In vitro embryo production using ovaries removed from culled cows

Transcription

1 SHORT COMMUNICATION In vitro embryo production using ovaries removed from culled cows G. Giritharan and R. Rajamahendran Faculty of Agricultural Sciences, University of British Columbia, 2357 Main mall, Vancouver, BC, Canada V6T 1Z4 ( Received 3 April 2001, accepted 5 September Can. J. Anim. Sci. Downloaded from by on 01/01/18 Giritharan, G. and Rajamahendran, R In vitro embryo production using ovaries removed from culled cows. Can. J. Anim. Sci. 81: The objective was to utilize in vitro maturation and fertilization to maximize embryo production from culled dairy cows. Ovaries were removed (via a colopotomy technique) from culled Holstein cows (n = 21) 2 d after standing estrus (T 1 ), 2 d after treatment with 40 mg follicle stimulating hormone (FSH) given at standing estrus (T 2 ), or at an unknown stage of the estrous cycle (T 3 ). Treatment with FSH increased (P < 0.05) oocyte recovery rate (mean ± SEM, 14.6 ± 2.2, 25.4 ± 3.4, and 11.1 ± 2.6 oocytes ovary 1 ), for groups T1, T2 and T3, respectively, and the number of blastocysts obtained (1.6 ± 0.5, 4.4 ± 0.4 and 1.6 ± 0.6 blastocysts ovary 1 ). Culled dairy cows of high genetic merit are potential embryo donors, with an average of six to eight transferable embryos obtained from each FSH-treated cow. Key words: FSH treatment, oocyte recovery, IVM, IVF, culled cows Giritharan, G. et Rajamahendran, R Production d embryons in vitro à partir des ovaires de vaches réformées. Can. J. Anim. Sci. 81: L expérience devait illustrer comment la maturation et la fécondation in vitro peuvent maximiser la production d embryons par les vaches laitières réformées. Les ovaires ont été prélevés (par colopotomie) de vaches Holstein réformées (n = 21) 2 jours après l œstrus (T 1 ), 2 jours après administration de 40 mg de folliculostimuline (FSH) à l œstrus (T 2 ) ou à un moment indéterminé du cycle œstral (T 3 ). L administration de folliculostimuline augmente (P < 0,05) le nombre d ovocytes récupérés (moyenne ± E.-T., 14,6 ± 2,2, 25,4 ± 3,4 et 11,1 ± 2,6 ovocytes par ovaire) chez les vaches T 1, T 2 et T 3, respectivement, et le nombre de blastocytes résultants (1,6 ± 0,5, 4,4 ± 0,04 et 1,6 ± 0,6 blastocytes par ovaire). Les vaches réformées d un grand intérêt génétique pourraient donc devenir des donneuses d embryons, chaque animal traité avec de la FSH pouvant en donner en moyenne six à huit utilisables. Mots clés: Administration de FSH, extraction des ovocytes, MIV, FIV, vaches réformées It is estimated that 25 to 30% of North American dairy cows are culled annually due to various factors (Bascom and Young 1998). With this culling rate, under traditional breeding systems, dairy cows produce an average of less than five calves during their lifetime. In the past 50 yr, a number of reproductive techniques have been developed to increase the number of calves that can be produced; these could be used to increase the number of calves produced by culled cows of high genetic merit. For example, multiple ovulation and embryo transfer is frequently done under farm conditions. However, this technique involves repeated administration of hormones and the number of transferable embryos obtained per flush is highly variable due to inconsistent responses to superovulatory treatments (Canseco et al. 1992; Armstrong 1993). The past decade witnessed considerable advancement in in-vitro maturation and fertilization, enabling the large-scale production of bovine embryos (Sivakumaran et al. 1993; Boediono et al. 1994). Super-stimulation followed by ultrasound-guided aspiration of follicles is a rapidly developing technique for collecting oocytes (from live cattle of high genetic merit) for in vitro embryo production (Bordignon et al. 1997; Ooe et al. 1997). However, this 589 technique requires expensive equipment and trained personnel. Alternatively, ovaries can be removed surgically or at slaughter and oocytes recovered. Based on ultrasonographic studies of ovarian follicular dynamics, removing ovaries during metestrus should optimize oocyte recovery and developmental competence (Rajamahendran et al. 1994). Furthermore, treatment with follicle stimulating hormone (FSH) during the endogenous FSH surge increased the number of viable oocytes and follicles by reducing atresia or by increasing the number of follicles recruited (Calder and Rajamahendran 1992). The objective of the present experiment was to maximize embryo production from culled dairy cows by using in vitro techniques for oocyte maturation and fertilization and for embryo culture. We hypothesized that removal of ovaries soon after emergence of the first follicular wave or giving exogenous FSH at the time of the endogenous FSH surge would increase the yield of viable oocytes and embryos compared to removing ovaries at random stages of the estrous cycle. Twenty-one cycling, lactating Holstein cows, 4 6 yr old and designated for culling, were randomly assigned to one

2 590 CANADIAN JOURNAL OF ANIMAL SCIENCE of three treatments. Seven cows were given an i.m. injection of 25 mg prostaglandin F 2α (PGF 2α ; Lutalyse, Pharmacia Animal Health, Orangeville, ON) to induce estrus and ovaries were removed 2 d after standing estrus (T 1 ). Seven additional cows were similarly treated with PGF 2α and were given an i.m. injection of 40 mg porcine FSH (Folltropin; Vetrepharm Canada Inc., Belleville, ON) at standing estrus and ovaries were removed 2 d later (T 2 ). The remaining seven cows were not treated, and ovaries were removed at an unknown stage of the estrous cycle (T 3 ). Under epidural anesthesia (Lidocaine HCL 2%; MTC Pharmaceuticals, Cambridge, ON), an incision was made in the vagina (colpotomy technique; Drost et al. 1992) and ovaries were removed with a chain ecraseur. Ovaries were placed in a vacuum flask containing sterile physiological saline (30 35 C) and brought to the laboratory within 4 h of collection. All animal procedures were conducted in accordance with the guidelines of the Canadian Council on Animal Care (1993). Oocytes from small follicles (< 7 mm) were collected into aspiration medium using an 18-gauge needle and a 10-mL syringe. The medium contained Dulbecco s phosphate buffered saline (GIBCO BRL; Canadian Life Technologies, Burlington, ON), 0.3% bovine serum albumin (Sigma- Aldrich Canada Ltd, Oakville, ON) and 50 µg ml 1 gentamicin (Sigma-Aldrich Canada Ltd). Oocytes with an evenly granulated cytoplasm and surrounded by more than three layers of cumulus cells were selected for maturation. These oocytes were cultured for 24 h at 38.5 C in humidified air containing 5% CO 2. The maturation medium consisted of TCM199 (Sigma-Aldrich Canada Ltd.), 0.01 mg ml 1 FSH, 5% superovulated cow serum (SCS; Boediono et al. 1994), and 50 µg ml 1 gentamicin. Frozen semen from a single bull was thawed at 37 C, washed twice by centrifugation at 500 g for 5 min, diluted to sperm ml 1 in Brackett and Oliphant medium (Brackett and Oliphant 1975), and supplemented with 2.5 mm caffeine sodium benzoate (Sigma-Aldrich Canada Ltd) and 20 µg ml 1 heparin (Sigma-Aldrich Canada Ltd). Sperm droplets (100 µl) were prepared under mineral oil and pre-incubated at 38.5 C in humidified air containing 5% CO 2 for 1 h. Twenty to thirty matured oocytes were placed in each of these semen droplets and incubated at 38.5 C in humidified air containing 5% CO 2 for h. The presumptive zygotes were then cultured in media prepared by mixing TCM-199, 5% SCS, 5 µg ml 1 insulin (Sigma-Aldrich Canada Ltd) and 50 µg ml 1 gentamicin (Boediono et al. 1994) in four-well culture dishes at 38.5 C in humidified air containing 5% CO 2. The culture medium was changed every 72 h. Cleavage and blastocyst formation were assessed 72 h after insemination and after 9 d of embryo culture, respectively. Analysis of variance was used to compare, among treatments, average number of oocytes, percent oocytes selected for maturation, percent inseminated oocytes that cleaved, percent cleaved embryos that developed to the blastocyst stage, and average number of blastocysts per ovary. An arcsine transformation was conducted on all percentage data prior to analysis and a Least Significant Difference test was used to locate differences among treatments. Table 1. Average number of oocytes, percent oocytes selected for maturation, percent inseminated oocytes that cleaved, percent embryos that developed to the blastocyst stage, and average number of blastocysts per ovary. Ovaries were removed from culled Holstein cows (n = 21) 2 d after standing estrus (T 1 ), 2 d after treatment with FSH at standing estrus (T 2 ) or at an unknown stage of the estrous cycle (T 3 ) Treatment T 1 T 2 T 3 Oocytes collected (mean ± SEM) z 14.6 ± 2.2b 25.4 ± 3.4a 11.1 ± 2.6b Oocytes selected (%) Oocytes cleaved (%) Blastocyst formation (%) Blastocysts (mean ± SEM) y 1.6 ± 0.5a 4.4 ± 0.4b 1.6 ± 0.6a z Mean and standard error of oocyte yield ovary 1. y Mean and standard error of blastocysts ovary 1. a,b Means with different letters within a row differ (P < 0.05). Results are shown in Table 1. Treatment with FSH increased (P < 0.05) the average number of oocytes and the average number of blastocysts per ovary. However, there was no significant effect of treatment group on percent oocytes selected for maturation, percent inseminated oocytes that cleaved, or percent cleaved embryos that developed to the blastocyst stage. The hypothesis that giving exogenous FSH at the time of the endogenous FSH surge would increase the yield of viable oocytes and embryos compared to removing ovaries at random stages of the estrous cycle was supported. Earlier studies have also demonstrated the beneficial effects of FSH treatment on in vitro (Ooe et al. 1997) and in vivo (Rajamahendran et al.1987) embryo production. However, the hypothesis that removal of ovaries soon after emergence of the first follicular wave would increase the yield of oocytes and embryos was not supported. This finding was similar to that of other reports failing to demonstrate any beneficial effect of superovulation soon after emergence of the first follicular wave on in-vivo embryo production (Goulding et al. 1990; Calder and Rajamahendran 1992). The oocyte recovery rate, cleavage rate and the blastocyst production rates obtained from ovaries removed from T 3 cows were in agreement with previous findings (Sivakumaran et al. 1993; Boediono et al. 1994). In conclusion, culled dairy cows were suitable embryo donors. Treatment with a low dose of FSH at the time of estrus and ovary removal 2 d later maximized oocyte and embryo yield, with an average of six to eight transferable embryos produced from each cow. Armstrong, D. T Recent advances in superovulation of cattle. Theriogenology 39: Bascom, S. S. and Young, A. J A summary of the reasons why farmers cull cows. J. Dairy Sci. 81: Boediono, A., Takagi, M., Saha, S. and Suzuki, T Influence of Day 0 and Day 7 superovulated cow serum during development of bovine oocytes in vitro. Reprod. Fertil. Dev. 6: Bordignon, V., Morin, N., Durocher, J., Bousquet, D. and Smith, L.C GnRH improves the recovery rate and the in vitro developmental competence of oocytes obtained by transvaginal follicular aspiration from super-stimulated heifers. Theriogenology 48:

3 GIRITHARAN AND RAJAMAHENDRAN EMBRYO PRODUCTION FROM CULLED COWS 591 Can. J. Anim. Sci. Downloaded from by on 01/01/18 Brackett, B. G. and Oliphant, G Capacitation of rabbit spermatozoa in vitro. Biol. Reprod. 12: Calder, M. and Rajamahendran, R Follicular growth, ovulation and embryo recovery in dairy cows given FSH at the beginning or middle of the estrous cycle. Theriogenology 38: Canadian Council on Animal Care Guide to the care and use of experimental animals. Vol. 1, 2nd ed. Canadian Council on Animal Care, Ottawa, ON. Canseco, R. S., Gwazdauskas, F. C., Toole R. J., Rajamahendran, R., Whittier, W. D. and Vinson, W. E A retrospective study on the effects of FSH and prostaglandin on superovulation responses in dairy cattle. Virginia J. Sci. 43: Drost, M., Savio, J. D., Barros, C. M., Badinga, L. and Thatcher, W. W Ovariectomy by colpotomy in cows. J. Am. Vet. Med. Assoc. 200(30): Goulding, D., Williams, D. H., Duffy, P., Boland, M. P. and Roche, J. F Superovulation in heifers given FSH initiated either at Day 2 or Day 10 of the estrous cycle. Theriogenology 34: Ooe, M., Rajamahendran, R., Boediono, A. and Suzuki, T Ultrasound guided follicle aspiration and IVF in dairy cows with FSH and dominant follicle removal. J. Vet. Med. Sci. 59: Rajamahendran, R., Canseco, R. S., Denbow, C., Gwazdauskas, F. C. and Vinson, W. E Effect of low dose of FSH given at the beginning of the estrous cycle and subsequent superovulatory response in Holstein cows. Theriogenology 28: Rajamahendran, R., Ambrose, J. D. and Burton, B Clinical and research applications of real-time ultrasonography in bovine reproduction. Can. Vet. J. 35: Sivakumaran, K., Rajamahendran, R., Calder, M. and Ambrose, J. D The influence of reproductive status of cows at slaughter on oocyte number and maturation in-vitro and the effect of co-culture systems on fertilization and subsequent development. World Rev. Anim. Prod. 28(4):

4

5 This article has been cited by: 1. Pavneesh Madan, Phillip J. Bridges, Carolyn M. Komar, Alexander G. Beristain, Rajadurai Rajamahendran, Joanne E. Fortune, Colin D. MacCalman Expression of Messenger RNA for ADAMTS Subtypes Changes in the Periovulatory Follicle after the Gonadotropin Surge and During Luteal Development and Regression in Cattle1. Biology of Reproduction 69:5, [Crossref]