Does the follicular fluid obtained from bovine follicles of different diameters influence oocyte development?/O fluido folicular obtido de foliculos de diferentes diametros influencia o desenvolvimento de oocitos bovinos?/El liquido folicular obtenido de foliculos de diferentes tamanos influencia el desarrollo de ovocitos bovinos?
The in vitro maturation (IVM) system is broadly applied for in vitro production (IVP) of embryos in domestic animals and humans. However, there is still a wide variation in blastocyst developmental rates of in vitro matured oocytes. This may be explained by inappropriate cytoplasmic maturation (I). It is known that the follicular fluid (FF) influences the ability of in v/iro-matured bovine oocytes to acquire developmental competence (2). The FF contains, among other specific components, steroids and glycoproteins synthesized by the theca and granulosa cells and it is believed to be the source of nutritional and developmental support of the oocytes (3). Follicular and oocyte maturation are parallel events that are functionally related (4). The concentration of FF, and the timing of addition of FF to the maturation medium could influence programming of the maturation process (5).
The aim of this study was to evaluate the effect of the supplementation of maturation media with FF aspirated from bovine follicles of different diameter, on developmental capacity of bovine oocytes fertilized in vitro.
MATERIALS AND METHODS
Ovaries were collected at an abattoir and transported within 2 hours after slaughter to the laboratory, at 30[degrees]C, in a 0.9% NaCl solution (Embryolab, UFSM, Santa Maria/RS Brazil) containing l00mg/mL Streptomycin and 50UI/mL Penicillin-G potassium salt.
Prior to aspiration of FF the follicles were dissected, the diameter measured by the use of a micrometer, and allocated into two groups according to the follicular diameter: small (<8 mm) and large (>8 mm) follicles. Aspiration of FF was performed using a 21-gauge needle connected to a vacuum pump (II mL/min, Nevoni-Equip.Med.Hosp.Ltda, Sao Paulo, SP, Brazil). The FF of several follicles was mixed to build a pool of each follicular diameter. Collins and Wright (6) reported that some factors in FF that improve bovine oocyte maturation may include heat-labile proteins inactivated by heat treatment or factors removed by filtration, thus the follicular fluid was only centrifuged and frozen at -20[degrees]C for later use in the maturation medium.
Oocyte collection was performed from follicles between 2 and 8mm in diameter. The follicles were aspirated using a 21-gauge needle in a 15 mL collection tube with suction provided by a vacuum pump. After collection, oocytes were held in FF for searching and selection under a stereomicroscope (Carl Zeiss Jena GmbH, Jena, Germany). Oocytes of quality I and II, with compact cumulus mass and uniform ooplasm were morphologically selected according to De Loos et al. (7). The oocytes were randomly allocated into one of five groups of 25 oocytes each. Oocytes were washed in 4 drops of TCM-HEPES [modified TCM199; (GIBCO BRL, 31100-27, Grand Island, NY, USA) with 5.95mg/mL HEPES (SIGMA, H7006), 0.025mg/mL sodium pyruvate (SIGMA, P4562), 2.2 mg/mL NaHC[0.sub.3] (SIGMA, S5761) and 10% Estrous Mare's Serum (EMS; Embryolab/UFSM, Santa Maria, RS, Brazil)] for the control group or 20% FF for each follicular diameter group. Unless otherwise indicated, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
In vitro maturation (IVM) was performed in 400uL drops of TCM-199 (GIBCO Cat. 31100-035), Hepes (SIGMA, H6I47) and 0.01UI rFSH-h/mL (L1930300; Serono Pharma S.p.a, Bari, Italy), using four-well culture plates (Nunc Brand Prod., Roskilde, Denmark). Oocytes were randomly distributed into five groups of maturation. In the group EMS (n=147) oocytes were matured for 24h with 10% EMS. In groups Small follicular fluid (SFF) (n=149) and Large follicular fluid (LFF) (n=147) oocytes were matured for 24h in the presence of 20% of SFF and LFF, respectively. In the group SFF+LFF (n=149) maturation was performed for 12h in the medium with SFF followed by 12h with LFF. In the group LFF+EMS (n=144) oocytes were matured for 12h in the medium with LFF and for 12h in the presence of EMS. Oocytes were matured at 39[degrees]C, in a humidified 5% CO2 in air atmosphere (W.C.Heraeus GmbH. Hanau, Germany) for 24 hours.
The in vitro fertilization and culture were performed according the methodology described by Rauber et al. (8). From the second day after fertilization onwards, the embryos were cultured in gasified plastic bags with 5%C02, 5%02 and 90% N2 held in an incubator (W.C Heraeus GmbH, Hanau, Germany) at 39[degrees]C. In vitro maturation, fertilization and culture were performed simultaneously in six replicates.
Cleavage, blastocyst and hatching rates were evaluated on Day 2, 7 and 9, respectively. Data were analyzed by GLM procedure (SAS 9.2). Percentages were submitted to the arcsine square root transformation before being analyzed through GLM procedure (SAS 9.2). Treatments were compared using the Tukey's test at a 5% probability level.
No treatment effect was observed on the maturation and fertilization. The cleavage and hatching rates on Day 2 and Day 9 were similar among treatments, respectively (Table 1). Blastocyst yield on Day 7 was lower for oocytes matured in medium supplemented with small follicular fluid compared to oocytes matured with EMS and to those matured in follicular fluid derived from large follicles (P < 0.05).
It has been reported that bovine oocytes maturation with 30% and 60% follicular fluid has a detrimental effect on embryonic development (5, 9). In addition, undiluted follicular fluid (100%) has been shown to be unsuitable for maturation of bovine oocytes (4, 10). On the other hand, follicular fluid added to the maturation media at 10% or 20% improves the developmental capacity of bovine oocytes (2, 5, 11).
In the present study, blastocyst development was higher when the maturation occurred with LFF compared to SFF (P < 0.05). This may be explained by the possibility that FF from follicles larger than 8mm in diameter contains substances that simulates the resumption of meiosis and cytoplasmic maturation. During follicular growth, the endocrine and other biochemical profiles (2, 12, 13) of the follicular fluid undergo important changes for oocytes maturation and achievement of developmental ability after fertilization (12).
The group of oocytes treated with SFF resulted in lower cleavage (P > 0.05) and blastocyst yield (P < 0.05) than LFF and EMS groups. Ali et al. (14) observed that supplementation of completely defined maturation medium with 5% of bovine FF derived from competent follicles (>8mm) led to a higher proportion of blastocysts compared to FF derived from small follicles (2 to 5mm) or BSA-V, suggesting that FF from follicles >8mm produces and releases signals that are favourable to the acquisition of developmental competence of immature oocytes. The hatching blastocyst yield was lower (P > 0.05) for oocytes fertilized in group treated with LFF. Although embryo development to the blastocyst stage was supported by diluted follicular fluid from large follicles, its influence was not superior to diluted serum for in vitro maturation of bovine oocytes, contrasting with Elmileik et al. (5) who observed significantly higher blastocyst and hatching yield of oocytes matured in the 10% FF group compared to the control group with 10%o estrous cow's serum.
The follicular diameter influenced the capacity of in vitro matured oocytes to acquire developmental competence. As the supplementation of maturation media with FF derived of small (<8 mm) and large follicles (>8mm) did not inhibit the developmental capacity of bovine oocytes, it could be used as an alternative to serum for in vitro production of embryos.
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(2.) Sirard MA, Roy F, Patrick B, Mermillod P, Guilbault LA. Origin of the follicular fluid added to the media during bovine IVM influences embryonic development. Theriogenology. 1995;44:85-94.
(3.) Gordon I. Oocytes recovery and maturation. In: Laboratory production of cattle embryo. Cambridge: CAB International, University Press; 1994. p.30-142.
(4.) Choi YH, Takagi M, Kamishita H, Wijayagunawardane MPB, Acosta TJ, Miyazawa K, et al. Developmental capacity of bovine oocytes matured in two kinds of follicular fluid and fertilized in vitro. Anim Reprod Sci. 1998;50:27-33.
(5.) Elmileik AMA, Maeda T, Terada T. Higher rates of development into blastocyst following the in vitro fertilization of bovine oocytes matured in a medium supplemented with the fluid from large bovine follicles. Anim Reprod Sci. 1995; 38:85-96.
(6.) Collins AR, Wright RW. Effects on embryo development of heat treatment and filtration of bovine follicular fluid used to supplement IVM medium. Theriogenology. 1995;43:89.
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(8.) Rauber LP, Alves DF, Figueiro DF, Brum DS, Hilgert F, Bernardi ML, et al. Desenvolvimento embrionario de oocitos bovinos mantidos em fluido folicular bovino de foliculos de diferentes diametros. Braz J Vet Res Anim Sci. 2003;40: 169-77.
(9.) Kim KS, Mitsumizo N, Fujita K, Utsumi K. The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos. Theriogenology. 1996;45:787-99.
(10.) Averi B, Strobech L, Jacobsen T, Bruck BI, Greve T. In vitro maturation of bovine cumulus-oocyte complexes in undiluted follicular fluid: effect on nuclear maturation, pronucleus formation and embryo development. Theriogenology. 2003;59:987-99.
(11.) Romero-Arredondo A, Seidel GE. Effects of follicular fluid during in vitro maturation of bovine oocytes on in vitro fertilization and early embryonic development. Biol Reprod. 1996;55:1012-6.
(12.) Einspainer R, Schuster H, Schams D. A comparison of hormone levels in follicle-luteincysts and in normal bovine ovarian follicles. Theriogenology. 1993;40:181-8.
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Recebido em: 19/11/2009
Aceito em: 29/11/2010
Denis Faustino Alves 
Carlos Antonio Mondino Silva 
Mara Iolanda Batistella Rubin  *
Lucio Pereira Rauber 
Gilson Antonio Pessoa 
Fabricio Desconsi Mozzaquatro 
Mari Lourdes Bernardi 
 Mestre Medicina Veterinaria (fisiopatologia da reproducao) pela Universidade Federal de Santa Maria. Professor na Universidade Catolica Dom Bosco - UCDB. rf73S9@ncdb.br
 Professor Adjunto Departamento de Clinica de Grandes Animais, Universidade Federal de Santa Maria. camondinos (?//yahoo.com.br
 Professora Titular Departamento de Clinica de Grandes Animais, Universidade Federal de Santa Maria.
* Endereco para correspondencia: Mara Iolanda Batistella Rubin. Laboratorio de Embriologia Animal - UFSM, Predio 97 Bloco 4 Sala 428 - Hospital Veterinario, Universidade Federal de Santa Maria-Av. Roraima, 1000 Bairro Camobi. 97.105900 Santa Maria-RS Fone: 55 55 3220 8501. email@example.com
 Doutor pela Universidade Ludwig-Maximilian (LMU) em Munique-Alemanlia. lucio.raub er(aiemail.com.  Pos-graduando em Medicina Veterinaria (fisi o patologia da reproducao) pela Universidade Federal de Santa Maria. gilonpessoavet@.ahoo.com.br.
 Doutor em medicina Veterinaria pela UFSM. fmozzaquatroCwyahoo.com.br.
 Doutora em Reproducao Animal, Professora Associada da Universidade Federal do Rio Grande do Sul, Brasil
Table 1. Embryo development after in vitro fertilization of bovine oocytes matured in medium with estrous mare serum or bovine follicular fluid derived from follicles with different diameter. Cleavage Blastocyst Groups of matured CCO (n) % (n) yield % (n) 10% EMS (1) (n=147) 89,8 (a) (132) 38.1 (a) (56) 20%SFF (2) (n=149) 82.5 (a) (123) 20.8 (c) (31) 20%LFF (3) (n=147) 92.5 (a) (136) 34.0 (ab) (50) 20% SFF + 20%LFF (4) (n=149) 91.3 (a) (136) 39.5 (a) (59) 20% LFF + 10% EMS (5) (n=l44) 88.8 (a) (128) 29.9 (abc) (43) Hatched Groups of matured CCO (n) blastocyst yield % (n) 10% EMS (1) (n=147) 30.6 (a) (45) 20%SFF (2) (n=149) 22.8 (a) (34) 20%LFF (3) (n=147) 20 (a) (29) 20% SFF + 20%LFF (4) (n=149) 25.5 (a) (38) 20% LFF + 10% EMS (5) (n=l44) 25.6 (a) (37) (a), (b), (c) Means wiihin columns of groups according stage of embryo development without common superscripts differ (PO.05). (1) EMS: 24h in 10% estrous mare serum (2) SFF: 24h in 20% follicular fluid of follicles [less than or equal to]8mm in diameter (3) LFF: 24h in 20% follicular fluid of follicles >8mm in diameter (4) SFF+LFF: 12h in 20% SFF + 12h in 20% LFF (5) LFF+EMS: 12h in 20% LFF + 12h in 10% EMS Table 1. Embryo development after in vitro fertilization of bovine oocytes matured in medium with estrous mare serum or bovine follicular fluid derived from follicles with different diameter. Cleavage Groups of matured CCO (n) % (n) 24h in 10% estrous mare serum (n=147) 89,8 (a) (132) 24h in 20% follicular fluid of follicles 82.5 (a) (123) [greater than or equal to]8mm in diameter (n=149) 24h in 20% follicular fluid of follicles 92.5 (a) (136) >8mm in diameter (n=147) 12h in 20% SFF+12h in 20% LFF (n=149) 91.3 (a) (136) 12h in 20% LFF+12h in 10% EMS (0=144) 88.8 (a) (128) Blastocyst Groups of matured CCO (n) yield % (n) 24h in 10% estrous mare serum (n=147) 38.1 (a) (56) 24h in 20% follicular fluid of follicles 20.8 (c) (31) [greater than or equal to]8mm in diameter (n=149) 24h in 20% follicular fluid of follicles 34.0 (ab) (50) >8mm in diameter (n=147) 12h in 20% SFF+12h in 20% LFF (n=149) 39.5 (a) (59) 12h in 20% LFF+12h in 10% EMS (0=144) 29.9 (abc) (43) Hatched Groups of matured CCO (n) blastocyst yield % (n) 24h in 10% estrous mare serum (n=147) 30.6 (a) (45) 24h in 20% follicular fluid of follicles 22.8 (a) (34) [greater than or equal to]8mm in diameter (n=149) 24h in 20% follicular fluid of follicles 20 (a) (29) >8mm in diameter (n=147) 12h in 20% SFF+12h in 20% LFF (n=149) 25.5 (a) (38) 12h in 20% LFF+12h in 10% EMS (0=144) 25.6 (a) (37) (a), (b), (c) Means within columns of groups according stage of embryo development without common superscripts differ (P0.05).