In ternationa l Scholars Journa ls African Journal of Pig Farming ISSN 2375-0731 Vol. 6 (11), pp. 001-007, November, 2018. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper Effect of cumulus-oocyte complexes (COCs) culture duration on IN VITRO maturation and parthenogenetic development of pig oocyte Zai-Dong Hua1, Xin-Min Zheng2*, Qing-Xin Wei2, Hou-Qiang Xu1, Ya-Gang Wang2, Xi- Mei Liu2, Li Li 2, Hong-Wei Xiao2 and Xian-Feng Qiao2 1 Guizhou Key Laboratory of Animal Genetics, Breeding and Reproduction, College of Animal Sciences, Guizhou University, Guiyang 550025, China. 2 Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Hubei Academy of Agricultural Sciences, Wuhan 430064, China. Accepted 27 July, 2018 We investigated and optimized the cumulus-oocyte complexes (COCs) culture duration for pig oocyte IN VITRO maturation and produced a number of high-quality metaphase-II (M-II) oocytes for generation of parthenotes. The present study graded the COCs into levels A, B and C according to layers of cumulus cells, which were cultured IN VITRO for 24, 32, 38, 44, 48 and 54 h, respectively. Subsequently, the oocytes with extruded polar body at different time point were electrically activated. The rates of cleavage and blastocyst formation were assessed on day 2 and 7, respectively. The maturation rates of COCs of both level A and B arrived at the highest point at 44 to 48 h, which were statistically insignificant (P > 0.05), while level C COCs needed longer maturation time (54 h), for most of the oocytes to become mature. COCs of different levels were cultured IN VITRO for the same term, the maturation rates of levels A and B were not significantly different (P > 0.05), but both were significantly higher (P < 0.05) than that of level C. After parthenogenetic activation, there were no significant differences between the cleavage rates in the groups of 38 to 54 h maturation duration, whereas the developmental rate to blastocysts after 44 h (16.1%) and 48 h (16.5%) maturation duration were significantly higher than those from the other term groups (0, 2.2, 9.8 and 12.4% for 24, 32, 38 and 54 h, respectively, P < 0.05). COCs of levels A and B were more suitable for oocytes preparation IN VITRO, which led to a high maturation rate: 44 to 48 h duration was beneficial for maturation, cleavage and blastocyst formation. As for 38 h or less maturation duration, rates of blastocyst were extremely low although some oocytes could be seen with the polar body and cleaved to some extent (64.5% for 3 8h IVM), indicating that oocytes were not really matured. Therefore, the developmental maturation of oocytes could not be judged only by excluding the first polar body. Key words: Cumulus-oocyte complexes (COCs), IVM, duration, polar body, activation. INTRODUCTION Since the cloned sheep “Dolly” was produced by the transplantation of nucleus of sheep mammary gland cell *Corresponding author. E-mail: anbit20@163.com. Tel/Fax: 0086-027-87380647. Abbreviations: COCs, Cumulus-oocyte complexes; M-II, metaphase-II; SCNT, somatic cell nuclear transfer; NT, nuclear transfer; pFF, pig follicular fluid; BSA, bovine serum albumin. into an enucleated oocyte (Wilmut et al., 1997), the deve- lopment of somatic cell nuclear transfer (SCNT) has made great progress and success in generating cloned offspring in mouse, cattle and goat (Cibelli et al., 1998; Wakayama et al., 1998; Baguisi et al., 1999; Galli et al., 1999; Wells et al., 1999). However, SCNT in pigs has little progress and has been proven to be more difficult than in other livestock, so the somatic cell nuclear transfer offspring could not be produced successfully until 2000 (Betthauser et al., 2000; Onishi et al., 2000; Figure 1. Pig oocytes graded. Polejaeva et al., 2000). Animal cloning by nuclear transfer (NT) was dependent upon a range of factors, oocyte maturation has been seen as the most important step in SCNT procedure, which directly impact the development of cloned embryos and the efficiency of SCNT (Wolf et al., 2001). Therefore, the maturation conditions, especial- ly the duration of culturing in vitro should be optimized. Without enough maturation culturing may result in cyto- plasm and nucleus premature and cleavage abnor- malities. In turn, too long duration is likely to lead to age and apoptosis of the oocytes. The aim of this study was to investigate whether cumu- lus cells improved developmental ability of pig oocytes and screen the best lasting time for oocytes maturation in vitro for production of parthenotes. The oocytes with extruded polar body at different term were electrically activated using an Electro Cell Manipulator 2001 (BTX Inc., San Diego). The best term of oocytes maturation in vitro was assessed based on developmental ability to the cleavage and blastocyst stage. MATERIALS AND METHODS Chemicals DPBS was purchased from Gibco Company. PMSG and hCG were from Hormone Products Factory in Ningbo, China and other chemi- cal reagents used for oocyte maturation, activation and embryo culture were purchased from Sigma Aldrich Chemical Co. (Budapest, Hungary) unless otherwise noted. Preparation of pig follicular fluid The pig follicular fluid (pFF) was collected from follicles (3 to 8 mm diameter) of pig ovaries using a 10-ml disposable syringe with a 16- gauge needle. After centrifugation at 1600 rpm for 20 min at 4°C, the suspension was filtered through 0.22 µm syringe filters and stored at -20°C until use. Cumulus-oocyte complexes (COCs) collection and classification Pig ovaries were obtained from prepubertal gilts at a local slau- ghterhouse and transported to the laboratory in a thermal container at 28 to 37°C in 0.9% NaCl solution supplemented with penicillin (200 IU/ml) and streptomycin (150 IU/ml). The ovaries were washed 3 to 5 times with saline solution (28 to 37°C) until they were aspiration. Pig follicular fluid was aspirated from follicles (3 to 8 mm diameter) using a 18-gauge needle fixed a 10 ml disposable syringe. COCs were washed three times in DPBS and graded into level A, B and C according to cumulus cells (A: cumulus cells of 5 or more layers, B: cumulus cells of 3 to 5 layers, C: cumulus cells of 1 to 2 layers) (Figure 1). IN VITRO culture of pig oocytes After COCs were divided into level A, B and C, they were washed three times with a specified maturation medium-mTCM199 or NCSU-23 with pFF and hormone, and transferred to a 500 µl drop of the same medium which had been previously covered with warm paraffin oil in a polystyrene culture dish and equilibrated at 39°C in atmosphere of 5% CO2 in air overnight, and cultured for 24, 32, 38, 44, 48 and 54 h, respectively. During the first 22 h of maturation, the medium contained 10 IU/ml PMSG and 10 IU/ml hCG. Culturing for the subsequent 22 h was performed in the same medium without hormone supplementation. The oocytes cultured in different terms were observed under stereo microscope at 4 × 10 times, and expanded cumulus cells were completely removed by treatment with 0.1% hyaluronidase and pipetted. The oocytes with uniform cytoplasm and emission of the first polar body (Figure 2) were used for parthenogenetic activation. As in vitro culturing lasted longer, cumulus cells fell off on their own, the perivitelline space of oocyte was obviously widened, and some oocytes excluded the second polar body (Figure 3). Activation and embryos culture Oocytes with the first polar body were washed three times with activation fluid (0.3 M mannitol, 1 mM CaCl2, 0.5 mM MgSO4 and 0.05 mg/ml bovine serum albumin, BSA), and transferred to a Figure 2. COCs after 44 h of IVM. chamber containing the same fluid. Activation were induced by application of an AC pulse of 10 V for 5 s followed by a single DC pulses of 1.6 kV/cm for 60 µs using an Electro Cell Manipulator 2001 (BTX Inc., San Diego). After each activation treatment, the embryos were washed five times with NCSU-23 containing 4 mg/ml BSA, and were then cultured in the same medium which had been previously covered with paraffin oil in a polystyrene culture dish and equilibrated at 38.5°C in an atmosphere of 5% CO2. The rates of cleavage and blastocyst formation were assessed on day 2 and 7, respectively (Figure 4). Statistical analysis All data were obtained from five replicates. Percentage data were analyzed by chi-square tests. A probability of P < 0.05 was considered statistically significant. RESULTS Effect of IN VITRO culture duration on maturation of COCs of level A Cumulus-oocyte complexes (COCs) of level A were cultured in vitro for 24, 32, 38, 44, 48 and 54 h, respec- tively, oocytes with extruded polar body were counted. The results showed that the maturation rates of oocytes cultured for 44 (79.4%), 48 (83.2%) and 54 h (74.2%) were significantly higher than those of those cultured for 38, 32 and 24 h (32.6, 15.0 and 4.0%, respectively, P < 0.05) (Table 1). Effect of IN VITRO culture duration on maturation of COCs of level B Only oocytes with extruded polar body were selected. From Table 2, the in vitro maturation duration of 44, 48 Figure 3. COCs after 54 h of IVM. and 54 h led to more matured oocytes (the rates were 61.4, 65.7 and 63.9%, respectively) than those led to by duration of 24 ~ 38 h (3.1, 13.9 and 30.0, respectively for rates, P < 0.05) (Table 2). Effect of IN VITRO culture duration on maturation of COCs of level C After culturing cumulus-oocyte complexes of level C for 24 to 60 h, the number of oocytes with extruded first polar body was very small. Clearly, under the same conditions, the maturation rates of level A and B were significantly higher than that of level C. Meantime, the time that the maturation rates reached maximum (30.3%) extended to 54 h (Table 3). The maturation rate of different levels of COCs In the same culture conditions, the maturation rates of levels A and B were not significantly different at the same time point, respectively (P > 0.05), but both were signifi- cantly higher when compared with level C (P < 0.05) (Table 4). Also, from Figure 1, we can clearly see that COCs of levels A and B had better maturation ability than the COCs of level C. Effect of maturation duration of COCs on parthenogenetic development Oocytes with extruded polar body were activated after maturation culture of COCs for 24, 32, 38, 44, 48 and 54 h, respectively. The rates of cleavage and blastocyst formation were assessed after activation on day 2 and 7, Figure 4. Embryos of parthenogenetic activation. Table 1. In vitro maturation of COCs of level A. IVM duration (h) Number of COCs of level A Number of matured oocytes (%) 24 600 24 (4.0) a 32 640 96 (15.0) b 38 614 200 (32.6) c 44 985 782 (79.4) d 48 856 712 (83.2) d 54 620 460 (74.2) d respectively (Table 5). The results showed that the cleavage rates of oocytes after IVM for 38 to 54 h were significantly higher than for 24 and 32 h. Although there were no significant differen- ces in the cleavage rates within 38 and 54 h IVM groups, the developmental rate to blastocysts in 44 (16.1%), 48 (16.5%) and 54 h (12.4%) groups were significantly higher than at 38 h groups, as well as at 24 and 32 h groups (9.8, 0 and 2.2%, respectively, P < 0.05). From Figure 2, we can clearly see the trend that the partheno- genetic development went and it was better with the IVM duration extending. Table 2. In vitro maturation of COCs of level B. IVM duration (h) No. of COCs of level B No. of matured oocytes (%) 24 860 27 (3.1) a 32 920 128 (13.9) b 38 875 262 (30.0) c 44 914 586 (64.1) d 48 880 578 (65.7) d 54 906 579 (63.9) d No. = Number. Within the same column, values with same superscripts are not significantly different (P > 0.05). Values with different superscripts are significantly different (P < 0.05). Table 3. In vitro maturation of COCs of level C. IVM duration(h) No. of COCs of level C No. of matured oocytes (%) 24 420 7 (1.7) a 32 480 45 (9.4) b 38 498 92 (18.5) c 44 590 138 (23.4) c 48 504 142 (28.2) d 54 479 145 (30.3) d 60 405 106 (26.2) d No. = Number. Within the same column, values with same superscripts are not significantly different (P > 0.05). Values with different superscripts are significantly different (P < 0.05). Table 4. The maturation rate of different levels of COCs. COCs level Percent of matured oocyte (%) 24 h 32 h 38 h 44 h 48 h 54 h A 4.0 a 15.0 a 32.6 a 79.4 a 83.2 a 74.2 a B 3.1 a 13.9 a 30.0 a 64.1 a 65.7 a 63.9 a C 1.7 b 9.4 b 18.5 b 23.4 b 28.2 b 30.3 b Table 5. Effect of maturation duration of COCs on parthenogenetic development. IVM duration (h) No. of embryos treated No. of embryos cleaved (%) No. of blastocyst (%) 24 58 15(25.9) a 0(0) a 32 272 104(38.2) b 6(2.2) a 38 572 370(64.5) c 56(9.8) b 44 1595 1247(78.2) c 256(16.1) c 48 1506 1230(81.7) c 248(16.5) c 54 1253 968(77.3) c 155(12.4) b No. = Number. DISCUSSION It had been documented that in vitro pig oocyte matu- ration was accompanied by cumulus cell proliferation, which could regulate the transporation of a variety of chemical substances through oocyte membranes, and inhibit oocyte degradation by discharging adverse factors (Chen et al., 2003; Shirazi et al., 2007). In turn, the （% ） 100 80 C le av ag e 60 40 20 0 24 32 38 44 48 54 In vitro culture time（h） Figure 5. The maturation rate of COCs level A, B and C. 100 % ） 80 60 R at e（ 40 20 0 24 32 38 44 48 54 In vitro culture time（h） Figure 6. The rates of embryos cleavage and the blastocysts. A-level B-level C-level cleavage blastocyst growth factors released by pig oocytes can promote the development of cumulus cells (Krisher et al., 2007; Ge et al., 2008; Zhang et al., 2010). However, details underlying this mutual in vitro process is yet to be discovered. Here, we aimed to investigate the effect of granule cells upon in vitro maturation of cumulus-oocyte complexes (COCs) by grading COCs into A, B and C levels based on layers of cumulus cells. Our results suggested that cumulus cells affected the maturation of pig oocyte in vitro. In the process of producing embryos by SCNT, the first polar body (PBl) presentation was used as a sign of blind suction enucleation. Under this kind of protocol, it was usually considered that oocytes developmental matura- tion can be judged only by excluding the first polar body. In our experiments, some oocytes could be seen with the polar body when IVM was cultured for 24 to 32 h, but the rates of cleavage and blastocyst were extremely low. Even 38 h IVM duration was not enough which led to significant less blastocyst than 44 h or more, although the cleavage percent for 38 h group was substantial, these indicated that oocytes cultured for less than 38 h were not really matured. Unmaturation of oocytes may signifi-cantly affect subsequent embryo development by reducing both the developmental rate and total cell number in blastocysts. Yajuan et al. (2004) found that among the different ages of pig, eggs matured oocytes partheno- genetically developed with a great difference in rate, 48 h eggs obtained a high rate of development. In our study, oocytes with extruded first polar body were activated after IVM for 24, 32, 38, 44, 48 and 54 h, respectively. The results showed that although there were no significant differences in the cleavage rates for COCs culture in vitro for 38 to 54 h, the developmental rate to blastocysts of 44 and 48 h oocytes were significantly higher than of the other term groups (P < 0.05). So, oocytes cultured in vitro for 44 to 48 h were the best maturation duration and provided a number of high- quality M-II oocytes for production of parthenotes. If PBl emission was delayed, in other words, COCs were cultured longer in vitro, and PBl and metaphase chromo- some location would be deviated which could lead to decrease of enucleation rate (Kono et al., 1991, 1992) in SCNT. On the one hand, this kind of aged oocytes may also compromise further development by inducing egg’s free Ca 2+ increase, which leads to a lower level of matu- ration promoting factor MPF, and then starting oocytes’ uncompleted pre-activation (Mtango et al., 2002; Somfai et al., 2007). Conclusion Layers of cumulus cells have some influence on oocyte maturation; COCs of level A and B were more suitable for oocytes preparation in vitro, obtained a high maturation and cleavage rate. cumulus-oocyte complexes were cul- tured in vitro for less than 44 h and the rates of cleavage and blastocyst were extremely low, except for the cleavage rate for 38 h, although some oocytes could be seen with the polar body, indicating that oocytes were not really matured. At the same time, oocytes of the develop- mental maturation could not be judged only by excluding the first polar body. Oocytes cultured in vitro for 44 to 48 h had the best maturation time and provided a number of high-quality metaphase-II (M-II) oocytes for production of parthenotes. ACKNOWLEDGEMENTS This project was supported by Subject of International Cooperation Foundation (No.2009BFA012) and National Biological Cultivation of New Varieties in China (No.2008ZX08006-003; No.2008ZX08006-002; No.2008Z X08010-003; No. 2008ZX08011-004; No.2009ZX08011- 030B). This study was supported from the Project from Hubei Key Laboratory (2010ZD127). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. REFERENCES Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overström EW, Echelard Y (1999). Production of goats by somatic cell nuclear transfer. Nat. Biotechnol. 17: 456- 461. 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