Effects of repetition, interval between treatments and season on the results from laparoscopic ovum pick-up in goats

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1 CSIRO PUBLISHING Reproduction, Fertility and Development, 2004, 16, Effects of repetition, interval between treatments and season on the results from laparoscopic ovum pick-up in goats J. Pierson A, B. Wang A, N. Neveu A, L. Sneek A, F. Côté A, C. N. Karatzas A and H. Baldassarre A,B A Nexia Biotechnologies Inc., 320 Chemin St Georges, St Telesphore, Quebec, J0P 1G0, Canada. B Corresponding author. hbaldassarre@nexiabiotech.com Abstract. The present study was conducted to evaluate the follicular response and oocyte yield following repeated gonadotrophin stimulation and laparoscopic aspiration in goats and to assess the effects of the time interval between procedures and season. A total of 98 adult goats were subjected to laparoscopic ovum pick-up (LOPU) five consecutive times in a transgenic production programme. Oestrus was synchronised by means of intravaginal sponges inserted for 10 days coupled with 125 µg cloprostenol 36 h before sponge removal and LOPU, and follicular development was stimulated with 80 mg follicle stimulating hormone and 300 IU equine chorionic gonadotrophin administered 36 h before LOPU. No difference was detected in the response for LOPUs 1, 2, 3 and 4. Although a small decrease in response was detected at LOPU 5 (P <0.05), the numbers of follicles aspirated and oocytes recovered were not different from those at LOPU 1 and LOPUs 1 and 4, respectively. With respect to time interval between LOPU and season, all intervals and seasons produced acceptable responses, with no difference in follicles aspirated and oocytes recovered between intervals and seasons. These results indicate that LOPU may be repeated up to five times in goats at different intervals and in different seasons with little or no important change in overall response. Extra keywords: folliculocentesis, gonadotrophin treatment, oocyte aspiration. Introduction Laparoscopic ovum pick-up (LOPU) is an effective and minimally invasive technique that permits the repeated collection of oocytes from the same donor animal and, therefore, extends the use of donor animal resources. The procedure was first described by Snyder and Dukelow (1974) in sheep and the technique was fully developed 20 years later in both sheep (Baldassarre et al. 1994; Tervit 1996) and goats (Graff et al. 1995; Baldassarre et al. 2002). Baldassarre et al. (1994) and Tervit (1996) refined LOPU by adjusting needle type and size and vacuum pressure, and related these parameters to oocyte recovery and quality and blastocyst production. Baldassarre et al. (1996, 2002) also studied the effect of different hormonal treatments and obtained similar results when comparing follicle-stimulating hormone (FSH) multi-injection regimens with FSH and equine chorionic gonadotrophin (ecg) oneshot regimens in both sheep and goats. Graff et al. (1995) showed that LOPU in goats enabled more of the available follicles to be aspirated, resulting in more oocytes recovered compared with ultrasound-guided transvaginal recovery. Laparoscopic ovum pick-up may be repeated several times because it is less invasive and results in fewer surgical adhesions than standard surgery (laparotomy) generally used for the recovery of in vivo-matured oocytes and embryos (Stangl et al. 1999; Baldassarre et al. 2003a). Repeated LOPU followed by in vitro production (IVP) has the potential to produce more offspring than traditional multiple ovulation embryo transfer (MOET) because it is less variable in results (Baldassarre and Karatzas 2004) and may avoid poor results caused by poor ovulation rates, early corpus luteum (CL) regression and poor fertilisation (Cognié et al. 2003; Baldassarre and Karatzas 2004). In addition, LOPU may be used in circumstances where MOET is not possible (prepubertal, pregnant and ageing animals; Baldassarre et al. 2002). This technique may also be applied to the recovery of oocytes for the generation of transgenic animals by somatic cell nuclear transfer (SCNT) and/or DNA pronuclear microinjection (PN-MI) of IVP zygotes (Baldassarre et al. 2002). In the present study, we examined the effects of repetition, time interval between procedures and season on the number of follicles aspirated and oocytes recovered by LOPU from hormonally treated dwarf goats. The present study represents the statistical analysis of data generated in a transgenic production programme in which LOPU was used to source oocytes for in vitro maturation (IVM) and in vitro fertilisation (IVF) followed by PN-MI and/or for use as recipient cytoplasts in SCNT. Oocytes were pooled prior to IVM; therefore, the information on oocyte quality and developmental competence cannot be evaluated with respect to repetition, interval length or season. CSIRO /RD /04/080795

2 796 Reproduction, Fertility and Development J. Pierson et al. Materials and methods Animal ethics All protocols used in the present study were approved by the Animal Care Committee of Nexia, in compliance with the guidelines from the Canadian Council on Animal Care. Animals A total of 98 non-lactating, non-pregnant adult Nigerian Dwarf (Breed Early Lactate Early (BELE )) goats (1 5 years old, with an average age of 1 year at LOPU 1) that were subjected to LOPU five times as part of a transgenic production programme were used in the present study. The BELE goats were used in the programme because they are sexually precocious, with first oestrus occurring at 3 4 months of age, and because their small physical size helps reduce feed and housing costs. The goats used in the present study were the result of an internal breeding programme and were not used in any other research protocol. Location and management The transgenic production programme was undertaken in St Telesphore, Quebec, Canada; a humid mid-latitude environment (45 N, 75 W).Animals were housed indoors in groups of 20 animals per pen, with exposure to natural daylight patterns. The natural daylight patterns were maintained throughout the year with programmed artificial lighting and with exposure to natural outdoor lighting through windows present in the housing area. The average ambient temperature was maintained between 13 and 16 C in the autumn, winter and spring, whereas in the summer the temperature ranged between 20 and 30 C. Animals were fed manually with good-quality hay, water and mineral blocks ad libitum. Hormone treatments All animals in the programme were synchronised by means of intravaginal sponges (Veramix ; Upjohn, Orangeville, Ontario, Canada) impregnated with 60 mg medroxyprogesterone acetate (MAP) inserted for 10 days coupled with a luteolytic injection of 125 µg cloprostenol (Estrumate ; Schering, Pointe Claire, Quebec, Canada) administered intramuscularly (i.m.) 36 h prior to sponge removal, which took place at the time of LOPU. Follicular development was stimulated in all animals with 80 mg FSH (Folltropin-V ; Vetrepharm, Belleville, Ontario, Canada) and 300 IU ecg (Novormon ; Vetrepharm) administered i.m. 36 h prior to sponge removal and LOPU. Anaesthesia Oocyte donors were deprived of food and water for 24 h prior to laparoscopy. Anaesthesia was induced with intravenous administration of 0.35 mg kg 1 bodyweight of valium (Diazepam ; Sabex, Boucherville, Quebec, Canada) and 5 mg kg 1 bodyweight of ketamine (Ketalean ; Bimeda-MTC, Cambridge, Ontario, Canada) and was maintained with isoflurane (Isoflo ; Abbott, Montreal, Quebec, Canada) via endotracheal intubation. Laparoscopic ovum pick-up As described previously (Baldassarre et al. 2003a), anaesthetised does were restrained in dorsal recumbence on a standard laparoscopy table and follicles were aspirated under laparoscopic observation using a 20-gauge short-bevel hypodermic needle mounted in a plastic pipette connected to a collection tube and vacuum line (Fig. 1). The vacuum pressure was regulated with a flow valve and adjusted to drops min 1. The collection medium used was TCM 199 (Sigma Chemical, St Louis, MO, USA) supplemented with 0.05 mg ml 1 heparin (Hepalean ; Organon Teknica, Scarborough, Ontario, Canada) and 1% (v/v) fetal calf serum and the collection tube contained 0.5 ml of this medium to receive the oocytes. Follicular contents were immediately Fig. 1. Laparoscopic photograph showing the left ovary of a gonadotrophin-stimulated goat during follicular aspiration. The column of follicular fluid is clearly visualised as aspirated through the aspiration pipette. transferred from the collection tube into a Petri dish and oocytes were located using a stereomicroscope. Statistical analysis The program used for the statistical analysis was GraphPad Prism (GraphPad Software, San Diego, CA, USA). The effect of repeated LOPU, in terms of follicles aspirated and oocytes recovered, was analysed using repeated-measures ANOVA and Tukey s multiplecomparison test. For the analysis of time interval between treatments, results from LOPUs 2, 3 and 4 were pooled. The interval between each LOPU varied from 5 to 32 weeks and the four intervals examined were 5 7 weeks (n = 67), 8 10 weeks (n = 136), weeks (n = 34) and 14 weeks (n = 57). For the analysis of season, results from LOPUs 1, 2, 3 and 4 were pooled. The four seasons examined were autumn (September December; n = 161), winter (January March; n = 83), spring (April June; n = 100) and summer (July August; n = 48). The effects of interval and season were analysed using ANOVA and Tukey s multiple-comparison tests. The effects of repetition, time interval and season were considered significantly different at P<0.05. Results During this programme, a total of 6996 follicles was aspirated (14.3 ± 5.3 follicles per donor) and 6305 oocytes were recovered (12.9 ± 5.6 oocytes per donor), resulting in an average recovery rate of 90%. No important surgical adhesions that could adversely affect further LOPU procedures were observed. Occasionally, adhesions of the omentum to the abdominal wall at the point of trocar entry were observed; however, these adhesions did not prevent further LOPUs from being performed successfully. As indicated in Table 1, no difference was detected in the number of follicles aspirated and oocytes recovered for LOPUs 1, 2, 3 and 4. A small decrease in response was observed at LOPU 5 (P <0.05); however, the numbers of follicles aspirated and oocytes recovered at LOPU 5 were not different from LOPU 1 and LOPUs 1 and 4 respectively.

3 Laparoscopic ovum pick-up in goats Reproduction, Fertility and Development 797 Table 1. Effect of repeated laparoscopic ovum pick-up on the number follicles aspirated and oocytes recovered (n = 98) LOPU 1 LOPU 2 LOPU 3 LOPU 4 LOPU 5 No. follicles aspirated 14.2 ± 5.3 a,b 15.1 ± 5.6 a 14.5 ± 5.0 a 14.6 ± 4.6 a 12.9 ± 4.6 b No. oocytes recovered 12.6 ± 5.6 a,b 13.5 ± 6.0 a 13.6 ± 5.3 a 13.0 ± 5.2 a,b 11.6 ± 5.1 b Recovery rate (%) a,b Within rows, values with different superscripts are different (P <0.05, repeated-measures ANOVA and Tukey s multiple-comparison test). LOPU, laparoscopic ovum pick-up. Table 2. Effect of time interval between laparoscopic ovum pick-ups on the number of follicles aspirated and oocytes recovered Pooled laparoscopic ovum pick-ups 2, 3 and 4 Interval between LOPUs (weeks) n No. follicles aspirated 14.6 ± ± ± ± 5.1 No. oocytes recovered 13.3 ± ± ± ± 5.3 Recovery rate (%) Within rows, values are not significantly different (P >0.05, ANOVA and Tukey s multiple-comparison test). LOPU, laparoscopic ovum pick-up. Table 3. Effect of season on the number of follicles aspirated and oocytes recovered Pooled laparoscopic ovum pick-ups 1, 2, 3 and 4 Season Autumn Winter Spring Summer (September December) (January March) (April June) (July August) n No. follicles aspirated 14.2 ± ± ± ± 5.0 No. oocytes recovered 13.1 ± ± ± ± 5.6 Recovery rate (%) Within rows, values are not significantly different (P >0.05, ANOVA and Tukey s multiple-comparison test). Because LOPUs 2, 3 and 4 were not different, data from these procedures were pooled to determine the effect of time interval between treatments. As indicated in Table 2, all intervals produced acceptable responses, with no difference in follicles aspirated and oocytes recovered between each interval. Laparoscopic ovum pick-ups 1, 2, 3 and 4 were pooled to study the effect of season. As with time interval, all seasons produced acceptable responses, with no difference in follicles aspirated and oocytes recovered between seasons (Table 3). Discussion The numbers of follicles aspirated and oocytes recovered by LOPU were similar to those reported previously in goats (Graff et al. 1995, 1999; Baldassarre et al. 2001, 2003a; Koeman et al. 2003). Our results indicate that repeated gonadotrophin stimulation and LOPU have no important effect on follicular response and oocyte yield. Although a small decrease in response was observed at LOPU 5, the decrease in follicles aspirated and oocytes recovered was not different from LOPU 1 and LOPUs 1 and 4 respectively.these results appear to contradict earlier reports using MOET with diminishing superovulatory response after three programmes (Remy et al. 1991; Cognié 1999). Previous studies by our group (Baldassarre et al. 2003a) involving up to four LOPUs indicate an increase in response at LOPU 2 compared with LOPUs 1, 3 and 4 that was not observed in the present study. This could be attributed to the small number of animals involved in the previous study (n = 10) compared with the present study (n = 98). Although occasional adhesions of the omentum to the abdominal wall were observed after LOPU,

4 798 Reproduction, Fertility and Development J. Pierson et al. no adhesions that could adversely affect or prevent further LOPU were reported (data not shown). Results from our production programme also indicate that many donors subjected to more than five LOPU procedures become pregnant and kid after insemination or natural breeding (H. Baldassarre et al., unpublished observations). This is in agreement with the results of Stangl et al. (1999), who reported that repeated LOPU had no negative effect on the fertility of donor sheep, even when repeated up to 20 times. Overall, these results indicate that hormonal treatment and LOPU can be repeated up to five times in the same donor goats with minimal surgical adhesions and no important change in overall response. Further studies should be performed to determine whether there is an important decrease in response when LOPU is repeated more than five times. With respect to time interval between gonadotrophin stimulation and LOPU, all four intervals produced acceptable results and no significant advantage was observed when this interval was extended. We can conclude from these results that the ovary recovers from LOPU in less than 5 weeks. It is important to note that LOPU is less traumatic to the ovary than ultrasound-guided ovum pick-up (OPU) because only the theca is perforated during aspiration, whereas during ultrasound-guided OPU the needle usually reaches the follicle following perforation of the ovarian stroma. To our knowledge, very few experiments have been conducted in goats to examine the effect of interval between repeated hormonal treatment and LOPU. In production programmes where large numbers of animals are available, it may be possible to use a longer interval; however, a period as short as 5 7 weeks between procedures may be advantageous in situations where donor animal resources are limited. Shorter intervals allow for improved management of animal resources and lower production costs by allowing one to do more with a given number of animals. Because LOPU can be repeated many times in the same donor within a short time period, it is possible to generate more offspring from genetically valuable animals using this technique. No significant difference was observed in the follicular response between seasons and this is in agreement with results of previous studies conducted in other mid-latitude environments (40 N) using MOET in sheep (Gonzalez- Bulnes et al. 2003) and goats (Pintado et al. 1998). Therefore, LOPU can be performed throughout the year with virtually no change in follicular response and oocyte yield, again facilitating the management of donor animal resources. Several groups have reported that repeated use of ecg results in poor fertility in fixed-time artificial insemination (AI) and results have been linked to the presence of antiecg antibodies developed as an immune response to previous treatments (Roy et al. 1999; Drion et al. 2001). A significant delay in the occurrence of oestrus, the luteinising hormone surge and ovulation accompany this antibody production and, therefore, indicate the importance of oestrus detection and modification of the timing of AI. Although the present study did not evaluate the same parameters, our results are encouraging because they indicate that repeated stimulation with both FSH and ecg has virtually no effect on follicular response and oocyte yield. Because LOPU was used in our transgenic production programme to source oocytes for IVM, IVF and PN-MI and nuclear transfer (NT), the pooling of oocytes prior to IVM and NT procedures prevented the analysis of oocyte quality and developmental competence with respect to repetition, interval length and season. With respect to the effect of season, we have reported previously a decrease in initial pregnancy rate and an increase in pregnancy loss in recipients transferred with NT embryos out of season (spring/summer) compared with recipients transferred during the reproductive season (autumn/winter; Baldassarre et al. 2003b). However, it was not possible to differentiate between developmental competence or seasonal differences in the ability of recipients to maintain pregnancy. In conclusion, LOPU is an extremely valuable tool for the production and propagation of genetically valuable animals. Repeated LOPU permits better use of donor animal resources and may overcome many of the problems encountered with traditional MOET. In previous reports (Baldassarre et al. 2002, 2003b) we have demonstrated the advantages of using LOPU-obtained oocytes in comparison with laparotomy sourced zygotes and oocytes for DNA microinjection and SCNT respectively. The results of the present study confirm LOPU as a very efficient method for such applications, given its repeatability and reliability as a source of immature oocytes. References Baldassarre, H., and Karatzas, C. N. (2004). Advanced assisted reproduction technologies (ART) in goats. Anim. Reprod. Sci , doi: /j.anireprosci Baldassarre, H., de Matos, D. G., Furnus, C. C., Castro, T. E., and Cabrera Fischer, E. I. (1994). Technique for efficient recovery of sheep oocytes by laparoscopic folliculocentesis. Anim. Reprod. Sci. 35, doi: / (94) Baldassarre, H., Furnus, C. C., de Matos, D. G., and Pessi, H. (1996). In vitro production of sheep embryos using laparoscopic folliculocentesis: alternative gonadotrophin treatments for stimulation of oocyte donors. Theriogenology 45, doi: / x(95) Baldassarre, H., Keefer, C. L., Gauthier, M., Bhatia, B., Begin, I., Pierson, J., Laurin, D., Trigg, T., Downey, B. R., and Karatzas, C. N. (2001). Laparoscopic ovum pick-up and zygote recovery in goats treated with deslorelin implants before superovulation. Theriogenology 55, 510. [Abstract] Baldassarre, H., Wang, B., Kafidi, N., Keefer, C. L., Lazaris, A., and Karatzas, C. N. (2002). Advances in the production and propagation of transgenic goats using laparoscopic ovum pick-up and in vitro embryo production technologies. Theriogenology 57, doi: /s x(01) Baldassarre, H., Wang, B., Kafidi, N., Gauthier, M., Neveu, N., et al. (2003a). Production of transgenic goats by pronuclear microinjection of in vitro produced zygotes from oocytes recovered

5 Laparoscopic ovum pick-up in goats Reproduction, Fertility and Development 799 by laparoscopy. Theriogenology 59, doi: /s x(02) Baldassarre, H., Keefer, C., Wang, B., Lazaris, A., and Karatzas, C. N. (2003b). Nuclear transfer in goats using in vitro matured oocytes recovered by laparoscopic ovum pick-up. Cloning Stem Cells 5, doi: / Cognié, Y. (1999). State of the art in sheep goat embryo transfer. Theriogenology 51, doi: /s x(98) Cognié, Y., Baril, G., Poulin, N., and Mermillod, P. (2003). Current status of embryo technologies in sheep and goat. Theriogenology 59, doi: /s x(02) Drion, P. V., Furtoss, V., Baril, G., Manfredi, E., Bouvier, F., et al. (2001). Four years of induction/synchronization of estrus in dairy goats: effect on the evolution of ecg binding in relation with the parameters of reproduction. Reprod. Nutr. Dev. 41, doi: /rnd: Gonzalez-Bulnes, A., Garcia-Garcia, R. M., Santiago-Moreno, J., Dominguez, V., Lopez-Sebastian, A., and Cocero, M. J. (2003). Reproductive season affects inhibitory effects from large follicles on the response to superovulatory FSH treatments in ewes. Theriogenology 60, doi: /s x(02) Graff, K. J., Meintjes, M., Paul, J. B., Dyer, V. W., Denniston, R. S., Ziomek, C. A., and Godke, R. A. (1995). Ultrasound-guided transvaginal oocyte recovery from FSH-treated goats for IVF. Theriogenology 43, 223. [Abstract] doi: / x(95)92377-l Graff, K. J., Meintjes, M., Dyer, V. W., Paul, J. B., Denniston, R. S., Ziomek, C. A., and Godke, R. A. (1999). Transvaginal ultrasoundguided oocyte retrieval following FSH stimulation of domestic goats. Theriogenology 51, doi: /s x (99) Koeman, J., Keefer, C. L., Baldassarre, H., and Downey, B. R. (2003). Developmental competence of prepubertal and adult goat oocytes cultured in semi-defined media following laparoscopic recovery. Theriogenology 60, doi: /s x(03) Pintado, B., Gutierrez-Adan, A., and Perez Llano, B. (1998). Superovulatory response of Murciana goats to treatments based on PMSG/Anti-PMSG or combined FSH/PMSG administration. Theriogenology 50, doi: /s x(98) Remy, B., Baril, G., Vallet, J. C., Dufour, R., Chouvet, C., Saumande, J., Chupin, D., and Beckers, J. F. (1991). Are antibodies responsible for a decreased superovulatory response in goats which have been treated repeatedly with pfsh? Theriogenology 36, doi: / x(91)90467-r Roy, F., Maurel, M. C., Combes, B., Vaiman, D., Cribiu, E. P., Lantier, I., Pobel, T., Delétang, F., Combarnous, Y., and Guillou, F. (1999). The negative effect of repeated equine chorionic gonadotrophin treatment on subsequent fertility in Alpine goats is due to a humoral immune response involving the major histocompatibility complex. Biol. Reprod. 60, Snyder, D. A., and Dukelow, R. (1974). Laparoscopic studies of ovulation, pregnancy diagnoses, and follicle aspiration in sheep. Theriogenology 2, Stangl, M., Kühholzer, B., Besenfelder, U., and Brem, G. (1999). Repeated endoscopic ovum pick-up in sheep. Theriogenology 52, doi: /s x(99) Tervit, H. R. (1996). Laparoscopy/laparotomy oocyte recovery and juvenile breeding. Anim. Reprod. Sci. 42, doi: / (96) Manuscript received 29 June 2004; revised and accepted 24 October