregnancy rate from embryos of the high-responding calves in the 4-mo cycle was nearly half that for the 2-mo cycle (12% vs. 21%), which appears to be due to an effect of repeated gonadotropin stimulation on the oocyte quality.
From the results of the present investigation it can be concluded that 1) some oocytes from gonadotropin-stimulated juvenile calves were fully competent for in vitro embryo development and for full-term pregnancies; 2) the number of antral follicles in juvenile calves appears to dictate the gonadotropin stimulation response, and this may be used as a means to screen high-responding oocyte or embryo donors; and 3) with appropriate care during and after the surgical collection of oocytes, the reproductive performance of donor calves of high genetic value may not be adversely affected by repeated oocyte collection sessions as demonstrated by their successful breeding at maturity. Further studies will involve a larger group of calves combined with hormonal and biochemical analyses in order to establish critical end points, as well as to understand the correlation between ovarian stimulation response and oocyte competence.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This research was supported in part by the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture, under Agreement No. 96-35203-3268. This is a scientific contribution No. 1911 of the Storrs Agricultural Experiment Station of the University of Connecticut. This research was also supported in part by the Genzyme Transgenic Corporation. 
2 Correspondence: Xiangzhong (Jerry) Yang, Department of Animal Science, 3636 Horsebarn Road Extension U-40, University of Connecticut, Storrs, CT 06269-4040. FAX: 860 486 4375; xiangzhong.yang{at}uconn.edu
Accepted: September 3, 1999.
Received: June 17, 1999.
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  R. A. Cushman, M. F. Allan, L. A. Kuehn, W. M. Snelling, A. S. Cupp, and H. C. Freetly Evaluation of antral follicle count and ovarian morphology in crossbred beef cows: Investigation of influence of stage of the estrous cycle, age, and birth weight J Anim Sci, June 1, 2009; 87(6): 1971 - 1980. [Abstract] [Full Text] [PDF]
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J.L.H. Ireland, D. Scheetz, F. Jimenez-Krassel, A.P.N. Themmen, F. Ward, P. Lonergan, G.W. Smith, G.I. Perez, A.C.O. Evans, and J.J. Ireland Antral Follicle Count Reliably Predicts Number of Morphologically Healthy Oocytes and Follicles in Ovaries of Young Adult Cattle Biol Reprod, December 1, 2008; 79(6): 1219 - 1225. [Abstract] [Full Text] [PDF]
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D. S. Burns, F. Jimenez-Krassel, J. L.H. Ireland, P. G. Knight, and J. J. Ireland Numbers of Antral Follicles During Follicular Waves in Cattle: Evidence for High Variation Among Animals, Very High Repeatability in Individuals, and an Inverse Association with Serum Follicle-Stimulating Hormone Concentrations Biol Reprod, July 1, 2005; 73(1): 54 - 62. [Abstract] [Full Text] [PDF]
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J.-Y. Jiang and B. K. Tsang Optimal Conditions for Successful In Vitro Fertilization and Subsequent Embryonic Development in Sprague-Dawley Rats Biol Reprod, December 1, 2004; 71(6): 1974 - 1979. [Abstract] [Full Text] [PDF]
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A. Oropeza, C. Wrenzycki, D. Herrmann, K.-G. Hadeler, and H. Niemann Improvement of the Developmental Capacity of Oocytes from Prepubertal Cattleby Intraovarian Insulin-Like Growth Factor-I Application Biol Reprod, June 1, 2004; 70(6): 1634 - 1643. [Abstract] [Full Text] [PDF]
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G. Ptak, M. Tischner, N. Bernabo, and P. Loi Donor-Dependent Developmental Competence of Oocytes from Lambs Subjected to Repeated Hormonal Stimulation Biol Reprod, July 1, 2003; 69(1): 278 - 285. [Abstract] [Full Text] [PDF]
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