277 In vitro oocyte transport through the oviduct of buffalo and crossbred beef cows Transporte de oócitos in vitro através do oviduto de búfalas e vacas de corte cruzadas Nelcio Antonio Tonizza de CARVALHO1; Júlia Gleyci SOARES2; Fernando da Silva VANNUCCI1; Magali D’ANGELO3; Andréa G. GALLUPO3; Gisele M. MELO3; Rosimeire J. SOUZA3; Marcílio NICHI2; Lindsay Unno GIMENES2; Manoel Francisco de SÁ FILHO2; Claudiney de Melo MARTINS2; Eduardo CASTRICCINI2; Pietro Sampaio BARUSELLI2 Unidade de Pesquisa e Desenvolvimento de Registro – Pólo Rgional do D.S.A. do Vale do Ribeira/APTA, Registro-SP, Brasil 2 Departamento de Reprodução Animal da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, São Paulo-SP, Brasil 3 Instituto Biológico, São Paulo-SP, Brasil 1 Abstract The present study was conducted to verify if the elevation of plasma concentrations of estradiol during superovulatory treatments affects the oocyte transport in buffalo females, as well as if the inferior quality of buffalo oocytes and/or some functional difference on the oviduct of these animals is responsible for the low embryo recovery rate in superovulated buffaloes when compared to cows subjected to the same treatment. Oviducts of 10 buffaloes and 15 of cows, treated to induce a single ovulation were used. The oviducts were placed on Petri dishes and received the following treatments: 5 buffalo oocytes with no E2 (G-BufBuf and G-BovBuf), 5 bovine oocytes with no E2 (G-BufBov and G-BovBov), 5 buffalo oocytes with E2 (G-BufE2Buf and G-BovE2Buf) and 5 bovine oocytes with E2 (G-BufE2Bov and G-BovE2Bov; factorial 2x2x2). Oocytes were incubated for 24h. Subsequently, oviducts were washed and oocytes were recovered and counted. Since no interactions were found between E2 treatment, oviducts and oocytes species, main effects were analyzed separately. Recovery rate and number of oocytes was higher on cattle compared to buffaloes (35.0+8.6% and 1.4+0.3 vs. 10.0±4.6% and 0.5±0.2, respectively; p<0.05); no effect of E2 treatment was observed on recovery rate and number of oocytes (29.8±9.0% and 1.3±0.4 vs. 16.9±6.1% and 0.7±0.2, respectively; p>0.05); the number of buffaloes and bovine oocytes recovered were similar (1.4±0.4 and 0.6±0.2, respectively; p>0.05). Oocytes recovery rate showed a trend (P=0.07) to be higher when buffalo oocytes were implanted when compared to bovine oocytes (35.2±9.2% vs. 12.9±5.4%). Present results suggest that oocyte transport by the oviduct of buffaloes and bovine was not dependent on oocytes species or E2 supplementation to the culture medium. Keywords: MOET. Oocytes. Estradiol. In vitro. Bufaloes. Resumo O presente estudo foi realizado para verificar se a elevação das concentrações plasmáticas de estradiol durante os tratamentos superovulatórios afeta o transporte dos oócitos em fêmeas bubalinas, bem como se a qualidade inferior dos oócitos de búfalos e/ou alguma diferença funcional no oviduto destes animais é responsável pela baixa taxa de recuperação de embriões em búfalas superovuladas quando comparadas a vacas submetidas ao mesmo tratamento. Foram utilizados 10 ovidutos de búfalas e 15 de vacas, tratadas para a indução de ovulação única. Os ovidutos foram colocados em placas de Petri e receberam os seguintes tratamentos: sem E2 e inseridos com 5 oócitos de búfalas (G-BufBuf e G-BovBuf); sem E2 e com 5 oócitos de vacas (G-BufBov e G-BovBov); com E2 e com 5 oócitos de búfalas (G-BufE2Buf e G-BovE2Buf); e com E2 e com 5 oócitos de vacas (G-BufE2Bov e G-BovE2Bov; fatorial 2x2x2). Posteriormente, foram incubados por 24h e, após esse período, foram lavados para a recuperação e contagem dos oócitos. Como não foi verificado efeito de interação, foram analisados os efeitos principais. O número e a taxa de recuperação de oócitos foi maior em ovidutos de vacas que de búfalas (1,4±0,3/35,0±8,6% vs. 0,5±0,2/10,0±4,6%; P<0,05). Foi verificado que o tratamento com ou sem E2 não interferiu no número e na taxa de recuperação de oócitos (1,3±0,4/29,8±9,0% vs. 0,7±0,2/16,9±6,1%; P>0,05). Não foi verificada diferença no número de oócitos de búfalas ou de vacas recuperados (1,4±0,4 e 0,6±0,2; P>0,05). Observou-se também que houve tendência (P=0,07) de maior taxa de recuperação de oócitos de búfalas que de vacas (35,2±9,2% vs. 12,9±5,4%). Os dados são indicativos de que o transporte de oócitos pelo oviduto de búfalas e de vacas independe da espécie do oócito e não é influenciado pelo E2. Palavras-chave: MOET. Oócitos. Estradiol. In vitro. Búfalos. Correspondent Author: Postal Code: 11900-000 – PoBox: 122 - Registro - SP Nelcio Antonio Tonizza de Carvalho Phone: (13) 3822-9068 / e-mail: nelcio@apta.sp.gov.br Unidade de Pesquisa e Desenvolvimento de Registro Pólo Regional do D.S.A. do Vale do Ribeira – DDD - APTA Received: 25/05/2010 Adress: Rodovia Regis Bittencourt (BR116), Km435, Bairro Ribeirão Vermelho Approved: 15/08/2012 Braz. J. Vet. Res. Anim. Sci., São Paulo, v. 49, n. 4, p. 277-284, 2012 278 and the endossalpinge ciliated cells during ovulations. Introduction The birth of buffaloes using Multiple Ovulation and Embryo Transfer (MOET) has been reported in Brazil and other countries1,2. However, in large-scale, the use of such technique is limited due to the low embryo recovery rate1,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21. Buffalo females respond to superovulatory treatments, however, less embryos are recovered comparing to bovine females. Baruselli22 verified that the buffalo species respond to the superovulatory treatments with ovulation rates of 62.8%, similarly to that observed by Stock, Ellington and Fortune23 in the bovine species. However, only 34.8% of ovulations of buffaloes subjected to superestimulation of the follicular growth resulted in the recovery of embryonic structures24. Conversely, Adams25 reported recovery rates between 63 and 80% in superovulated bovine females. These differences between the embryo recovery rates in these species may be associated to failure in the capture process and/or oocytes transport by the oviduct24,26. Bellve and McDonald27 verified in superovulated The absence of this interaction promotes failure in the oocytes capture, because, according to Hunter29, during the ovulation, the oviduct fluid flows directly to the abdominal cavity. Therefore, the understanding of physiological processes that involve E2 profile, as well as the quality and transport of oocytes, is extremely important in order to improve MOET efficiency in buffaloes. The aim of the present study was to test the hypothesis that in vitro oocyte transport through the oviduct of buffalo and crossbred beef cows is influenced by oviduct species and/or by gamete species and/or by the presence of E2 in culture medium. Towards this end, buffalo and bovine oviducts collected during the post-ovulatory period (i.e. period of higher development and activity of oviduct cilia29) and incubated in culture medium supplemented or not with E2, received bovine or buffalo oocytes inserted through the infundibulum. Material and Method small ruminant that oviduct retro peristalsis occurred This project is in accordance with Ethical Principles due to high concentration of estradiol (E2), which in Animal Research adopted by Bioethic Commision could explain the low embryo recovery rate found in of the Faculty of Veterinary Medicine and Animal buffaloes submitted to the same treatment. Addition- Science of University of São Paulo and was approved ally, Kolle et al. found that oviductal epithelium is “ad referendum” (protocol number 175/2002). The able to select live oocytes (mature and immature) in first part of the experiment was performed at the Ca- mares; however, as soon as the oocyte is degenerated, choeirinha livestock (Poços de Caldas - MG, Brazil) it floats in the oviductal lumen. This implies that the in July 2005 using six years old crossbred bovine fe- oviduct is able to select live oocytes. Moreover, the males (n=15; Bos indicus x Bos taurus), showing body fragile connection between oocytes and granulosa score condition > 4.0 (scale of 1–5, where 1=very thin cells may negatively influence transport of these gam- and 5=very fat). The second part of the experiment etes. According to Hunter , Lam et al. and Talbot, was conducted in January 2006, at the Barra do Cap- Shur and Myles31, the pick up process by the infun- inzal farm (Registro - SP, Brazil), using six years old dibulum, as well as the transport of oocytes across the Murrah buffalo cows (n=10), also showing body score oviduct depend on the quality of the female gamete. condition > 4.0. These animals were maintained on a 28 29 30 The elevation on the estradiol/progesterone proportion may damage the interaction between the oocytes Braz. J. Vet. Res. Anim. Sci., São Paulo, v. 49, n. 4, p. 277-284, 2012 Brachiaria decumbens pasture with free access to water and mineralized salt. 279 The experiment was performed in a 2x2x2 factorial arrangement; the factors were oviduct species (buffalo vs. cattle), E2 treatment (with vs. without) and oocyte species (buffalo vs. cattle). Estrous cycles synchronization was performed in all animals using an intravaginal progesterone device (P4; DIB; MSD Animal Health, Brazil) and 2mg of Estradiol Benzoate (IM; Benzoato de Estradiol; Tecnopec, Brazil) on Day 0 (AM). Devices were removed after 8 days (Day 8, AM), when animals received a single injection of 0.15mg of prostaglandin (IM; dcloprostenol; Prolise; Tecnopec, Brazil) and 400IU of eCG (IM; Novormon; MSD Animal Health, Brazil). Twenty four hours later (Day 9, AM), animals received 25mg of GnRH (IM; Lecirelina; Gestran-Plus; Tecnopec, Brazil). Females were slaughtered 48 hours after ovulation induction (Day 11, AM; Figure 1). Bovine females were slaughtered at the Frigonossa Abattoir (Poços de Caldas - MG), and buffalo females were slaughtered at the Frivale Abattoir (Cajati - SP). Females were eviscerated and the reproductive tract removed. The ipsilateral and contralateral oviducts to the ovary containing the ovulatory follicle were recovered from the ovaries containing one or more CLs and placed into an insulated container in D-PBS solution (glucose 1000 mg, sodium pyruvato 36 mg, penicilin 1.000.000 IU, streptomycin 50 mg and amphotericin B 250 µg), at 38.0oC. The container was transported to the laboratory in less than 5 hours, which allowed the temperature to remain between 37.0 and 38.0oC. In order to optimize and reduce the number of slaughtered animals, both oviducts, ipsilateral and contralateral to the ovulated ovary, were used. According to Havlicek et al.32, the oviduct contralateral to the ovulated ovary may fulfill the necessary requirements to the embryo culture in the bovine species. Therefore, functional modifications of the ipsilateral oviduct may occur also in the contralateral oviduct since the activities of both oviducts are partially coordinated by hormonal mechanisms affected by systemic concentrations of E2 and P433. Bovine oocytes were collected from 20 ovaries obtained from abattoirs; buffalo oocytes were collected by in vivo follicular aspiration from 15 buffaloes. In the laboratory (Unidade de Biologia Celular, Centro de Sanidade Animal, Instituto Biológico, São Paulo - SP), both buffalo and cattle cumulus oocyte complexes (COCs) were washed using TCM199 culture medium with HEPES added to 10% bovine fetal serum, 10µl of piruvate, 0.2mM and 25µl of gentamicine. COCs were then matured in 4.5ml of TCM199 medium with bicarbonate added to 10% bovine fetal serum, 10µl of piruvate, 0.2mM and 25µl of gentamicine, 5µl of estradiol, 5µl of FSH and 50µl of LH for 24 hours in a stove/oven with 38.5ºC; 5% of CO2 and 92% of humidity. Only matured oocytes (i.e. oocytes showing expanded cumulus cells) were used, totalizing 80 cattle and 70 buffalo oocytes. The considered criteria for the CCOs evaluation were the number of layers and the compactation level of the Cumulus cells, as Figure 1 - Schematic diagram of treatments to synchronize ovulation in buffaloes. EB = 2.0 mg Estradiol Benzoate; PGF2a = 0.15 mg d-cloprostenol; eCG = 400 IU equine Chorionic Gonadotropin; GnRH = 25 µg Buserelin acetate Braz. J. Vet. Res. Anim. Sci., São Paulo, v. 49, n. 4, p. 277-284, 2012 280 well the cytoplasm aspect as much as the color, homo- ment (E2 vs. control), as well as the second and third geneity and integrity . order interactions, were determined by PROC GLM. 34 In the laboratory, oviducts were washed in Hanks Differences between treatments were analyzed us- balanced solution (HBSS) supplemented with 25mM ing parametric tests (GLM procedure for each factor of HEPES. Afterwards, each oviduct was placed in separately or LSD when combining factors) and non- a Petri dish (150 x 25mm) and, in the anterior por- parametric tests (Wilcoxon), according to the residue tion of the infundibulum, 5 matured buffalo oocytes normality (Gaussian distribution) and variance homo- (G-BufBuf, G-BufE2Buf, G-BovBuf and G-BovE- geneity. Number of recovered oocytes and recovery 2Buf) and 5 matured cattle oocytes (G-BufBov, G- rate did not have randomly distributed residuals and BufE2Bov, G-BovBov and G-BovE2Bov), together no transformation was effective; therefore, data were with 50ml of maturation medium, were inserted us- analyzed using non-parametric procedures. A prob- ing a 1ml syringe connected to a Tom Cat catheter. ability value of p<0.05 was considered significant. Re- All oviducts were immersed in 100ml of maturation sults were reported as untransformed means ± S.E.M. medium, supplemented (G-BufE2Buf, G-BufE2Bov, G-BovE2Buf and G-BovE2Bov) or not (G-BufBuf, GBufBov, G-BovBuf and G-BovBov) with 10ng/mL of 17b-estradiol (Sigma, Brazil; Table 1). Results Recovery rate and number of oocytes were higher in Each Petri dish, containing the oviducts with the oo- Group G-BovE2Buf (p<0.05; Table 2) when compared cytes, was incubated for 24 hours, at 38.5°C in a hu- to Groups G-BufBuf, G-BufBov, G-BufE2Bov. On the midified 5% CO2 incubator. After incubation, oviducts other hand, no differences were found between the first were washed with 40ml of washing medium (D-PBS) and Groups G-BufE2Buf, G-BovBuf and G-BovE2Bov supplemented with 1% bovine fetal serum inserted in (p>0.05; Table 2). the infundibulum with an intramammary cannula. The Considering that no interactions were found be- effluent of each oviduct was collected separately, placed tween oviducts species, oocytes species and E2 treat- into 100 x 20mm Petri dishes, and evaluated through ment for any variables, the main effects of each factor stereomicroscope (SMZ-2T) at 20-30X magnification was tested. The oocytes number and recovery rates in order to quantify the number of oocytes. were higher on bovine oviducts when compared to All data were evaluated using SAS System for Win- the buffalo (p<0.05; Table 2). However, buffalo oo- dows35. The effects of oviduct species (buffalo vs. cat- cytes tended to be recovered (p=0.07) in higher rates tle), oocytes species (buffalo vs. cattle) and E2 treat- than cattle oocytes. No effect of E2 supplementation Table 1 – Experimental groups according to species, treatment and number of oocytes Groups G-BufBuf G-BufBov G-BufE2Buf G-BufE2Bov G-BovBuf G-BovBov G-BovE2Buf G-BovE2Bov Specie (number of oviducts) buffaloes (n=3) buffaloes (n=4) buffaloes (n=3) buffaloes (n=4) cattle (n=4) cattle (n=4) cattle (n=4) cattle (n=4) Braz. J. Vet. Res. Anim. Sci., São Paulo, v. 49, n. 4, p. 277-284, 2012 Treatments without E2 without E2 with E2 with E2 without E2 without E2 with E2 with E2 Number of oocytes matured (animal species) 5 (buffaloes) 5 (cattle) 5 (buffaloes) 5 (cattle) 5 (buffaloes) 5 (cattle) 5 (buffaloes) 5 (cattle) 281 G-BufE2Bov (n=4) 1.5±0.5a.b G-BovBuf (n=4) 0.5±0.3b G-BovBov (n=4) 2.2±0.8a GBovE2Buf (n=4) 1.5±0.64a.b G-BovE2Bov (n=4) 0.03 0.09 0.07 0.63 0.71 0.63 0.43 cytes transit in buffalo cows could not be confirmed in vitro. Results of the present experiment do not agree with previous experiments showing differences on the Table 2 - Number and recovery rate of buffalo and cattle of oocytes introduced (in vitro) through the oviduct of buffaloes and cows and cultured in the presence or abscence of E2 in the culture medium G-BufE2Buf (n=3) 0.2±0.2b Therefore, our hypothesis that E2 would influence oo- Effects (P > F) G-BufBov (n=4) 1.3±0.9a.b independently on the oocytes and oviducts species. Mean ± SEM G-BufBuf (n=3) 0.2±0.2b effect on the rate and number of recovered oocytes, Specie Treatment Oocyte Sp.*Treat. Sp.*Ooc. Treat.*Ooc. Sp.*Treat.*Ooc. 0.3±0.3b 0.46 falo and cattle oocytes. E2 supplementation to the culture medium had no Dependent variables Number of oocytes recovered 0.80 ducts, leading to the low recovery rates of both buf- 0.41 which could have caused modifications on the ovi- 0.74 could be explained by the culture interval (24 hours), 0.06 expected a recovery rate similar to this value. This 0.19 insemination (FTAI) are close to 50%37,38,39,40,41,42, we 0.02 synchronization of ovulation for fixed time artificial 31.7±18.3a.b vine and buffalo females submitted to protocols for 55.0±22.2a lower than expected. Because conception rates of bo- 10.0±5.8b (35.0±8.6% and 10.0±4.6%, respectively), results were MAIN EFFECTS in the cattle oviducts when compared to the buffalo’s 43.3±15.7a.b Oocyte of Cattle Despite the higher oocyte recovery rate observed 5.0±5.0b Oocyte of Buffalo oocytes recovery. 26.7±17.6a.b With Estradiol 0.6±0.2 flushing, resulting in decreased number and rate of 5.0±5.0b Without Estradiol 1.4±0.4 the disintegration of some oocytes during the oviduct 6.7±6.7b Oviduct of Cattle 1.3±0.4 ing of the uterine ostium. This, in turn, could cause Oocyte recovery rate (%) Oviduct of Buffalo 0.7±0.2 pared to bovine cows, which could cause the narrow- 1.4±0.3a buffaloes showed a thicker muscular layer when com- 0.5±0.2b a histology examination, verified that the isthmus of Number of oocytes recovered ter in vitro flushing of buffalo oviducts. Carvalho36, in 12.9±5.4y which could have compromised oocyte recovery af- 35.2±9.2x difference between cattle and buffalo oviducts exist, 29.8±9.0 (Table 2). It is likely that anatomic and/or functional 16.9±6.1 buffalo; regardless E2 treatment and oocytes species 35.0±8.6a the oviducts of bovine females when compared to the 10.0±4.6b the number and rate of oocyte recovery were higher in Oocyte recovery rates (%) Results of the present experiment demonstrated that Means within a line with different superscripts differ (p<0.05) Means within a line with different superscripts indicate a trend (p=0.07) Discussion a. b and rate of oocyte recovery (p>0.05; Table 2). x. y to the culture medium was observed for both number Braz. J. Vet. Res. Anim. Sci., São Paulo, v. 49, n. 4, p. 277-284, 2012 282 quality of oocytes depending on the species. According to Gasparrini43, due to a more fragile connection between the granulosa cells found in buffaloes COCs, buffalo oocytes are considered to exhibit an inferior quality when compared to cattle oocytes. Therefore, we expected a higher recovery rate of cattle oocytes, instead of the trend found for a higher recovery rate for buffalo oocytes in the present experiment. We believe that oocyte transit throughout the oviduct of both cattle and buffalo cows do not depend on oocyte quality. Probably, oocyte quality would play an important role on oocyte transport after follicular collapse and further pick up by the infundibulum in vivo. Baruselli et al.44 observed increased embryonic structures recovery rates in superovulated buffaloes previously treated with rBST, which could be related to an improved oocyte quality. On the other hand, Carvalho et al.45 found low embryonic structures recovery rate two and five days after the first AI in buffaloes submitted to a MOET protocol. The authors suggested that oocyte pick up by the infundibulum but not the oocyte transport through the oviduct would be the key factor to the lower recovery rate in buffaloes. Possibly, within the oviduct, the number of cumulus cells, as well as the degree of adhesion between those cells and the oocyte, renders no influence on oocyte transport. To support this possibility, according to Lam et al.30, the cumulus cells are eliminated during oocyte transit from the infundibulum to the fecundation point. Conclusion Results of the present experiment indicate that, opposite to our hypothesis, in vitro oocyte transport through the oviduct of buffalo and crossbred beef cows is not influenced by gamete species or by the presence of 17b-estradiol in the culture medium. Acknowledgments We thank the Barra do Capinzal and Cachoeirinha farms for providing the animals used in the present experiment and FAPESP (Fundação de Apoio à Pesquisa do Estado de São Paulo) for the financial support for research trials and scientific development in buffalo breeding. References 1. BARUSELLI, P. S. Basic requirements for artificial insemination and embryo transfer in buffaloes. 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