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Scholars
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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.  

Betthauser J, Forsberg E, Augenstein M, Childs L, Eilertsen K, Enos J, 
Forsythe T, Golueke P, Jurgella G, Koppang R, Lesmeister T, Mallon 
K, Mell G, Misica P, Pace M, Pfister-Genskow M, Strelchenko N, 
Voelker G, Wat S, Thompson S, Bishop M (2000). Production of 
cloned pigs from in vitro systems. Nat. Biotechnol. 18: 1055-1059. 

  
  

 
 

 
Chen XY, Liu D, Li QW (2003). Collection method and in vitro 

maturation of pig acolytes of ovaries derived from slaughterhouse[J]. 
Yunnan J. Anim. Sci. Vet. Med. 19: 75-78.  

Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce 
de León FA, Robl JM(1998). Cloned transgenic calves produced from 
nonquiescent fetal fibroblasts. Science, 280: 1256-1258.  

Galli C, Duchi R, Moor RM, Lazzari G (1999). Mammalian leukocytes 
contain all the genetic information necessary for the development of 
a new individual. Cloning, 1: 161-170.  

Ge L, Sui HS, Lan GC, Liu N, Wang JZ, Tan JH (2008). Coculture with 
cumulus cells improves maturation of mouse oocytes denuded of the 
cumulus oophorus: observations of nuclear and cytoplasmic events. 
J. Fertil. Steril. 90: 2376-2388.  

Krisher RL, Brad AM, Herrick JR, Sparman ML, Swain JE (2007). A 
comparative analysis of metabolism and viability in porcine oocytes 
during in vitro maturation. Anim. Reprod. Sci. 98: 72-96.  

Kono T, Kwon OY, Ogawa M, Nakahara T (1991). Development of 
mouse oocytes receiving embryonic nuclei and thymocytes, J. 
Theriogenol. pp. 22-35.  

Kono T, Kwon OY, Watanabe T, Nakahara T (1992). Development of 
mouse enucleated oocytes reciving embryonic a nucleus from 
different stages of the second cell cycle. J. Reprod. Fetil. 96: 275-
278.  

Mtango NR, Varisanga MD, Yajuan D, Suzuki T (2002). Development to 
blastocyst stage of pig oocytes matured fertilized and 
electroactivated in vitro J. Arch. Tierz. Dummerstorf. 232: 178-183.  

Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada 
H, Perry AC (2000). Pig cloning by microinjection of fetal fibroblast 
nuclei. Scinece, 289: 1188-1190.  

Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, 
Boone J, Walker S, Ayares DL, Colman A, Campbell KH (2000). 
Cloned pigs produced by nuclear transfer form adult somatic cell. 
Nature, 407: 85-90.  

Shirazi A, Sadeghi N (2007). The effect of ovine oocyte diameter on 
nuclear maturation. J. Small Rumin. Res. 69: 103-107.  

Somfai T, Ozawa M, Noguchi J, Kaneko H, Kuriani Karja NW, Farhudin 
M, Dinnyés A, Nagai T, Kikuchi K (2007). Developmental 
competence of in vitro-fertilized porcine oocytes after in vitro 
maturation and solid surface vitrification: effect of cryopreservation on 
oocyte antioxidative system and cell cycle stage. J. Cryobiol. 55: 115-
126.  

Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R 
(1998). Full-term development of mice from enucleated oocytes 
injected with cumulus cell nuclei. Nature, 394: 369-374.  

Wells DN, Misica PM, Tervit HR (1999). Production of cloned calves 
following nuclear transfer with cultured adult mural granulosa cells. 
Biol. Rep. 60: 996-1005.  

Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH (1997). Viable 
offspring derived from fetal and adult mammalian cells. Nature, 385: 
810-813.  

Wolf DP, Mitalipov S, Norgren Jr. RB (2001). Nuclear transfer 
technology in mammalian cloning. Arch. Med. Res. 32: 609-613.  

Zhang X, Miao YL, Zhao JG, Spate L, Bennett MW, Murphy CN, 
Schatten H, Prather RS (2010). Porcine oocytes denuded prior to 
maturation can develop the to the blastocyst stage if provided a 
cumulous cell-derived co-culture system. J. Anim. Sci. 9: 25-27. 


