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Reproductive Technology |
Dipartimento di Scienze Biomediche Comparate,3 Università di Teramo, 64100 Teramo, Italy
Department of Animal Reproduction,4 University of Agriculture, Krakòw, Pol
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, embryo, in vitro fertilization, ovary, ovum pick-up/transport
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In ruminants, the variability in follicular response and capacity for embryos can be caused by several environmental factors like nutrition [7–10], management of animals [1, 11], and season or photoperiod [2]. Another factor influencing developmental competence of oocytes is the timing of hormonal stimulation relative to the stage of estrus cycle [12]. Several studies suggest that growth and ovulation rate of follicles as well as oocyte developmental capacity are genotype dependent [13, 14].
The aim of numerous studies was to optimize embryogenic competence of oocytes collected from prepubertal calves [15–17] and lambs [15, 18, 19]. Others [20, 21] have sought to define morphofunctional cause(s) of differences in developmental potential between prepubertal and adult-derived oocytes. Those experiments were conducted using genetically heterogeneous material, different nutritional and seasonal conditions, or even unspecified sources of ovarian material.
Environmental [7], genetic [13, 22], and ovarian cycle effects [12] notwithstanding, the causes of diversity in donor response remain the subject of considerable speculation. Thus, our aim was to exclude, as far as possible, the aforementioned factors to verify the competence of oocytes in homogenous groups of animals. To do this, we used Sarda breed females, born within 48 h of each other and reared under identical conditions. To eliminate any variation depending on timing of the onset of puberty or differences in estrus cycle, the first oocyte recovery was performed as early as possible. We have previously demonstrated full developmental capability of oocytes from 1-mo-old lambs [3]; oocyte competence was compared among females from this age. The primary aim of this work was to identify differences in oocyte competence among individuals. To assess these differences, three groups of prepubertal ewes were stimulated at various ages, using a number of gonadotropin treatments. Once an optimal hormonal regimen was identified, we investigated whether the effect of an individual donor persisted and was observed between genetically similar animals. For this purpose we compare follicular response and subsequent embryogenic capability of sibling lambs born on the same day and kept under identical environmental conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal experiments were performed in accordance with DPR 27/1/1992 (Animal Protection Regulations of Italy) in conformity with European Community regulation 86/609 and in adherence with guidelines established in the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the Society for the Study of Reproduction.
Nineteen Sarda breed female lambs born 25–26 October and eight adult (3–4 yr old) artificially inseminated Sarda ewes were maintained under field conditions at the Institute of Zootecnics Research Farm, Sassari. Progesterone treatment was done by insertion of Norgestomet s.c. implants (Crestar, Intervet) on Day 0. Animals were randomly divided into different groups and hormonally stimulated as outlined in Table 1. There were essentially three treatments. The first (A) consisted of two different gonadotropin preparations (FSH + eCG). The amounts of hormone administered were altered slightly (A') to reflect the increase in weight of 7-mo-old animals. The second treatment (B) consisted of multiple injections of low doses of FSH previously determined in our study [3]. The third treatment (C), of higher gonadotropin dosage, was routinely used in our laboratory for oocyte recovery from adult sheep [23].
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Oocyte Retrieval
The ovaries were exposed by paramedian laparotomy in ewes anesthetized with acepromazine maleate (0.05 mg/kg body weight [BW]) and pentothal sodium (10 mg/kg BW). Follicular oocytes were aspirated with a 5-ml syringe fitted with a 20-gauge needle. The aspiration medium consisted of Hepes-buffered tissue culture medium 199 (Gibco Laboratories, Grand Island, NY) supplemented with 5% calf serum, 0.05 mg/ml heparin, and 0.05 mg/ml gentamicin sulfate. To avoid any adhesions, the reproductive organs were extensively washed with saline after the puncture of follicles. Moreover, the surface of the ovary was covered with hydrocortisone acetate ointment (Cortison Chemicetina, Pharmacia & Upjohn, Milan, Italy). Oocytes from each donor were maintained separately throughout all in vitro procedures.
All chemicals, unless otherwise indicated, were obtained from Sigma Chemical Co. (St. Louis, MO).
In Vitro Maturation, Fertilization, and Culture
Methods of in vitro embryo production were adapted from those previously described [3]. All recovered oocytes were matured in vitro (IVM) in bicarbonate-buffered TCM-199 (Gibco) (275 mOsm) containing 2 mM glutamine, 100 µM cysteamine, 0.3 mM sodium pyruvate, 10% fetal bovine serum (FBS) (Gibco), 5 µg/ml FSH (Ovagen), 5 µg/ml LH, and 1 µg/ml estradiol. Oocytes from individual donors were incubated in 0.4 ml of medium in 4-well culture plates (Nunclon, Roskilde, Denmark) in a humidified atmosphere of 5% CO2 in air at 39°C for 24 h.
The in vitro fertilization (IVF) medium used was bicarbonate-buffered synthetic oviduct fluid (SOF) [24] enriched with 20% (v:v) heat-inactivated estrous sheep serum, 2.9 mM calcium lactate, and 16 µM isoproterenol. Fresh semen, obtained from a black ram of proven fertility (Sarda breed) was used throughout the study. Fertilization was carried out in 50-µl drops, at a concentration of 1 x 106 sperm/ml, at 39°C in a humidified atmosphere of 5% CO2 in air for 20–22 h.
Presumptive zygotes were transferred to 20-µl drops consisting of SOF enriched with 1% (v:v) basal medium Eagle (BME) essential amino acids (Gibco), 1% (v:v) minimum essential medium (MEM) nonessential amino acids (Gibco), 1 mM glutamine, and 8 mg/ml fatty acid-free BSA (Gibco). Cultures were carried out in a humidified atmosphere of 5% CO2, 7% O2, and 88% N2 at 39°C. On Day 3 and Day 5 of culture (Day 0 = day of fertilization), 5% charcoal-stripped FBS (Gibco) was added to the medium. After 7 or 8 days of culture, blastocysts were transferred for development to term.
Embryo Transfer
Blastocysts were surgically transferred in pairs to recipient ewes 7 days after the onset of natural estrus. Pregnancy status was determined by ultrasonography at 40, 60, and 80 days after ovulation (7.5 MHz high-resolution linear probe; Aloka, Assago, Italy). Parturition was induced by a single injection of estradiol benzoate (2 mg, Estradiolo, AMSA, Milano, Italy) followed by 4 i.m. injections of betamethasone (Bentelan; Glaxo Wellcome, Alges, Portugal) at 12-h intervals (1 mg per 10 kg BW) as described previously [25].
Developmental Competence of the Oocytes Collected from Siblings
To compare quantity and quality of oocytes collected from genetically homogenous groups of females (daughters of donor no. 18), lambs were stimulated at 1 mo of age, and collected oocytes were subjected to in vitro procedures as described above. Developed blastocysts were transferred to synchronized recipient sheep.
Statistical Analysis
Statistical analysis was performed using chi-square analysis of all data (SAS/STAT User's Guide, 6.03 Edition, SAS Institute Inc., Cary, NC) and by a one-way analysis of variance using the statistical package SPSS (SPSS, Inc., Chicago, IL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries from lambs were characterized by various morphological criteria (Fig. 1, a, b, c, d, and e). Lambs responding to FSH + eCG treatment generally had follicles of greater dimensions (5–6 mm), compared with follicular response after pure FSH treatment (3–4 mm), independent of the dose of FSH used for stimulation. Of 19 lambs (groups 1, 2, and 3) stimulated at 1 mo of age, four (22%) did not respond to the hormonal treatment (only a few follicles of reduced dimensions < 2 mm). In subsequent sessions the number of lambs with poor follicular response increased, that is, nine (47%) animals did not respond to the second treatment and 14 (74%) to the third. Fourteen lambs (groups 1 and 2) were stimulated for a fourth time and of these, eight (57%) did not respond, and among five lambs (group 1) stimulated five times, two (40%) did not respond to the final treatment. In the adult test group (eight ewes), two (25%) had very poor follicular response (three to five follicles of reduced dimensions, < 2 mm) during all sessions of oocytes recovery, and the number of recovered oocytes was quite constant among the rest of the donors (Fig. 1f). Follicles from donors with poor response were aspirated, and data from recovery of oocytes are included. It should be noted that no blastocysts developed from these oocytes. The follicular response as measured by number of oocytes recovered from lambs (groups 1, 2, and 3) and adults is presented in Figure 2a. In all groups, follicular response was highest in the first session, declining thereafter until the fourth session when a small improvement in yield was obtained. Although the analysis of variance revealed no significant differences between groups in the mean oocytes number (13.92 ± 15.04 from group 1 vs. 12.13 ± 8.48 from group 2 vs. 11.53 ± 9.45 from group 3 vs. 10.74 ± 2.39 [adults]), the standard deviation (in some cases higher from the mean value) indicated very high variability in mean number of oocytes collected within the groups. Oocyte recovery rates were within the range of 75%–85%. The proportion of oocytes selected for culture was similar for lambs (68%) and ewes (73%) stimulated with six doses of FSH, but a lower percentage of oocytes was selected from lambs stimulated with FSH + eCG (48%).
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Influence of Age, Type, and Number of Stimulation on Oocyte Competence
The highest follicular response (P < 0.001) was obtained from 1-mo-old lambs and the lowest at 3 mo of age; however, important differences in follicle number were observed among 2, 5, and 7 mo of age (Fig. 3). In correlation to follicular response, the proportion of blastocysts produced from prepubertal oocytes was also related to age, although this was not significant because of low number of embryos. Statistically significant differences were observed for follicular response with respect to the type of hormonal treatment, and no statistically valid effect of hormonal stimulation was observed on comparison of embryo development (Fig. 4b). No significant effect of number of stimulations on either oocytes recovery or blastocysts development was revealed among any of the groups (Fig. 2, a and b).
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Donor-Dependent Embryogenic Competence
Only 8 of 19 1-mo-old lambs (42%) gave oocytes progressing to the blastocyst stage in vitro following the stimulation (Fig. 5, a–c). The majority of these donors continued to generate good-quality oocytes, developing to embryos following further stimulation treatments. Generally, repeatability of individual embryo response was observed in all groups, either lambs or adult ewes (Fig. 5, a–d), notwithstanding a few blastocysts produced in a unique session (from lamb 5 at the second session and lamb 9 at the third session). Statistical analysis (chi-square test) revealed significant differences in overall blastocyst production among individual donors from all groups (data not shown). The development to blastocyst varied among individuals in all sessions. Particularly at first stimulation, a significant donor effect (P < 0.001, P < 0.005) on blastocysts production was revealed. Substantial variability among lambs persisted in following sessions; however, it became less pronounced (P < 0.05) because of generally lower numbers of oocytes per lamb. Observed differences among donors can be attributed to individuals, and present independent of age of lambs and type or number of gonadotropin stimulation.
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Ovarian Response and Oocyte Competence of Daughters of High Responders
As a consequence of the results we obtained, revealing donor-dependent competence of oocytes, we decided to compare developmental ability of oocytes between genetically similar animals. Consequently in the second generation, five sister lambs (Fig. 6), were stimulated at 1 mo of age (as their genetic mother: lamb 18). Lamb 18 had the highest rate of embryo development, differing most significantly (P < 0.001) from all other individuals in its group. Figure 7 reports oocyte recovery and numbers of embryos that developed from oocytes collected from five donors and their mother on hormonal stimulation at 1 mo of age. Ovarian response in terms of follicular size and number was consistent for all five lambs. In fact, no significant differences in both oocyte recovery and embryo production were observed between sisters (chi-square test), although a number of them differed significantly from their mother.
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Pregnancies
Blastocysts (n = 107) derived on IVF from lamb oocytes and 50 of 125 blastocysts developed from adult sheep oocytes were transferred to recipient ewes. Twenty blastocysts developed following first oocyte collection from lamb 18 were transferred to 10 recipient ewes. Data about the pregnancy progression determined by ultrasonography scanning and embryo survival to term are presented in Figure 8. Because the second part of the experiment was based solely on daughters of lamb 18, data about development posttransfer of embryos derived from this female are included in Figure 8. Statistical analysis revealed no differences in pregnancy progression between recipient ewes carrying lamb and adult ewe-derived embryos; however, the effect of lamb-derived embryos vs. adult ones was significant (P < 0.001) comparing the proportion of offspring obtained from both groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a continuation of our previous experiments, in which the variability of response among prepubertal lambs was already demonstrated [3], here we show that the repeated stimulation of a very homogenous group of lambs allows for a way to study individual reproductive differences within groups. The efficiency of repeated ovarian stimulation has already been shown with adult and prepubertal oocyte donors [22, 30, 31]. Moreover, full developmental ability of lamb oocytes with several offspring originated from prepubertals was also proved in our previous experiments [3–5]. This capability offers an excellent tool for studying the acquisition of oocyte embryogenic competence under various conditions. According to careful description of the ovarian structure in prepubertal lambs [32], the most favorable time for follicular stimulation seems to be 4 wk of age when a maximum number of vesicular follicles is present on the surface of the ovary. Morphology of the ovaries from prepubertal lambs observed in our study corresponds to the ovarian structure described in the study of Kennedy et al. [32] in which development of ovaries between birth and 33 wk of age was examined at monthly intervals in Merino lambs. We confirm their report showing the largest number of follicles was obtained from 1-mo-old lambs. In both studies there was a marked decline in numbers of follicles between the first and third month (Fig. 3). Moreover, the study reports only small numbers of macroscopically visible follicles from 2 mo of age onward.
Similarly, in our work 50%–78% of 2- to 7-mo-old lambs did not produce sufficient number and size of follicles reasonable for embryo production. Furthermore, the presence of many fresh atretic scars previously observed in the 10- to 12-wk ovaries [32, 33] can explain the poorest follicular response (1.6 oocytes/donor) obtained in our study with 3-mo-old donors. This gradual, age-dependent fall in number of available oocytes has been previously described [6]. On the other hand, the increased growth rate of ovarian follicles was noted in lambs close to puberty (Fig. 3). Recent reports suggest that the developmental competence of prepubertal oocytes is also acquired in an age-dependent manner [34, 35]. In our study, oocyte number and competence were donor dependent throughout the lifetime of the ewe. The age-dependent response (P < 0.01) was not influenced significantly by the number of treatments. Gonadotropin stimulation of prepubertal lambs, irrespective of the uniformity of the experimental group, shows a substantial variability between individuals in connection to ovarian morphology (see Follicular Response in Results). Highly significant differences (P < 0.001) between individual donors were observed either in quantity of recovered oocytes or following development of in vitro fertilized embryos (data not shown). Such variability among donors in follicular and embryo response has been already reported in adult [11, 36–39] and prepubertal bovine [6, 40]. Its economic as well as scientific relevance triggered numerous studies trying to explain the variability in animal response by several environmental factors, stage of the estrus cycle, or differences in age of stimulated females [12, 26, 41]. However, apart from the fact that a heterogeneous animal model was used in those studies, the novelty of our research is that although marked differences were noted between the uniform group of individuals, a similar embryo yield was observed within individual donors (Fig. 5, a–d). The individual repeatability of response in adults revealed by former studies [42–44] and here extended for early prepuberty indicates that the number of oocytes possible to recover is preset for each female.
In recent years there have been a number of publications reporting development of oocytes recovered from prepubertal donors. Most authors claim that the developmental ability of juvenile female oocytes is lower than adults, although the explanations for this vary [3, 20, 21]. Generally, the low competence of prepubertally derived oocytes can not be attributed only to the age of donor because oocytes derived from 1-mo-old lambs were of similar or higher competence than those from adult oocytes. It is noteworthy that similar numbers of embryos were repeatedly produced from some individuals, but others were never able to produce competent oocytes (Fig. 5). Low developmental potential was associated to improper (low) follicular response following gonadotropin treatment. Interestingly, such poor responders constantly produce developmentally noncompetent oocytes. Taneja et al. [6] report that oocytes capable to full development are recovered solely from highly responding donors. In fact, the best donor (lamb 18) produced the highest number of embryos and had the highest rate of pregnancies. The pregnancy status of foster mothers carrying embryos developed from lamb 18 oocytes is included in Figure 8, and as can be noted, the mean pregnancy and survival rate is significantly higher from prepubertals donors but also from adult oocyte donors. In contrast, there are no differences in the proportion of pregnancies between prepubertal and adult donors. The effect of the origin of the embryo (lamb or adult), although not significant during the pregnancy, was highlighted later by their survival to term (P < 0.001). These results are in agreement with our previous study [3] in which the pregnancies had similar values independent of adult or prepubertal origin of transferred embryos, but the number of lambs born was dependent (P < 0.05) on the source of embryos.
Excluding all environmental influences, the uniformity of breed, age, and general management together with a high number of ovine embryos obtained during the experiment (more than 200 blastocysts produced, 42 lambs born) allowed us to postulate the individual donor-mediated response. Repeated stimulation of a homogenous group of prepubertal and adult sheep showed significant differences in donor embryo yield. In the comparison between the best, in terms of embryo production donor, in lamb 18 and her five daughters, the level of variability substantially decreased. In fact, all of them can be classified as highly responding donors, although the ovarian output of some lambs differed from their mother. It could be attributed to different environmental conditions at the time of stimulation and a minor resemblance between mother and daughter. What is most relevant is that a direct comparison of animals with a higher level of homogeneity (performed among daughters of lamb 18) shows statistically similar response in all individuals. We would like to emphasize that following stimulation, no differences were observed among sister lambs, either in oocyte number or embryo competence in vitro. Such homogeneity in animal response, achieved for the first time in this report, could be ascribed to substantial similarity of genotypes in the second generation of lambs (the same mother and father); thereby it reinforces the idea that paternally inherited characteristics have an influence on oocyte viability and developmental competence.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Grazyna Ptak, Dipartimento di Scienze Biomediche Comparate, Università di Teramo, Piazza A. Moro 45, 64100 Teramo, Italy; gptak{at}tiscali.it
Received: 23 September 2002.
First decision: 15 October 2002.
Accepted: 3 February 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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