The development potential of bovine embryo co-culture with Vero and Vero / BRL cells

The objective of this study was to evaluate the effect of two bovine embryo co-culture systems on calving rates. The embryos were co-cultured with Vero or Vero/BRL cells (mixed co-culture) until the blastocyst stage. A higher percentage of blastocysts was observed on Vero/BRL cells, 42.85%, compared with 30.41% on Vero cells (P≤0.05). The blastocysts from Vero/BRL and Vero cells were transferred to recipients. A higher rate of calving was obtained after transfer of embryos co-cultured with Vero cells (37.50%), compared with Vero/BRL cells (13.16%) (P≤0.01). A greater loss of pregnancy after transfer of embryos co-cultured with Vero/BRL cells was observed between 35 and 65 days. All calves were born naturally, healthy, and with normal weight. Vero cells better support cattle prenatal development than Vero/BRL cells. Probably Vero/BRL cells lead to epigenetic modifications that are responsible for early foetal resorption.


INTRODUCTION
In vitro production of bovine embryos has applications both in cattle breeding and in biotechnology for cloning, transgenics and creation of chimeras (Gordon, 2003).There are three frequently used systems for culture of bovine embryos from zygote to blastocyst.The most common system relies on sequential culture in several different media: SOFaa (Holm et al., 1999), KSOM (Liu and Foote, 1995), CR1 (Rosenkranz and First, 1994) and CZB (Rehman et al., 1994).Another system employs co-culture with different kinds of cells such as Vero cells (Pegoraro et al., 1998), BRL cells (Reed et al., 1994) or a combination of both (so-called "mixed co-culture") (Duszewska et al., 2000).A third system uses medium preconditioned by the above cells (Maed et al., 1996), but this system is rarely used.All of these systems have similar advantages and disadvantages (Gordon, 2003, review: Orsi andReischl, 2007).Although they are well-established, there is a need to determine the effect of each system on the developmental potential of embryos by transfer to recipients (Hansen, 2006).
In this study, two co-culture systems were compared to determine the developmental potential of bovine embryos after transfer to recipients.The first system was based on co-culture of embryos with Vero cells and the second coculture with both Vero and BRL cells (mixed co-culture).
Vero is a kidney epithelial cell line from the green monkey (Cercopithecus aetiops).The cells are derived from the kidney, which shares a common mesodermal origin with the genital tract.BRL cells are rat liver epithelial and parenchyma-like cells (Rattus norvegicus), which are of endodermal origin.
Bovine embryos have been successfully co-cultured with Vero cells, but the percentage reaching the blastocyst stage is lower than that of embryos co-cultured with BRL cells (Duszewska et al., 2000).According to some authors, the pregnancy rate after transfer of embryos developing on Vero cells is high (Menck et al., 1997).Hence, Vero and BRL cells were combined in an effort to enrich the environment for embryonic development and also to improve prenatal development.

In vitro maturation of bovine oocytes
Bovine ovaries were obtained from a slaughterhouse and transported within 2 h to the laboratory in phosphate-buffered solution (PBS) with 100 IU/ml streptomycin and 100 IU/ml penicillin at approximately 30°C.Cumulus-oocyte complexes (COCs) were collected by aspiration from follicles (2 to 6 mm in diameter) using a syringe with an 18-gauge needle and an air pump system.COCs were washed 3 times in TCM199 buffered with 25 mM Hepes supplemented with 10% FBS, 50 µmg/ml gentamicin sulphate, 100 IU penicillin and 50 µmg/ ml streptomycin, and adjusted to pH 7.4.The COCs were matured in TCM199 buffered with 25 mM Hepes supplemented with 10% FBS, 0.02 IU/ml pFSH, 1µmg/ml 17βb-oestradiol, 0.2 mM Na pyruvate and 50 µmg/ml gentamicin sulphate.A group of COCs (20 immature oocytes) was placed in one well of a 4-well dish and matured in 450 µml TCM199 buffered with 25 mM Hepes and supplemented with 10% FBS, 0.02 IU/ml pFSH, 1µmg/ml 17βb-oestradiol, 0.2 mM Na pyruvate and 50 µmg/ml gentamicin sulphate.The COCs were matured for 24 h at 38.5°C in 5% CO 2 in humidified air (Marguant-Le Guienne et al., 1989).

In vitro culture of bovine embryos
At 20 h post-insemination, the cumulus cells were removed and the zygotes washed in Menezo B2 medium (ART of CCD) supplemented with 10% FBS (GIBCO) and placed in 40-µml drops of Menezo B2 medium supplemented with 10% FBS under mineral oil.The zygotes were randomly allocated to one of the experimental groups: Vero and Vero/BRL.
Embryos were co-cultured until day 7, i.e. 168 h post insemination, at 38.5°C in 5% CO 2 in humidified air.During culture the medium was renewed twice (at 48 h, 20 µml of medium was removed and 20 µml of Menezo B2 supplemented with 10% FBS was added; and at 144 h, 20 µml of medium was removed and 20 µml of Menezo B2 without serum was added).

Culture of Vero and BRL cells
Frozen samples of established Vero and BRL cell lines (ATCC, Maryland, USA) were used to prepare respective Vero cell monolayers and BRL cell monolayers.Cell lines were thawed and cells seeded at a concentration of 1x10 6 /ml in 25 cm 2 culture flasks in the TCM 199 medium supplemented with 10% FBS, 50 µmg/ml gentamicin sulphate, 100 IU penicillin, and 50 µmg/ml streptomycin and cultured at 38.5ºC in 5% CO 2 in humidified air.The medium from both the Vero and BRL cultures was removed and replaced with fresh medium after 2 days.BRL cells (after 4 days of culture) and Vero cells (after 7 days of culture) were trypsinized (0.25% trypsin) for passages to prepare the monolayers and microdrops for culture of embryos.

Preparation of Vero cells in drops under mineral oil
Vero cells were placed at a concentration of 2x10 3 /10 µml into a microdrop (40 µml) of Menezo B2 (ART of CCD) supplemented with 10% FBS under mineral oil and cultured at 38.5ºC in 5% CO 2 in humidified air.The next day, the zygotes were placed in a microdrop.

Preparation of Vero/BRL cells in drops under mineral oil
Vero cells were first placed at a concentration of 1x10 3 /10µml into a microdrop (40 µml) of Menezo B2 (ART of CCD) supplemented with 10% FBS under mineral oil.After 30 min, BRL cells were added at the same concentration to the same drop, which was then cultured at 38.5ºC in 5% CO 2 in humidified air.One day later, the zygotes were placed in the microdrop.

Embryo transfer and offspring evaluation
Oestrus synchronization of the recipients was induced by injecting 2 ml (0.5 mg) of the prostaglandin F 2α -analogue, Cloprostenol (Bioestrovet-Vetoquinol, Gorzów, Poland) every 11 days.Seven to eight days after the standing heat, one fresh embryo was transferred to the uterine horn ipsilateral to the ovary displaying a corpus luteum.Embryos were transferred to recipients in Embryo Transfer Medium (BioLife Transfer Medium, Agtech Inc., USA).Recipients were monitored daily for heat behaviour and examined by ultrasound after 5 weeks and then monthly to confirm pregnancy.After calving, offspring were weighed and examined by veterinary doctors.

Statistical analysis
Rates of cleavage and blastocyst formation per zygote and rate of development after transfer to recipients were analysed by Chi-square tests.Birth weight is given as the mean value ± standard deviation.Probabilities (P-values) of less than 0.05 were regarded as statistically significant and less than 0.01 as highly statistically significant.

RESULTS
The development of zygotes co-cultured with Vero or Vero/BRL cells was compared on day 2 (48 h post insemination) and on day 7 (168 h post insemination) (Table 1).On day 2 no significant differences were observed in embryo development (cleaved embryos) between the two co-culture systems.On day 7, a higher percentage of successfully developed blastocysts was observed on mixed co-culture -Vero/BRL cells (42.85%), whereas a lower percentage of blastocysts was observed on Vero cells (30.41%).The difference between Vero and Vero/BRL was statistically significant (P≤ 0.05).
The results of embryo transfer are presented in Table 2. On day 35, no significant difference was observed in prenatal development of embryos between Vero and Vero/BRL cells.On day 65, a higher percentage of prenatal development was found on Vero cells (37.50%), whereas a lower percentage, on Vero/BRL cells (15.78%).The difference between the two co-culture systems was statistically significant (P≤0.05).A higher calving rate was observed from embryos co-cultured with Vero cells (37.50%) as compared with Vero/BRL cells (13.16%).The difference between groups was highly statistically significant (P≤0.01).All calves were born naturally and healthy.Independent of culture system, the birth weight of all calves was similar.

DISCUSSION
Bovine embryos developed more efficiently to the blastocyst stage on Vero/ BRL cells: 42.85% (mixed co-culture) than on Vero cells: 30.41% (Table 1).This supports our previous results, in which the difference between the two coculture systems was similar: 40.50% on Vero/BRL cells vs 27.25% on Vero cells (Duszewska et al., 2000).Also in other studies, a lower percentage of blastocysts was obtained from co-culture of embryos with both Vero cells, about 30% (Menck et al., 1997;Pegoraro et al., 1998) and BRL cells, about 35% (Farin et al., 1995;Hernandez-Ledezma et al., 1996).
Somatic cells may support embryo development through two possible mechanisms.First, somatic cells could remove deleterious components from the culture medium, protect against agents of oxidative stress and/or modulate the physico-chemical conditions of the medium.Some authors suggest that Vero cells may specifically assist by metabolizing or absorbing inhibitory substances from the culture medium.Second, somatic cells may also secrete embryotrophic factors (review: Orsi and Reischl, 2007).Both Vero and BRL cells secrete many such factors, including colony stimulating factor (CSF), epidermal growth factors (EGFs), fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), insulin growth factors (IGF-I and -II), interleukins (ILs), leukaemia inhibitory factor (LIF), platelet-derived growth factor (PDGF), stem cell factor (SCF) and transforming growth factors (TGF-α, -β 1 and -β 2 ) (Duszewska et al., 2000).
The aim of this research was to compare the developmental potential of embryos co-cultured with Vero and Vero/BRL cells after transfer to recipients.Generally, both embryo yield and embryo quality contribute to embryo transfer efficiency (Rizos 2002).In vitro conditions during maturation of oocytes, fertilization and embryo development can lead to a short-term response and long-term consequences for prenatal and postnatal development.Altered intracellular signalling, metabolic stress, changes in gene expression, apoptosis, cell proliferation, chromosomal abnormality and epigenetic modifications may all have short-term effects.By contrast, long-term consequences of in vitro culture can include reduced implantation capacity, unbalanced foetal/placental allocation, altered maternal nutrient provision and abnormal foetal growth rate (Lonergan et al., 2006).In farm animals, these long-term consequences lead to abnormal birth weight and postnatal growth abnormality, referred to as Large Offspring Syndrome (LOS) (van Wagtgendonk et al., 2000;Bertolini et al., 2002).Sometimes, but not usually, LOS has been associated with in vitro production of embryos, including nuclear transfer and pronuclear injection (Young and Fairburn 2000;review: van Wagtgendonk et al., 2000;Renard et al., 2002;Duszewska et al., 2003Duszewska et al., , 2004)).
In our experiment more offspring were obtained after transfer of embryos co-cultured with Vero cells (37.5%) than with Vero/BRL (13.16%) (Table 2).Generally, the calving rate after transfer of cultured embryos to recipients is lower (about 30-40 %) than of embryos obtained in vivo (about 70%) (Pomar et al., 2005).However a calving rate of 13.16% after transfer of embryos co-cultured with Vero/BRL cells is drastically lower.The high pregnancy loss occurred between days 35 and 65.
However, embryos developed better to the blastocyst stage with Vero/BRL cells than with Vero cells, but after transfer, embryos co-cultured with Vero cells developed better.The difference between co-culture systems could be explained by excessive concentrations of some growth factors and proteins in the mixed co-culture system (Vero/BRL).These results conflict with the opinion that mixed co-culture is more effective than systems using a single cell line (Orsi and Reischl, 2007).
Probably, Vero/BRL cells lead to changes in epigenetic modifications during development.Epigenetic modifications of DNA and chromatin are important for genome function during prenatal and postnatal development.Environmental factors during in vitro development, as well as oocyte maturation and possibly fertilization, can alter epigenetic modifications and thus have short-and longterm effects on development (Reik et al., 2003).

CONCLUSIONS
Vero cells better support bovine prenatal development than Vero/BRL cells.It is likely that Vero/BRL cells led to epigenetic modifications that manifested later in development and were responsible for early foetal resorption.

Table 1 .
In vitro development of bovine embryos co-cultured with Vero and Vero/BRL cells

Table 2 .
The results of embryo transfer to recipients