Introduction
For in vitro production (IVP), technology has been improved significantly in cattle and sheep, but the quality of the embryos produced is impaired in comparison with their in vivo counterparts (Niemann et al., Reference Niemann, Wrenzycki, Lucas-Hahn, Brambrink, Kues and Carnwath2002).
The influence of various in vitro procedures on embryo survival and the production of normal offspring has been investigated in different species (Kazuhiro et al., Reference Kazuhiro, Akira, Naomi, Masaki, Junko, Hiroyuki, Tomiji and Takashi2002; Lazzari et al., Reference Lazzari, Wrenzycki, Herrmann, Duchi, Kruip, Niemann and Galli2002; Dattena et al., Reference Dattena, Mara, Bin and Cappai2007). The birth of unusually large lambs has been reported to be probably due to an inappropriate environment in which the embryo is stored for a relatively long time or to extensive manipulation in the laboratory. Several factors give rise to the heavy calf or lamb and they include asynchronous transfer, progesterone treatment (Kleeman et al., Reference Kleeman, Walker and Seamark1994), nuclear cloning of embryos (Willadsen et al., Reference Willadsen, Janzen, McAlister, Shea, Hamilton and McDermand1991), and in vitro culture (Holm et al., Reference Holm, Walker and Seamark1996; Walker et al., Reference Walker, Hartwich and Seamark1996).
Several authors have reported an increase in birth weight of lambs following transfer of cultured embryos (Walker et al., Reference Walker, Heard and Seamark1992; Thompson et al., Reference Thompson, Gardner, Pugh, McMillan and Tervit1995; Lazzari et al., Reference Lazzari, Wrenzycki, Herrmann, Duchi, Kruip, Niemann and Galli2002; Dattena et al., Reference Dattena, Mara, Bin and Cappai2007). Our laboratory had reported previously normal birth weights of Sarda lambs born from blastocysts produced in vitro and cultured with the addition of 5% charcoal-stripped FBS added only on the third and fifth days of culture (Ptak et al., Reference Ptak, Dattena, Loi, Tischner and Cappai1999).
It is well known that a considerable proportion of the offspring born from transfer of IVP embryos is affected by large offspring syndrome (LOS). This syndrome is characterized by a variety of pathologies, including abnormal phenotypes, placental and organ defects, increased gestation length, elevated abortion and increased perinatal death, but the most important feature is a significant increase in birth weight (Walker et al., Reference Walker, Hartwich and Seamark1996; Sinclair et al., Reference Sinclair, McEvoy, Carolan, Maxfield, Maltin, Young, Wilmut, Robinson and Broadbent1998; Young et al., Reference Young, Sinclair and Wilmut1998; Niemann & Wrenzycki, Reference Niemann and Wrenzycki2000; Lazzari et al., Reference Lazzari, Wrenzycki, Herrmann, Duchi, Kruip, Niemann and Galli2002).
In this paper we have considered five groups of embryos produced in vitro in different culture media, vitrified and transferred into recipient ewes. The groups are indicated as BSA4, BSA8, BSA8-HA, BSA8-CH and serum. The rationale of these five groups was the following: in previous paper Dattena et al. (Reference Dattena, Mara, Bin and Cappai2007) showed that bovine serum albumin (BSA; 8 mg/ml) gives heavy lambs, for this reason we decided to halve the dose from 8 to 4 mg/ml. In previous studies other authors have reported that the use of hyaluronan (HA) improves embryo quality in general (Furnus et al., Reference Furnus, de Matos and Martínez1998; Kano et al., Reference Kano, Miyano and Kato1998; Lane et al., Reference Lane, Maybach, Hooper, Hasler and Gardner2003; Dattena et al., Reference Dattena, Mara, Bin and Cappai2007), for this reason we used BSA with HA during the culture period. The BSA8-CH group follows the protocol used traditionally in our laboratory (Accardo et al., Reference Accardo, Dattena, Pilichi, Mara, Chessa and Cappai2004) and replaced the first protocol used in our laboratory (Ptak et al., Reference Ptak, Dattena, Loi, Tischner and Cappai1999) represented by the serum group.
The aim of the present study was to compare body weight and lambing rate of lambs born from five different in vitro culture systems used in the last 10 years in our laboratories and to investigate if different culture systems can lead to differences in body birth weight and in lambing rate.
Materials and methods
Except when otherwise indicated, all chemicals were obtained from Sigma-Aldrich.
Collection of ovaries and oocytes
Ovaries of adult Sarda sheep were obtained from local slaughterhouses and transported to the laboratory within 2–3 h in saline solution at a temperature of ~35–37°C. Oocytes were collected by cutting the ovaries. Only the follicular oocytes covered by at least two layers of granulosa cells and an even cytoplasm were selected for in vitro maturation.
In vitro maturation (IVM)
Oocytes were washed in HEPES-buffered TCM199 (H-TCM199) supplemented with 0.4% bovine serum albumin (BSA). The medium used for maturation was bicarbonate-buffered TCM199 that contained 2 mM glutamine, 100 μM cysteamine, 0.3 mM sodium pyruvate, 1 μg/ml estradiol-17β, 0.1 i.u./ml follicle stimulating hormone (r-FSH) and 0.1 i.u./ml luteinizing hormone (LH) supplemented with 0.4% BSA (BSA4, BSA8, BSA8-HA and BSA8-CH groups) or with 10% fetal bovine serum (FBS; serum group). The oocytes were incubated in 400 μl of medium in 4-well dishes (Nunc, Nunclon Denmark), each well contained 20–30 oocytes covered with mineral oil. In vitro maturation (IVM) conditions were 5% CO2 in humidified air at 39°C for 24 h.
In vitro fertilization (IVF)
After maturation, the oocytes were partially denuded of granulosa cells with 300 i.u./ml of hyaluronidase in H-TCM199. Fresh semen from a Sarda breed ram of proven fertility was collected and kept at room temperature for up to 2 h and then washed in synthetic oviduct fluid (SOF) supplemented with 0.4% BSA. The fertilization medium consisted of SOF (Tervit et al., Reference Tervit, Whittingham and Rowson1972) enriched with 20% heat inactivated estrous sheep serum. A maximum of 10–15 oocytes per drop were fertilized in 50 μl with 1 × 106 sperm/ml at 39°C and 5% CO2 in humidified air.
In vitro culture (IVC)
On the day after fertilization, presumptive zygotes were washed in SOF to remove spermatozoa debris. The presumptive zygotes were allocated to 20-μl culture drops (5–6 embryos/drop) and cultured for 6–7 days until blastocyst stage in medium that consisted of SOF supplemented with 1% basal Eagle's medium (BME), 1% minimum essential medium (MEM), 1 mM glutamine and 8 mg/ml fatty acid-free BSA (BSAfaf), for BSA8, BSA8 + HA, BSA8-CH and serum groups, or 4 mg/ml fatty acid-free BSA for the BSA4 group. On the third and fifth days of culture (day 0: day of fertilization) 5% of charcoal-stripped FBS was added to the medium of the BSA8-CH and serum groups, whilst 8 or 4 mg/ml fattyacid-free BSA was added in BSA8 and BSA8-HA or BSA4 groups respectively and 6 mg/ml HA was added to the BSA8-HA group. Embryos were incubated in humidified air that consisted of 7% O2, 5% CO2, 88% N2 and at 39°C (see Table 1).
Table 1 Groups of culture media with different supplements of BSA and serum
BSA, bovine serum albumin; CH, charcoal stripped; FBS, fetal bovine serum; HA, hyaluronan.
Vitrification and warming of embryos
Dulbecco's phosphate-buffered saline (PBS) supplemented with 0.3 mM Na pyruvate and 20% FBS was used as the basic vitrification solution. Blastocysts were exposed at room temperature to 10% ethylene glycol (EG) and 10% dimethyl sulphoxide (DMSO) for 5 min, then to 20% EG, 20% DMSO, and 0.5 M sucrose for <30 s. Blastocysts were loaded into open pulled straws (OPS) and immediately plunged into liquid N2. The straws were warmed in a water bath at 37°C and the contents expelled into a Petri dish, mixed in 0.5 M sucrose for 3–5 min and moved into a drop of H-TCM199 and 20% FBS for the transfer to recipient ewes, in accordance with Dattena et al. (Reference Dattena, Accardo, Pilichi, Isachenko, Mara, Chessa and Cappai2004).
Embryo transfer
Only embryos of high quality (Q1) were utilized for embryo transfer. In total, 240 in vitro produced embryos (Q1) were used; 240 blastocysts (48 from the BSA4 group, 51 from the BSA8 group, 50 from the BSA8-HA group, 46 from the BSA-CH group and 45 from the serum group) were transferred in pairs into synchronized sheep by inguinal mini-laparatomy with the help of a Tom-cat.
Lambing
At day 146 of pregnancy, ewes were allocated indoor to monitor the onset of lambing. The lambs were weighed and identified within 6 h of birth.
Results
Lambing rate
When lambing rate was compared within groups statistical difference was found between the BSA groups and the BSA-CH or serum groups (BSA4 and BSA8-HA versus BSA8-CH P < 0.05, BSA8 versus BSA8-CH P < 0.001, BSA8 and BSA8-HA versus serum P < 0.05). There was no significant difference between the BSA groups or between the BSA8-CH and serum groups (see Table 2).
Table 2 Lambing rates and heavy lamb percentage of embryos produced in vitro after culture with different supplements, vitrified and transferred into recipient ewes
Values in the same column with different letters differ: a,bP < 0.05, a,cP < 0.001.
BSA, bovine serum albumin; CH, charcoal stripped; HA, hyaluronan.
Birth weight
The body weight of Sardinian lambs was considered heavy when it was ≥ 4.5 kg. Thus heavy body weight was found for lambs in BSA8 (5/24, 20.8%) and BSA8-HA (4/22, 18.2%), but only between BSA4 and BSA8 groups was there a significant difference (BSA4 versus BSA8 and BSA8-HA P < 0.05). Whilst no heavy lambs were produced in the BSA4, BSA8-CH and serum groups (Table 2).
Discussion
The results presented in this study demonstrated that the use of different supplements can affect lamb body weight. Although it was not the aim of this work to report the embryo cryotolerance, and thus the lambing rates of the vitrified IVP embryos, it has been reported for completeness.
In BSA8-CH and serum groups, the lambing rate was lower when compared with the BSA groups with significant differences between groups BSA4 and BSA8-CH, BSA8 and BSA8-CH, BSA8-HA and BSA8-CH, BSA8 and BSA8-HA versus serum (P < 0.05, P < 0.001, P < 0.05, P < 0.05 and P < 0.05 respectively). Thus, embryos produced with BSA instead of serum demonstrated a higher cryotolerance. These data are similar to those reported by Dattena et al. (Reference Dattena, Mara, Bin and Cappai2007) and Mara et al. (Reference Mara, Mayorga, Sanna and Dattena2009) in sheep and by Lazzari et al. (Reference Lazzari, Wrenzycki, Herrmann, Duchi, Kruip, Niemann and Galli2002) in cows.
In our experience, the addition of HA improved neither development to the blastocyst stage nor pregnancy and lambing rates in vitrified embryos when compared with BSA8 (Dattena et al., Reference Dattena, Mara, Bin and Cappai2007). When compared with the BSA8-CH and serum groups, the lambing rate in the BSA8-HA group was significantly improved, although the increase was less than that of the BSA groups. These results are difficult to compare with previous studies because most of the authors did not transfer the embryos. These results are in agreement with other studies in which an enhanced lambing rate was reported when this macromolecule was used to substitute serum (Lane et al., Reference Lane, Maybach, Hooper, Hasler and Gardner2003; Dattena et al., Reference Dattena, Mara, Bin and Cappai2007; Mara et al., Reference Mara, Mayorga, Sanna and Dattena2009).
As reported by previous authors, in vitro culture (Walker et al., Reference Walker, Heard and Seamark1992; Holm et al., Reference Holm, Walker and Seamark1996) and vitrification of embryos (Young et al., Reference Young, Sinclair and Wilmut1998; Sinclair et al., Reference Sinclair, McEvoy, Maxfield, Maltin, Young, Wilmut, Broadbent and Robinson1999) may result in fetuses that are heavier than usual. In this study there were significant differences among the groups analyzed in term of birth weight, in both the BSA8 and BSA8-HA groups in which heavy lambs were born.
Similarly, Lazzari et al. (Reference Lazzari, Wrenzycki, Herrmann, Duchi, Kruip, Niemann and Galli2002), Dattena et al. (Reference Dattena, Mara, Bin and Cappai2007) and Mara et al. (Reference Mara, Mayorga, Sanna and Dattena2009) reported the presence of heavy born animal from blastocysts produced in vitro with BSA. In contrast, other studies reported reduction of large offspring when BSA was added in place of serum during culture (Thompson et al., Reference Thompson, Gardner, Pugh, McMillan and Tervit1995; van Wagtendonk-de Leeuw et al., Reference van Wagtendonk-de Leeuw, Mullaart, de Roos, Merton, den Daas, Kemp and de Ruigh2000). These conflicting findings underscore that incidence of heavy lambs varies greatly and that these conditions are difficult to reproduce (Young et al., Reference Young, Sinclair and Wilmut1998). In addition, serum or BSA doses, number of batches and the culture system used varied greatly from laboratory to laboratory, making data difficult to compare.
Thus, in order to reduce the heavy lamb syndrome, keeping good cryotolerance, we decided to halve the dose of BSA from 8 mg/ml to 4 mg/ml. It has been reported by Lazzari et al. (Reference Lazzari, Wrenzycki, Herrmann, Duchi, Kruip, Niemann and Galli2002) that doses of BSA between 8 mg/ml and 16 mg/ml are considered high. Indeed, in this study when 4 mg/ml BSA treatment was used no heavy lambs were born.
In conclusion, the different culture media used in this study affected the body weight and the quality of embryos in term of cryotolerance.
Acknowledgements
The authors thank Giampero Camoglio and Antonio Pintadu for assistance in the care and handling of animals.
This work was supported by the Regione Autonoma della Sardegna Biodiversità APQ5/2007.
