- Email: [email protected]
Developmental competence of prepubertal and adult goat oocytes cultured in semi-defined media following laparoscopic recovery
Theriogenology 60 (2003) 879–889
Developmental competence of prepubertal and adult goat oocytes cultured in semi-defined media following laparoscopic recovery Jennifer Koemana, Carol L. Keeferb, Hernan Baldassarreb, Bruce R. Downeya,* a
Department of Animal Science, McGill University, Macdonald Campus 21, 111 Lakeshore Rd., Ste Anne de Bellevue, Que., Canada H9X 3V9 b Nexia Biotechnologies Inc., 1000 St. Charles Ave., Block B, Vaudreuil-Dorion, Que., Canada J7V 8P5 Received 22 May 2002; accepted 4 December 2002
Abstract With an increased interest in transgenic animal production, the caprine species offers many advantages, and the prepubertal goat is a potential source of large numbers of oocytes for in vitro embryo production. The aim of the present study was to evaluate the follicular response and recovery of oocytes from prepubertal and adult goats following ovarian stimulation and laparoscopic recovery, and their developmental competence following culture in semi-defined media. Oocytes were collected over a 15-week period from prepubertal goats (3–7 months old) and adult controls (2–4 years old) that had been subjected to estrus synchronization and ovarian stimulation. Following insemination, zygotes were cultured for 96 h in G1.2 followed by an additional 120 h in G2.2. Morulae and blastocysts were scored using light microscopy on Days 7 and 9 followed by fluorescent staining for cell counts on Day 9 (216 h postinsemination). The mean numbers of follicles aspirated and oocytes recovered were significantly greater for prepubertal than for adult goats (P < 0:01). The number of oocytes recovered from prepubertal goats was observed to decline significantly with increasing age of the animals (P < 0:05). The proportion of oocytes that matured and cleaved did not differ significantly between prepubertal and adult goats. Furthermore, no significant differences in morulae development (percentage of those cleaved), 5% versus 4%, or blastocyst development, 6% versus 7%, were observed for prepubertal and adult derived oocytes (P > 0:1), respectively. Mean cell number per blastocyst also did not differ significantly. In conclusion, higher yields of oocytes were obtained from gonadotrophinprimed, prepubertal does than from adults, while in vitro development was similar. # 2003 Elsevier Science Inc. All rights reserved. Keywords: In vitro fertilization; Oocyte; Embryo; Culture; Goat
* Corresponding author. Tel.: þ1-514-398-7798; fax: þ1-514-398-7964. E-mail address: [email protected] (B.R. Downey).
0093-691X/$ – see front matter # 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0093-691X(03)00090-6
880
J. Koeman et al. / Theriogenology 60 (2003) 879–889
1. Introduction In vitro production of goat embryos is a rapidly advancing field. The production of valuable transgenic goats, capable of producing substances of pharmaceutical and technological value in their milk, has encouraged the development of techniques able to support the propagation of these animals. Application of in vitro techniques (in vitro maturation, fertilization, and culture) to species such as the caprine would facilitate the production of large numbers of embryos from a single genetically valuable animal. The use of prepubertal animals presents added benefits, in that the collection of oocytes at an earlier age would reduce the generation interval and, consequently, accelerate the propagation of genetically valuable animals. Prepubertal animals represent a vast untapped supply of oocytes, with yields, at least in the sheep, higher from prepubertal than from adult animals [1]. The production of embryos using oocytes derived from prepubertal cattle [2–7], sheep [7–12] and goats [13–17] has been achieved successfully in vitro. Several studies have indicated similar rates of maturation and fertilization following IVM–IVF of prepubertal and adult goat oocytes [13,15,18,19]. Furthermore, a few studies [13,16] have reported no differences in the rates of cleavage and blastocyst development between oocytes obtained from prepubertal and adult goats. To date, goat oocytes have been harvested principally from ovariectomized [20,21] or slaughtered does [13,15,16,22–24]. Laparoscopic ovum pick-up (LOPU) has provided an efficient and relatively noninvasive method for the collection of oocytes from small ruminants (sheep [25,26], goats [17,27], calves [2,3,28]) in which other techniques may not be feasible (transvaginal ultrasound-guided) or desirable (slaughterhouse, laparotomy). With this in mind, the objectives of the present research were to compare the follicular response and oocyte recovery from hormonally stimulated prepubertal versus adult goats and, using a semi-defined culture system, to evaluate and compare the developmental competence of oocytes recovered from prepubertal and adult goats.
2. Materials and methods 2.1. Experimental design Following synchronization of estrus in prepubertal and adult goats, follicular response, numbers of oocytes recovered by LOPU and their developmental competence following culture in semi-defined media were assessed. Conducted over a 15-week period during the nonbreeding season (June to September), the study compared the performance of adult animals with that of young growing goats during the latter one-half of the prepubertal period. Twenty four adult goats, ranging in age from 2 to 4 years, and 28 prepubertal goats born 3 months prior to the onset of the experiment were randomly allocated to one of ten oocyte collection sessions throughout the experimental period. Three adult goats and three to five prepubertal goats were used for each collection session. Hence, prepubertal animals were approximately 90 days of age at the time of the first collection and close to 200 days old by the tenth and final collection session. Upon aspiration from the follicles, oocytes were processed immediately for in vitro culture in order to assess rates of developmental
J. Koeman et al. / Theriogenology 60 (2003) 879–889
881
competence. Oocytes from four sessions were lost due to incubator failure (Sessions 1 and 2) and technical difficulties (Sessions 5 and 6) so that development data from only six sessions are reported here. 2.2. Ovarian stimulation and oocyte collection Estrus was synchronized in adult goats with intravaginal sponges (Veramix1, Upjohn Co., Orangeville, Ont., Canada), containing 60 mg medroxyprogesterone acetate, inserted for 10 days prior to oocyte collection. At 36 h before oocyte collection, all prepubertal and adult goats were given 125 mg cloprostenol i.m. (Estrumate1, Schering Canada Inc., Pointe-Claire, Que., Canada), 300 IU eCG i.m. (Folligon1, Intervet Canada Ltd., Whitby, Ont., Canada) and 80 mg NIH-FSH-PI i.m. (Folltropin1-V, Vetrepharm Canada Inc., London, Ont., Canada). Oocytes were collected via LOPU as previously described by Baldassarre et al. [25]. Oocytes were aspirated from all follicles >2 mm in diameter visible on the surface of the ovaries. The aspiration medium consisted of Tissue Culture Medium 199 (TCM-199; Life Technologies, Burlington, Ont., Canada) supplemented with 100 IU/ml penicillin-G, 0.1 mg/ml streptomycin, 0.05 mg/ml kanamycin, 0.5% fatty acid free-bovine serum albumin (FAF-BSA) and 0.05 mg/ml heparin (Sigma–Aldrich Canada, Oakville, Ont., Canada). 2.3. In vitro maturation, fertilization and culture Follicular aspirates were processed immediately upon recovery from each individual animal. Recovered oocytes were washed once through aspiration medium without heparin and transferred to maturation medium consisting of TCM-199 supplemented with 0.02 IU bovine luteinizing hormone (bLH), 0.002 IU bovine follicle stimulating hormone (bFSH; Sioux Biochemical Inc., Sioux Center, IA, USA), 1 mg/ml 17-b estradiol, 0.2 mM pyruvic acid, 50 mg/ml kanamycin, and 10% heat inactivated goat serum (Sigma–Aldrich). The cumulus investments of each oocyte were recorded as Grade A (3 cumulus layers), Grade B (1–2 cumulus layers), Grade C (denuded), or Grade D (expanded cumulus). Following the recovery of oocytes from the last animal in each collection, oocytes were pooled for each treatment group (i.e. adult goats and prepubertal goats). Oocytes were then allocated, in groups of 10–15, to 50 ml droplets of maturation medium under mineral oil (Sigma–Aldrich) and matured for 26 h at 38.5 8C in a humidified atmosphere of 5% CO2 in air. Following maturation, oocytes were partially denuded of their cumulus investment (50%) by repeated pipetting, washed once through EmCare (Immuno-Chemical Products, New Zealand) supplemented with 1 mg/ml BSA, and placed in 40 ml droplets of fertilization medium under oil. Fertilization medium consisted of Tyrode’s Albumin Lactate Pyruvate Solution supplemented with 20% estrus goat serum (heat inactivated) and 27.5 mg/ml pyruvic acid. Fresh spermatozoa were collected from a buck of proven fertility. Following incubation at room temperature for 2–3 h in the dark, viable sperm were separated by centrifugation through a two-layer (45 and 90%) Percoll gradient. The loose pellet of spermatozoa was washed once by centrifugation with modified defined medium (mDM) consisting of defined medium [29] supplemented with 6 mg/ml FAF-BSA, 3.1 mg/ml NaHCO3, 0.138 mg/ml
882
J. Koeman et al. / Theriogenology 60 (2003) 879–889
pyruvic acid, and 50 mg/ml gentamicin (Sigma–Aldrich). The remaining sperm pellet was resuspended in a capacitating solution consisting of 1 ml of mDM supplemented with 500 mM 8-bromoadenosine 30 :50 cyclic monophosphate sodium (8-bromo-cAMP) and 100 nM ionomycin calcium salt (Sigma–Aldrich) (Parrish JJ, personal communication). Semen was capacitated for 15 min at 38.5 8C. Following capacitation, oocytes were incubated with 1 106 sperm/ml for 18–22 h under the same atmosphere and temperature conditions as previously described for IVM. Following IVF, presumptive zygotes were freed from loosely attached sperm by washing through EmCare containing 1 mg/ml BSA prior to in vitro culture. A semi-defined two-step culture system, G1.2/G2.2, was utilized in this experiment as it has provided good results in the cow [30] and the goat [31]. Presumptive zygotes, in groups of 15, were transferred to 25 ml droplets of G1.2 culture medium and placed at 38.5 8C in a humidified atmosphere of 7% O2, 7% CO2 and 86% N2. Approximately 48 h postinsemination, uncleaved ova (1-cell) were removed and stained (Hoechst 33324; Sigma–Aldrich) for the assessment of the nuclear stage of arrest. Cleaved ova (2 cells) were transferred to fresh droplets of G1.2. On Day 5 (120 h postinsemination), ova were transferred to G2.2 media and cultured for four additional days under the same conditions as described above. Morulae and blastocysts were scored morphologically on Days 7 and 9 of culture, followed by staining (Hoechst 33342; Sigma–Aldrich) for cell counts on Day 9. 2.4. Morphological and nuclear evaluation of morulae and blastocysts Morulae and blastocysts were classified morphologically according to the following criteria: embryos appearing as a compacted mass of cells (16 cells) at magnification 50 under the stereomicroscope were classified as morulae; embryos exhibiting the presence of a blastocoel were classified as blastocysts; embryos containing a blastocoel and either emerging from the zona pellucida or lacking a zona pellucida were classified as hatched blastocysts. The nuclear status of uncleaved ova and the number of cells in each morula and blastocyst were assessed by fluorescence microscopy. The nuclear status of uncleaved ova was recorded as germinal vesicle, metaphase I, metaphase II, fertilized (two pronuclei), abnormal (activated, penetrated, polypronuclei/multinucleated), or degenerated (data not shown). Embryos scored morphologically as morulae on Day 9 of culture, but observed to have less than 16 nuclei following cell counts, were reclassified as pseudo-morulae. Morulae which had greater than 16 nuclei remained classified as morulae. Similarly, blastocysts scored morphologically on Day 9 of culture but observed to have less than 32 nuclei following cell counts were reclassified as pseudo-blastocysts. Blastocysts which had greater than 32 nuclei remained classified as blastocysts. 2.5. Statistical analysis Follicle aspiration and oocyte recovery data were analyzed by ANOVA (Mixed Models Procedure, SAS/STAT [32]). The relationship between the age of prepubertal goats and the number of oocytes recovered (P < 0:05) was also analyzed using the Mixed Models Procedure of the Statistical Analysis System. Proportional data were compared using Fisher’s exact test (P < 0:01).
J. Koeman et al. / Theriogenology 60 (2003) 879–889
883
3. Results 3.1. Follicular aspiration and oocyte recovery Data pooled from each of the ten collections indicate that the mean numbers of follicles aspirated (32 3:7 versus 18:4 1:1) and oocytes recovered (25 2:8 versus 16 1:2) were significantly greater for prepubertal than for adult goats (P < 0:01), respectively. Oocyte recovery rates (82 and 88%) were similar for both groups. Because a similar oocyte recovery rate was observed for prepubertal and adult goats, oocyte recovery data were utilized to assess a relationship between the age of prepubertal goats and the number of oocytes recovered (Fig. 1). A linear regression (slope S:E:) was noted for data from both prepubertal (1:37 0:59) and adult (0:04 0:27) goats. Linear regressions for prepubertal and adult data were significantly different (P < 0:05). Moreover, the slope determined using prepubertal data was significantly different from zero (P < 0:05), indicating that the number of oocytes recovered from prepubertal goats declined significantly with increasing age of the animals. No significant changes in the number of oocytes recovered from adult goats were observed throughout the experiment. Eightyseven percent of the cumulus oocyte complexes recovered from prepubertal, and 82% of those recovered from adult goats were classified as Grade A or B. 3.2. In vitro embryo production The number of oocytes cleaved (% of oocytes cultured) did not differ significantly between prepubertal (46.4%) and adult (50.7%) goats. The incidence of ova arrested during early cleavage was relatively small for oocytes derived from both prepubertal and adult goats, 3.5% versus 6.2%, respectively. As previously described, morulae and blastocysts scored morphologically on Days 7 and 9 of culture were further subdivided into pseudomorulae (<16 cells), morulae (16 cells), pseudo-blastocysts (<32 cells) and blastocysts
Fig. 1. Mean (S.E.M.) number of oocytes recovered from prepubertal (&) or adult (&) goats per collection. The numbers of oocytes recovered from prepubertal animals declined significantly with increasing age (P < 0:05).
884
J. Koeman et al. / Theriogenology 60 (2003) 879–889
Fig. 2. (a) Two adult-derived pseudo-blastocysts. While not apparent under the epifluorescence microscope, both ova were scored morphologically as blastocysts based on the presence of a visible blastocoel. (b) Three prepubertal-derived blastocysts, containing 180–220 nuclei, after 9 days of culture.
(32 cells), following cell counts on Day 9 of culture. Fig. 2a illustrates the characteristic appearance of pseudo-blastocysts using fluorescence microscopy. Prepubertal-derived blastocysts containing between 180 and 200 nuclei are shown in Fig. 2b. Percentages of pseudo-morulae (59.9% versus 54.9%), morulae (4.7% versus 4.4%), pseudo-blastocysts (22.1% versus 23.9%), and blastocysts (6.4% versus 7.1%) did not differ significantly between prepubertal and adult goats, respectively. The distribution of morulae cell numbers determined following cell counts is presented in Fig. 3. Morulae scored morphologically on Days 7 and 9 of culture were observed to contain between 2 and 56 nuclei following cell counts. Similarly, blastocysts scored morphologically on Days 7 and 9 of culture were observed to contain between 2 and 350 nuclei following cell counts
Fig. 3. Morula cell numbers (% of cleaved embryos) determined on Day 9 for oocytes from prepubertal (&) and adult (&) goats. Differences between treatment groups were not significant.
J. Koeman et al. / Theriogenology 60 (2003) 879–889
885
Fig. 4. Blastocyst cell numbers (% of cleaved embryos) determined on Day 9 for oocytes from prepubertal (&) and adult (&) goats. Differences between treatment groups were not significant.
(Fig. 4). The mean cell number (S.E.M.) per blastocyst (32 cells) did not differ significantly between prepubertal and adult goats (120 32 versus 117 30).
4. Discussion This study clearly demonstrated that more follicles can be stimulated to develop and more oocytes can be recovered from prepubertal than from adult goats, although the difference declines substantially as the prepubertal animals increase in age. The developmental competence of oocytes from prepubertal and adult goats appeared to be similar. 4.1. Follicular aspiration and oocyte recovery Laparoscopic ovum pick-up (LOPU) was utilized in this research as a minimally invasive method for the recovery of oocytes from prepubertal and adult goats. Large numbers of oocytes can be recovered from gonadotrophin-stimulated prepubertal goats by LOPU, as reported for other prepubertal ruminants (calves [33], sheep [8], goats [17]). Furthermore, the majority of oocytes recovered from prepubertal and adult goats, 87 and 82%, respectively, were classified as Grade A or B oocytes. The results of the present study indicate that LOPU is an efficient technique for the recovery of high quality oocytes from both prepubertal and adult goats. A significant relationship between the age of prepubertal goats and the number of oocytes recovered was also observed in the present experiment. A significant linear decline in the number of oocytes recovered (slope S:E:; 1:37 0:59) was associated with increasing age of the prepubertal goats (3–7 months) during the 15 weeks experimental period. In a separate study, a similar decline in the number of oocytes recovered was
886
J. Koeman et al. / Theriogenology 60 (2003) 879–889
observed with increasing age between two different age groups of prepubertal goats. Recovery of oocytes by LOPU from gonadotrophin-stimulated prepubertal goats resulted in a significantly higher yield from 60 to 90-day old goats than from 90 to 150-day old goats [17]. In the present study, despite a decline in the number of oocytes recovered with increasing age of the prepubertal goats, the mean number of oocytes recovered from prepubertal animals using data pooled from ten collections was significantly higher than that observed for adult goats (25 2:8 versus 16 1:2, respectively). The enhanced follicular response of prepubertal goats found in this study justifies the utility of collecting oocytes from animals of genetic value at an early age. 4.2. In vitro embryo production The present study documents the similar developmental capacity of prepubertal and adult goat oocytes to the blastocyst stage following IVM–IVF–IVC. These results are in agreement with results previously reported [13], in which the developmental capacities of oocytes derived from prepubertal and adult goats, with or without hormonal stimulation, were compared. Izquierdo et al. [15,16] also obtained comparable rates of blastocyst development using oocytes derived from prepubertal and adult goats. Furthermore, the rates of development to the blastocyst stage for adult goats in the present study were similar to those reported by Izquierdo et al. [16], but lower than those reported by others [23,24]. While the exact factors underlying the relatively low rate of blastocyst development achieved in the present experiment remain unknown, the reclassification of blastocysts based on cell counts (6% versus 7%, respectively) as compared to morphological appearance (25% versus 24%, respectively) resulted in a considerable reduction of the reported rate. The criteria used to classify morulae and blastocysts (morphological appearance versus cell counts) are often not reported which presents difficulties when comparing rates of blastocyst development among different laboratories. The ranges of morula and blastocyst cell numbers observed following cell counts are particularly intriguing as they reveal that morphological appearance does not necessarily indicate the true developmental status of the goat embryo. Further research is required to evaluate the full developmental potential of blastocysts following transfer; however, studies in which zygotes or early cleavage stage embryos were transferred indicate that embryos derived from prepubertal goats are as viable as those obtained from adults [17]. The reduced in vitro developmental competence of oocytes observed in the present study may be due to deficiencies in the in vitro production system. Maturation treatments, sperm preparation and culture systems are all known variables that can affect overall success rates [22–24]. In a recent study, Izquierdo et al. [16] did not observe any improvement in proportion of embryos developing to the blastocyst stage following in vivo culture. It may be that the in vitro maturation system is inadequate, as Crozet et al. [22] observed significant differences in blastocyst development following recovery of oocytes from small, medium, and large follicles, and from the oviduct. Seasonal effects also can not be excluded as this study was performed during the summer months (June to early September). Decreased rates of development are commonly observed in bovine in vitro production systems during the summer, while significant reductions in the developmental competence of ovine oocytes collected during anestrus have also been noted [34].
J. Koeman et al. / Theriogenology 60 (2003) 879–889
887
In conclusion, the mean number of oocytes recovered from prepubertal animals was significantly higher than that observed for adult goats (25 2:8 versus 16 1:2, respectively), while developmental competence was similar. The identification of optimal experimental conditions able to support development of a reasonable proportion of goat oocytes to the blastocyst stage (i.e. higher than 30%) is required before similarities (or differences) in the developmental competence of prepubertal and adult goat oocytes can be fully recognized. The successful development of a small number of prepubertal goat oocytes to the blastocyst stage achieved in this experiment is encouraging for the application of IVP techniques to prepubertal goats. Similarly high mean cell number of prepubertal- and adult-derived blastocysts (121 32 and 117 30, respectively) further emphasizes the developmental potential of prepubertal goat oocytes. The utilization of IVP conditions specifically designed to meet the requirements of the prepubertal goat oocyte/ embryo may enhance rates of blastocyst development. Successful production of embryos derived from prepubertal goats would, in turn, facilitate the rapid propagation of genetically valuable animals.
Acknowledgements We would like to thank J-T Chung, M. Drolet, K. Gaudreau-Provost, D. Laurin, J. Pierson, J. Pika, and H. Watt for their technical assistance throughout this experiment. This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), Nexia Biotechnologies Inc. and the Conseil des recherches en peˆ che et en agro-alimentaire du Que´ bec (CORPAQ). Jennifer Koeman was the recipient of an NSERC Postgraduate Scholarship.
References [1] Ptak G, Loi P, Dattena M, Tichner M, Cappai P. Offspring from one-month-old lambs: studies on the developmental capability of prepubertal oocytes. Biol Reprod 1999;61:1568–74. [2] Armstrong DT, Holm P, Irvine BJ, Petersen BA, Stubbings RB, McLean D, et al. Pregnancies and live birth from in vitro fertilization of calf oocytes collected by laparoscopic follicular aspiration. Theriogenology 1992;38:667–78. [3] Armstrong DT, Irvine BJ, Earl CR, McLean D, Seamark RF. Gonadotropin stimulation regimens for follicular aspiration and in vitro embryo production from calf oocytes. Theriogenology 1994;42:1227–36. [4] Revel F, Mermillod P, Peynot N, Renard JP, Heyman Y. Low developmental capacity of in vitro matured and fertilized oocytes from calves compared with that of cows. J Reprod Fertil 1995;103:115–20. [5] Damiani P, Fissore RA, Cibelli JB, Long CR, Balise JJ, Robi JM, et al. Evaluation of developmental competence, nuclear and ooplasmic maturation of calf oocytes. Mol Reprod Dev 1996;45:521–34. [6] Taneja M, Bols PEJ, Van de Velde A, Ju J, Schreiber D, Tripp MW, et al. Developmental competence of juvenile calf oocytes in vitro and in vivo: influence of donor animal variation and repeated gonadotropin stimulation. Biol Reprod 2000;62:206–13. [7] Palma GA, Tortonese DJ, Sinowatz F. Developmental capacity in vitro of prepubertal oocytes. Anat Histol Embryol 2001;30:295–300. [8] Earl CR, Irvine BJ, Kelly JM, Rowe JP, Armstrong DT. Ovarian stimulation protocols for oocyte collection and in vitro embryo production from 8 to 9 week old lambs. Theriogenology 1995;43:203 [abstract].
888
J. Koeman et al. / Theriogenology 60 (2003) 879–889
[9] O’Brien JK, Beck NFG, Maxwell WMC, Evans G. Effect of hormone pre-treatment of prepubertal sheep on the production and developmental capacity of oocytes in vitro and in vivo. Reprod Fertil Dev 1997;9: 625–31. [10] O’Brien JK, Dwarte D, Ryan JP, Maxwell WMC, Evans G. Developmental capacity, energy metabolism and ultrastructure of mature oocytes from prepubertal and adult sheep. Reprod Fertil Dev 1996;8: 1029–37. [11] Ledda S, Boliolo L, Calvia P, Leoni G, Naitana S. Meiotic progression and developmental competence of oocytes collected from juvenile and adult ewes. J Reprod Fertil 1997;109:73–8. [12] Ledda S, Bogliolo L, Leoni G, Naitana S. Production and lambing rate of blastocysts derived from in vitro matured oocytes after gonadotropin treatment of prepubertal ewes. J Anim Sci 1999;77:2234–9. [13] Mogas T, Palomo MJ, Izquierdo MD, Paramio MT. Developmental capacity of in vitro matured and fertilized oocytes from prepubertal and adult goats. Theriogenology 1997;47:1189–203. [14] Izquierdo D, Villamediana P, Palomo MJ, Mogas T, Paramio MT. Effect of sperm capacitation and fertilization media on IVF and early embryo development of prepubertal goat oocytes. Theriogenology 1998;49:1501–13. [15] Izquierdo D, Villamediana P, Paramio MT. Effect of culture media on embryo development from prepubertal goat IVM–IVF oocytes. Theriogenology 1999;52:847–61. [16] Izquierdo D, Villamediana P, Lopez-Bejar M, Paramio MT. Effect of in vitro and in vivo culture on embryo development from prepubertal goat IVM–IVF oocytes. Theriogenology 2002;57:1431–41. [17] Baldassarre H, Wang B, Kafidid N, Keefer C, Lazaris A, Karatzas CN. Advances in the production and propagation of transgenic goats using laparoscopic ovum pick-up and in vitro embryo production technologies. Theriogenology 2002;57:275–84. [18] Martino A, Mogas T, Palomo MJ, Paramio MT. Meiotic competence of prepubertal goat oocytes. Theriogenology 1994;41:969–80. [19] Martino A, Mogas T, Palomo MJ, Paramio MT. In vitro maturation and fertilization of prepubertal goat oocytes. Theriogenology 1995;43:473–85. [20] Younis AI, Zuelke KA, Harper KM, Oliveira MAL, Brackett BG. In vitro fertilization of goat oocytes. Biol Reprod 1991;44:1177–82. [21] Keskintepe L, Darwish GM, Kenimer AT, Brackett BG. Term development of caprine embryos derived from immature oocytes in vitro. Theriogenology 1994;42:527–35. [22] Crozet N, Ahmed-Ali M, Dubos MP. Developmental competence of goat oocytes from follicles of different size categories following maturation, fertilization and culture in vitro. J Reprod Fertil 1995;103:293–8. [23] Keskintepe L, Luvoni GC, Rzucidlo SJ, Brackett BG. Procedural improvements for in vitro production of viable uterine stage caprine embryos. Small Rumin Res 1996;20:247–54. [24] Pawshe CH, Palanisamy A, Taneja M, Jain SK, Totey SM. Comparison of various maturation treatments on the in vitro maturation of goat oocytes and their early embyronic development and cell numbers. Theriogenology 1996;46:971–82. [25] Baldassarre H, de Matos DG, Furnus CC, Castro TE, Cabrera Fischer EI. Technique for efficient recovery of sheep oocytes by laparoscopic folliculocentesis. Anim Reprod Sci 1994;35:145–50. [26] Baldassarre H, Furnus CC, de Matos DG, Pessi H. In vitro production of sheep embryos using laparoscopic folliculocentesis: alternative gonadotrophin treatments for stimulation of oocyte donors. Theriogenology 1996;45:707–17. [27] Graff KJ, Meintjes M, Gyer VW, Paul JB, Denniston RS, Ziomek C, et al. Transvaginal ultrasound- guided oocyte retrieval following FSH stimulation of domestic goats. Theriogenology 1999;51:1099–119. [28] Baldassarre H. Ovum pick-up followed by in vitro production of embryos in sheep and cattle. In: Proceedings of the 4th Swedish International Program on Animal Reproduction and Biotechnology for Latin America. Belem, Para, Brazil, 1998; p. 62–70. [29] Brackett B, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod 1975;12:260–74. [30] Krisher RL, Lane M, Bavister BD. Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media. Biol Reprod 1999;60:1345–52. [31] Ongeri EM, Bormann CL, Butler RE, Melican D, Gavin WG, Echelard Y, et al. Development of goat embryos after in vitro fertilization and parthenogenetic activation by different methods. Theriogenology 2001;55:1933–45.
J. Koeman et al. / Theriogenology 60 (2003) 879–889
889
[32] SAS/STAT1 Software. Changes and enhancements through Release 6.12. Cary, NC: SAS Institute Inc.; 1997. [33] Armstrong DT, Kotaras PJ, Earl CR. Advances in production of embryos in vitro from juvenile and prepubertal oocytes from the calf and lamb. Reprod Fertil Dev 1997;9:333–9. [34] Mitchell LM, Staines ME, McCallum GJ, Kuran M, McEvoy TG. Season affects in vitro developmental competence of ovine oocytes. Theriogenology 2000;53:362 [abstract].
Developmental competence of prepubertal and adult goat oocytes cultured in semi-defined media following laparoscopic recovery Jennifer Koemana, Carol L. Keeferb, Hernan Baldassarreb, Bruce R. Downeya,* a
Department of Animal Science, McGill University, Macdonald Campus 21, 111 Lakeshore Rd., Ste Anne de Bellevue, Que., Canada H9X 3V9 b Nexia Biotechnologies Inc., 1000 St. Charles Ave., Block B, Vaudreuil-Dorion, Que., Canada J7V 8P5 Received 22 May 2002; accepted 4 December 2002
Abstract With an increased interest in transgenic animal production, the caprine species offers many advantages, and the prepubertal goat is a potential source of large numbers of oocytes for in vitro embryo production. The aim of the present study was to evaluate the follicular response and recovery of oocytes from prepubertal and adult goats following ovarian stimulation and laparoscopic recovery, and their developmental competence following culture in semi-defined media. Oocytes were collected over a 15-week period from prepubertal goats (3–7 months old) and adult controls (2–4 years old) that had been subjected to estrus synchronization and ovarian stimulation. Following insemination, zygotes were cultured for 96 h in G1.2 followed by an additional 120 h in G2.2. Morulae and blastocysts were scored using light microscopy on Days 7 and 9 followed by fluorescent staining for cell counts on Day 9 (216 h postinsemination). The mean numbers of follicles aspirated and oocytes recovered were significantly greater for prepubertal than for adult goats (P < 0:01). The number of oocytes recovered from prepubertal goats was observed to decline significantly with increasing age of the animals (P < 0:05). The proportion of oocytes that matured and cleaved did not differ significantly between prepubertal and adult goats. Furthermore, no significant differences in morulae development (percentage of those cleaved), 5% versus 4%, or blastocyst development, 6% versus 7%, were observed for prepubertal and adult derived oocytes (P > 0:1), respectively. Mean cell number per blastocyst also did not differ significantly. In conclusion, higher yields of oocytes were obtained from gonadotrophinprimed, prepubertal does than from adults, while in vitro development was similar. # 2003 Elsevier Science Inc. All rights reserved. Keywords: In vitro fertilization; Oocyte; Embryo; Culture; Goat
* Corresponding author. Tel.: þ1-514-398-7798; fax: þ1-514-398-7964. E-mail address: [email protected] (B.R. Downey).
0093-691X/$ – see front matter # 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0093-691X(03)00090-6
880
J. Koeman et al. / Theriogenology 60 (2003) 879–889
1. Introduction In vitro production of goat embryos is a rapidly advancing field. The production of valuable transgenic goats, capable of producing substances of pharmaceutical and technological value in their milk, has encouraged the development of techniques able to support the propagation of these animals. Application of in vitro techniques (in vitro maturation, fertilization, and culture) to species such as the caprine would facilitate the production of large numbers of embryos from a single genetically valuable animal. The use of prepubertal animals presents added benefits, in that the collection of oocytes at an earlier age would reduce the generation interval and, consequently, accelerate the propagation of genetically valuable animals. Prepubertal animals represent a vast untapped supply of oocytes, with yields, at least in the sheep, higher from prepubertal than from adult animals [1]. The production of embryos using oocytes derived from prepubertal cattle [2–7], sheep [7–12] and goats [13–17] has been achieved successfully in vitro. Several studies have indicated similar rates of maturation and fertilization following IVM–IVF of prepubertal and adult goat oocytes [13,15,18,19]. Furthermore, a few studies [13,16] have reported no differences in the rates of cleavage and blastocyst development between oocytes obtained from prepubertal and adult goats. To date, goat oocytes have been harvested principally from ovariectomized [20,21] or slaughtered does [13,15,16,22–24]. Laparoscopic ovum pick-up (LOPU) has provided an efficient and relatively noninvasive method for the collection of oocytes from small ruminants (sheep [25,26], goats [17,27], calves [2,3,28]) in which other techniques may not be feasible (transvaginal ultrasound-guided) or desirable (slaughterhouse, laparotomy). With this in mind, the objectives of the present research were to compare the follicular response and oocyte recovery from hormonally stimulated prepubertal versus adult goats and, using a semi-defined culture system, to evaluate and compare the developmental competence of oocytes recovered from prepubertal and adult goats.
2. Materials and methods 2.1. Experimental design Following synchronization of estrus in prepubertal and adult goats, follicular response, numbers of oocytes recovered by LOPU and their developmental competence following culture in semi-defined media were assessed. Conducted over a 15-week period during the nonbreeding season (June to September), the study compared the performance of adult animals with that of young growing goats during the latter one-half of the prepubertal period. Twenty four adult goats, ranging in age from 2 to 4 years, and 28 prepubertal goats born 3 months prior to the onset of the experiment were randomly allocated to one of ten oocyte collection sessions throughout the experimental period. Three adult goats and three to five prepubertal goats were used for each collection session. Hence, prepubertal animals were approximately 90 days of age at the time of the first collection and close to 200 days old by the tenth and final collection session. Upon aspiration from the follicles, oocytes were processed immediately for in vitro culture in order to assess rates of developmental
J. Koeman et al. / Theriogenology 60 (2003) 879–889
881
competence. Oocytes from four sessions were lost due to incubator failure (Sessions 1 and 2) and technical difficulties (Sessions 5 and 6) so that development data from only six sessions are reported here. 2.2. Ovarian stimulation and oocyte collection Estrus was synchronized in adult goats with intravaginal sponges (Veramix1, Upjohn Co., Orangeville, Ont., Canada), containing 60 mg medroxyprogesterone acetate, inserted for 10 days prior to oocyte collection. At 36 h before oocyte collection, all prepubertal and adult goats were given 125 mg cloprostenol i.m. (Estrumate1, Schering Canada Inc., Pointe-Claire, Que., Canada), 300 IU eCG i.m. (Folligon1, Intervet Canada Ltd., Whitby, Ont., Canada) and 80 mg NIH-FSH-PI i.m. (Folltropin1-V, Vetrepharm Canada Inc., London, Ont., Canada). Oocytes were collected via LOPU as previously described by Baldassarre et al. [25]. Oocytes were aspirated from all follicles >2 mm in diameter visible on the surface of the ovaries. The aspiration medium consisted of Tissue Culture Medium 199 (TCM-199; Life Technologies, Burlington, Ont., Canada) supplemented with 100 IU/ml penicillin-G, 0.1 mg/ml streptomycin, 0.05 mg/ml kanamycin, 0.5% fatty acid free-bovine serum albumin (FAF-BSA) and 0.05 mg/ml heparin (Sigma–Aldrich Canada, Oakville, Ont., Canada). 2.3. In vitro maturation, fertilization and culture Follicular aspirates were processed immediately upon recovery from each individual animal. Recovered oocytes were washed once through aspiration medium without heparin and transferred to maturation medium consisting of TCM-199 supplemented with 0.02 IU bovine luteinizing hormone (bLH), 0.002 IU bovine follicle stimulating hormone (bFSH; Sioux Biochemical Inc., Sioux Center, IA, USA), 1 mg/ml 17-b estradiol, 0.2 mM pyruvic acid, 50 mg/ml kanamycin, and 10% heat inactivated goat serum (Sigma–Aldrich). The cumulus investments of each oocyte were recorded as Grade A (3 cumulus layers), Grade B (1–2 cumulus layers), Grade C (denuded), or Grade D (expanded cumulus). Following the recovery of oocytes from the last animal in each collection, oocytes were pooled for each treatment group (i.e. adult goats and prepubertal goats). Oocytes were then allocated, in groups of 10–15, to 50 ml droplets of maturation medium under mineral oil (Sigma–Aldrich) and matured for 26 h at 38.5 8C in a humidified atmosphere of 5% CO2 in air. Following maturation, oocytes were partially denuded of their cumulus investment (50%) by repeated pipetting, washed once through EmCare (Immuno-Chemical Products, New Zealand) supplemented with 1 mg/ml BSA, and placed in 40 ml droplets of fertilization medium under oil. Fertilization medium consisted of Tyrode’s Albumin Lactate Pyruvate Solution supplemented with 20% estrus goat serum (heat inactivated) and 27.5 mg/ml pyruvic acid. Fresh spermatozoa were collected from a buck of proven fertility. Following incubation at room temperature for 2–3 h in the dark, viable sperm were separated by centrifugation through a two-layer (45 and 90%) Percoll gradient. The loose pellet of spermatozoa was washed once by centrifugation with modified defined medium (mDM) consisting of defined medium [29] supplemented with 6 mg/ml FAF-BSA, 3.1 mg/ml NaHCO3, 0.138 mg/ml
882
J. Koeman et al. / Theriogenology 60 (2003) 879–889
pyruvic acid, and 50 mg/ml gentamicin (Sigma–Aldrich). The remaining sperm pellet was resuspended in a capacitating solution consisting of 1 ml of mDM supplemented with 500 mM 8-bromoadenosine 30 :50 cyclic monophosphate sodium (8-bromo-cAMP) and 100 nM ionomycin calcium salt (Sigma–Aldrich) (Parrish JJ, personal communication). Semen was capacitated for 15 min at 38.5 8C. Following capacitation, oocytes were incubated with 1 106 sperm/ml for 18–22 h under the same atmosphere and temperature conditions as previously described for IVM. Following IVF, presumptive zygotes were freed from loosely attached sperm by washing through EmCare containing 1 mg/ml BSA prior to in vitro culture. A semi-defined two-step culture system, G1.2/G2.2, was utilized in this experiment as it has provided good results in the cow [30] and the goat [31]. Presumptive zygotes, in groups of 15, were transferred to 25 ml droplets of G1.2 culture medium and placed at 38.5 8C in a humidified atmosphere of 7% O2, 7% CO2 and 86% N2. Approximately 48 h postinsemination, uncleaved ova (1-cell) were removed and stained (Hoechst 33324; Sigma–Aldrich) for the assessment of the nuclear stage of arrest. Cleaved ova (2 cells) were transferred to fresh droplets of G1.2. On Day 5 (120 h postinsemination), ova were transferred to G2.2 media and cultured for four additional days under the same conditions as described above. Morulae and blastocysts were scored morphologically on Days 7 and 9 of culture, followed by staining (Hoechst 33342; Sigma–Aldrich) for cell counts on Day 9. 2.4. Morphological and nuclear evaluation of morulae and blastocysts Morulae and blastocysts were classified morphologically according to the following criteria: embryos appearing as a compacted mass of cells (16 cells) at magnification 50 under the stereomicroscope were classified as morulae; embryos exhibiting the presence of a blastocoel were classified as blastocysts; embryos containing a blastocoel and either emerging from the zona pellucida or lacking a zona pellucida were classified as hatched blastocysts. The nuclear status of uncleaved ova and the number of cells in each morula and blastocyst were assessed by fluorescence microscopy. The nuclear status of uncleaved ova was recorded as germinal vesicle, metaphase I, metaphase II, fertilized (two pronuclei), abnormal (activated, penetrated, polypronuclei/multinucleated), or degenerated (data not shown). Embryos scored morphologically as morulae on Day 9 of culture, but observed to have less than 16 nuclei following cell counts, were reclassified as pseudo-morulae. Morulae which had greater than 16 nuclei remained classified as morulae. Similarly, blastocysts scored morphologically on Day 9 of culture but observed to have less than 32 nuclei following cell counts were reclassified as pseudo-blastocysts. Blastocysts which had greater than 32 nuclei remained classified as blastocysts. 2.5. Statistical analysis Follicle aspiration and oocyte recovery data were analyzed by ANOVA (Mixed Models Procedure, SAS/STAT [32]). The relationship between the age of prepubertal goats and the number of oocytes recovered (P < 0:05) was also analyzed using the Mixed Models Procedure of the Statistical Analysis System. Proportional data were compared using Fisher’s exact test (P < 0:01).
J. Koeman et al. / Theriogenology 60 (2003) 879–889
883
3. Results 3.1. Follicular aspiration and oocyte recovery Data pooled from each of the ten collections indicate that the mean numbers of follicles aspirated (32 3:7 versus 18:4 1:1) and oocytes recovered (25 2:8 versus 16 1:2) were significantly greater for prepubertal than for adult goats (P < 0:01), respectively. Oocyte recovery rates (82 and 88%) were similar for both groups. Because a similar oocyte recovery rate was observed for prepubertal and adult goats, oocyte recovery data were utilized to assess a relationship between the age of prepubertal goats and the number of oocytes recovered (Fig. 1). A linear regression (slope S:E:) was noted for data from both prepubertal (1:37 0:59) and adult (0:04 0:27) goats. Linear regressions for prepubertal and adult data were significantly different (P < 0:05). Moreover, the slope determined using prepubertal data was significantly different from zero (P < 0:05), indicating that the number of oocytes recovered from prepubertal goats declined significantly with increasing age of the animals. No significant changes in the number of oocytes recovered from adult goats were observed throughout the experiment. Eightyseven percent of the cumulus oocyte complexes recovered from prepubertal, and 82% of those recovered from adult goats were classified as Grade A or B. 3.2. In vitro embryo production The number of oocytes cleaved (% of oocytes cultured) did not differ significantly between prepubertal (46.4%) and adult (50.7%) goats. The incidence of ova arrested during early cleavage was relatively small for oocytes derived from both prepubertal and adult goats, 3.5% versus 6.2%, respectively. As previously described, morulae and blastocysts scored morphologically on Days 7 and 9 of culture were further subdivided into pseudomorulae (<16 cells), morulae (16 cells), pseudo-blastocysts (<32 cells) and blastocysts
Fig. 1. Mean (S.E.M.) number of oocytes recovered from prepubertal (&) or adult (&) goats per collection. The numbers of oocytes recovered from prepubertal animals declined significantly with increasing age (P < 0:05).
884
J. Koeman et al. / Theriogenology 60 (2003) 879–889
Fig. 2. (a) Two adult-derived pseudo-blastocysts. While not apparent under the epifluorescence microscope, both ova were scored morphologically as blastocysts based on the presence of a visible blastocoel. (b) Three prepubertal-derived blastocysts, containing 180–220 nuclei, after 9 days of culture.
(32 cells), following cell counts on Day 9 of culture. Fig. 2a illustrates the characteristic appearance of pseudo-blastocysts using fluorescence microscopy. Prepubertal-derived blastocysts containing between 180 and 200 nuclei are shown in Fig. 2b. Percentages of pseudo-morulae (59.9% versus 54.9%), morulae (4.7% versus 4.4%), pseudo-blastocysts (22.1% versus 23.9%), and blastocysts (6.4% versus 7.1%) did not differ significantly between prepubertal and adult goats, respectively. The distribution of morulae cell numbers determined following cell counts is presented in Fig. 3. Morulae scored morphologically on Days 7 and 9 of culture were observed to contain between 2 and 56 nuclei following cell counts. Similarly, blastocysts scored morphologically on Days 7 and 9 of culture were observed to contain between 2 and 350 nuclei following cell counts
Fig. 3. Morula cell numbers (% of cleaved embryos) determined on Day 9 for oocytes from prepubertal (&) and adult (&) goats. Differences between treatment groups were not significant.
J. Koeman et al. / Theriogenology 60 (2003) 879–889
885
Fig. 4. Blastocyst cell numbers (% of cleaved embryos) determined on Day 9 for oocytes from prepubertal (&) and adult (&) goats. Differences between treatment groups were not significant.
(Fig. 4). The mean cell number (S.E.M.) per blastocyst (32 cells) did not differ significantly between prepubertal and adult goats (120 32 versus 117 30).
4. Discussion This study clearly demonstrated that more follicles can be stimulated to develop and more oocytes can be recovered from prepubertal than from adult goats, although the difference declines substantially as the prepubertal animals increase in age. The developmental competence of oocytes from prepubertal and adult goats appeared to be similar. 4.1. Follicular aspiration and oocyte recovery Laparoscopic ovum pick-up (LOPU) was utilized in this research as a minimally invasive method for the recovery of oocytes from prepubertal and adult goats. Large numbers of oocytes can be recovered from gonadotrophin-stimulated prepubertal goats by LOPU, as reported for other prepubertal ruminants (calves [33], sheep [8], goats [17]). Furthermore, the majority of oocytes recovered from prepubertal and adult goats, 87 and 82%, respectively, were classified as Grade A or B oocytes. The results of the present study indicate that LOPU is an efficient technique for the recovery of high quality oocytes from both prepubertal and adult goats. A significant relationship between the age of prepubertal goats and the number of oocytes recovered was also observed in the present experiment. A significant linear decline in the number of oocytes recovered (slope S:E:; 1:37 0:59) was associated with increasing age of the prepubertal goats (3–7 months) during the 15 weeks experimental period. In a separate study, a similar decline in the number of oocytes recovered was
886
J. Koeman et al. / Theriogenology 60 (2003) 879–889
observed with increasing age between two different age groups of prepubertal goats. Recovery of oocytes by LOPU from gonadotrophin-stimulated prepubertal goats resulted in a significantly higher yield from 60 to 90-day old goats than from 90 to 150-day old goats [17]. In the present study, despite a decline in the number of oocytes recovered with increasing age of the prepubertal goats, the mean number of oocytes recovered from prepubertal animals using data pooled from ten collections was significantly higher than that observed for adult goats (25 2:8 versus 16 1:2, respectively). The enhanced follicular response of prepubertal goats found in this study justifies the utility of collecting oocytes from animals of genetic value at an early age. 4.2. In vitro embryo production The present study documents the similar developmental capacity of prepubertal and adult goat oocytes to the blastocyst stage following IVM–IVF–IVC. These results are in agreement with results previously reported [13], in which the developmental capacities of oocytes derived from prepubertal and adult goats, with or without hormonal stimulation, were compared. Izquierdo et al. [15,16] also obtained comparable rates of blastocyst development using oocytes derived from prepubertal and adult goats. Furthermore, the rates of development to the blastocyst stage for adult goats in the present study were similar to those reported by Izquierdo et al. [16], but lower than those reported by others [23,24]. While the exact factors underlying the relatively low rate of blastocyst development achieved in the present experiment remain unknown, the reclassification of blastocysts based on cell counts (6% versus 7%, respectively) as compared to morphological appearance (25% versus 24%, respectively) resulted in a considerable reduction of the reported rate. The criteria used to classify morulae and blastocysts (morphological appearance versus cell counts) are often not reported which presents difficulties when comparing rates of blastocyst development among different laboratories. The ranges of morula and blastocyst cell numbers observed following cell counts are particularly intriguing as they reveal that morphological appearance does not necessarily indicate the true developmental status of the goat embryo. Further research is required to evaluate the full developmental potential of blastocysts following transfer; however, studies in which zygotes or early cleavage stage embryos were transferred indicate that embryos derived from prepubertal goats are as viable as those obtained from adults [17]. The reduced in vitro developmental competence of oocytes observed in the present study may be due to deficiencies in the in vitro production system. Maturation treatments, sperm preparation and culture systems are all known variables that can affect overall success rates [22–24]. In a recent study, Izquierdo et al. [16] did not observe any improvement in proportion of embryos developing to the blastocyst stage following in vivo culture. It may be that the in vitro maturation system is inadequate, as Crozet et al. [22] observed significant differences in blastocyst development following recovery of oocytes from small, medium, and large follicles, and from the oviduct. Seasonal effects also can not be excluded as this study was performed during the summer months (June to early September). Decreased rates of development are commonly observed in bovine in vitro production systems during the summer, while significant reductions in the developmental competence of ovine oocytes collected during anestrus have also been noted [34].
J. Koeman et al. / Theriogenology 60 (2003) 879–889
887
In conclusion, the mean number of oocytes recovered from prepubertal animals was significantly higher than that observed for adult goats (25 2:8 versus 16 1:2, respectively), while developmental competence was similar. The identification of optimal experimental conditions able to support development of a reasonable proportion of goat oocytes to the blastocyst stage (i.e. higher than 30%) is required before similarities (or differences) in the developmental competence of prepubertal and adult goat oocytes can be fully recognized. The successful development of a small number of prepubertal goat oocytes to the blastocyst stage achieved in this experiment is encouraging for the application of IVP techniques to prepubertal goats. Similarly high mean cell number of prepubertal- and adult-derived blastocysts (121 32 and 117 30, respectively) further emphasizes the developmental potential of prepubertal goat oocytes. The utilization of IVP conditions specifically designed to meet the requirements of the prepubertal goat oocyte/ embryo may enhance rates of blastocyst development. Successful production of embryos derived from prepubertal goats would, in turn, facilitate the rapid propagation of genetically valuable animals.
Acknowledgements We would like to thank J-T Chung, M. Drolet, K. Gaudreau-Provost, D. Laurin, J. Pierson, J. Pika, and H. Watt for their technical assistance throughout this experiment. This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), Nexia Biotechnologies Inc. and the Conseil des recherches en peˆ che et en agro-alimentaire du Que´ bec (CORPAQ). Jennifer Koeman was the recipient of an NSERC Postgraduate Scholarship.
References [1] Ptak G, Loi P, Dattena M, Tichner M, Cappai P. Offspring from one-month-old lambs: studies on the developmental capability of prepubertal oocytes. Biol Reprod 1999;61:1568–74. [2] Armstrong DT, Holm P, Irvine BJ, Petersen BA, Stubbings RB, McLean D, et al. Pregnancies and live birth from in vitro fertilization of calf oocytes collected by laparoscopic follicular aspiration. Theriogenology 1992;38:667–78. [3] Armstrong DT, Irvine BJ, Earl CR, McLean D, Seamark RF. Gonadotropin stimulation regimens for follicular aspiration and in vitro embryo production from calf oocytes. Theriogenology 1994;42:1227–36. [4] Revel F, Mermillod P, Peynot N, Renard JP, Heyman Y. Low developmental capacity of in vitro matured and fertilized oocytes from calves compared with that of cows. J Reprod Fertil 1995;103:115–20. [5] Damiani P, Fissore RA, Cibelli JB, Long CR, Balise JJ, Robi JM, et al. Evaluation of developmental competence, nuclear and ooplasmic maturation of calf oocytes. Mol Reprod Dev 1996;45:521–34. [6] Taneja M, Bols PEJ, Van de Velde A, Ju J, Schreiber D, Tripp MW, et al. Developmental competence of juvenile calf oocytes in vitro and in vivo: influence of donor animal variation and repeated gonadotropin stimulation. Biol Reprod 2000;62:206–13. [7] Palma GA, Tortonese DJ, Sinowatz F. Developmental capacity in vitro of prepubertal oocytes. Anat Histol Embryol 2001;30:295–300. [8] Earl CR, Irvine BJ, Kelly JM, Rowe JP, Armstrong DT. Ovarian stimulation protocols for oocyte collection and in vitro embryo production from 8 to 9 week old lambs. Theriogenology 1995;43:203 [abstract].
888
J. Koeman et al. / Theriogenology 60 (2003) 879–889
[9] O’Brien JK, Beck NFG, Maxwell WMC, Evans G. Effect of hormone pre-treatment of prepubertal sheep on the production and developmental capacity of oocytes in vitro and in vivo. Reprod Fertil Dev 1997;9: 625–31. [10] O’Brien JK, Dwarte D, Ryan JP, Maxwell WMC, Evans G. Developmental capacity, energy metabolism and ultrastructure of mature oocytes from prepubertal and adult sheep. Reprod Fertil Dev 1996;8: 1029–37. [11] Ledda S, Boliolo L, Calvia P, Leoni G, Naitana S. Meiotic progression and developmental competence of oocytes collected from juvenile and adult ewes. J Reprod Fertil 1997;109:73–8. [12] Ledda S, Bogliolo L, Leoni G, Naitana S. Production and lambing rate of blastocysts derived from in vitro matured oocytes after gonadotropin treatment of prepubertal ewes. J Anim Sci 1999;77:2234–9. [13] Mogas T, Palomo MJ, Izquierdo MD, Paramio MT. Developmental capacity of in vitro matured and fertilized oocytes from prepubertal and adult goats. Theriogenology 1997;47:1189–203. [14] Izquierdo D, Villamediana P, Palomo MJ, Mogas T, Paramio MT. Effect of sperm capacitation and fertilization media on IVF and early embryo development of prepubertal goat oocytes. Theriogenology 1998;49:1501–13. [15] Izquierdo D, Villamediana P, Paramio MT. Effect of culture media on embryo development from prepubertal goat IVM–IVF oocytes. Theriogenology 1999;52:847–61. [16] Izquierdo D, Villamediana P, Lopez-Bejar M, Paramio MT. Effect of in vitro and in vivo culture on embryo development from prepubertal goat IVM–IVF oocytes. Theriogenology 2002;57:1431–41. [17] Baldassarre H, Wang B, Kafidid N, Keefer C, Lazaris A, Karatzas CN. Advances in the production and propagation of transgenic goats using laparoscopic ovum pick-up and in vitro embryo production technologies. Theriogenology 2002;57:275–84. [18] Martino A, Mogas T, Palomo MJ, Paramio MT. Meiotic competence of prepubertal goat oocytes. Theriogenology 1994;41:969–80. [19] Martino A, Mogas T, Palomo MJ, Paramio MT. In vitro maturation and fertilization of prepubertal goat oocytes. Theriogenology 1995;43:473–85. [20] Younis AI, Zuelke KA, Harper KM, Oliveira MAL, Brackett BG. In vitro fertilization of goat oocytes. Biol Reprod 1991;44:1177–82. [21] Keskintepe L, Darwish GM, Kenimer AT, Brackett BG. Term development of caprine embryos derived from immature oocytes in vitro. Theriogenology 1994;42:527–35. [22] Crozet N, Ahmed-Ali M, Dubos MP. Developmental competence of goat oocytes from follicles of different size categories following maturation, fertilization and culture in vitro. J Reprod Fertil 1995;103:293–8. [23] Keskintepe L, Luvoni GC, Rzucidlo SJ, Brackett BG. Procedural improvements for in vitro production of viable uterine stage caprine embryos. Small Rumin Res 1996;20:247–54. [24] Pawshe CH, Palanisamy A, Taneja M, Jain SK, Totey SM. Comparison of various maturation treatments on the in vitro maturation of goat oocytes and their early embyronic development and cell numbers. Theriogenology 1996;46:971–82. [25] Baldassarre H, de Matos DG, Furnus CC, Castro TE, Cabrera Fischer EI. Technique for efficient recovery of sheep oocytes by laparoscopic folliculocentesis. Anim Reprod Sci 1994;35:145–50. [26] Baldassarre H, Furnus CC, de Matos DG, Pessi H. In vitro production of sheep embryos using laparoscopic folliculocentesis: alternative gonadotrophin treatments for stimulation of oocyte donors. Theriogenology 1996;45:707–17. [27] Graff KJ, Meintjes M, Gyer VW, Paul JB, Denniston RS, Ziomek C, et al. Transvaginal ultrasound- guided oocyte retrieval following FSH stimulation of domestic goats. Theriogenology 1999;51:1099–119. [28] Baldassarre H. Ovum pick-up followed by in vitro production of embryos in sheep and cattle. In: Proceedings of the 4th Swedish International Program on Animal Reproduction and Biotechnology for Latin America. Belem, Para, Brazil, 1998; p. 62–70. [29] Brackett B, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod 1975;12:260–74. [30] Krisher RL, Lane M, Bavister BD. Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media. Biol Reprod 1999;60:1345–52. [31] Ongeri EM, Bormann CL, Butler RE, Melican D, Gavin WG, Echelard Y, et al. Development of goat embryos after in vitro fertilization and parthenogenetic activation by different methods. Theriogenology 2001;55:1933–45.
J. Koeman et al. / Theriogenology 60 (2003) 879–889
889
[32] SAS/STAT1 Software. Changes and enhancements through Release 6.12. Cary, NC: SAS Institute Inc.; 1997. [33] Armstrong DT, Kotaras PJ, Earl CR. Advances in production of embryos in vitro from juvenile and prepubertal oocytes from the calf and lamb. Reprod Fertil Dev 1997;9:333–9. [34] Mitchell LM, Staines ME, McCallum GJ, Kuran M, McEvoy TG. Season affects in vitro developmental competence of ovine oocytes. Theriogenology 2000;53:362 [abstract].