Reprod Dom Anim 49, 306–314 (2014); doi: 10.1111/rda.12275 ISSN 0936–6768

Lipid Droplet Analysis Using In Vitro Bovine Oocytes and Embryos EA Ordo~nez-Leon1, H Merchant2, A Medrano1, M Kjelland3 and S Romo1 1 Universidad Nacional Autonoma de Mexico (Facultad de Estudios Superiores–Cuautitl an), Cuautitlan, Mexico; 2Universidad Nacional Autonoma de Mexico (Instituto de Investigaciones Biomedicas), Mexico City, Mexico; 3Conservation Genetics & Biotech, LLC, Vicksburg, MS, USA

Contents The aim of this study was to quantify the content of lipid droplets in bovine oocytes and embryos from Bos indicus (Bi), Bos taurus (Bt) and Bos indicus 9 Bos taurus (Bi 9 Bt). Oocytes were aspirated post-mortem and subjected to in vitro maturation, in vitro fertilization and in vitro development; the medium employed at each stage (TCM-199, TALP, SOF) was supplemented with (i) serum replacement (SR), (ii) foetal calf serum (FCS) or (iii) oestrous cow serum (ECS). The structure and distribution of the lipid droplets were established using electron microscopy, but were quantified using an optical microscope on semi-fine toluidine blue-stained sections. The highest percentage of embryos corresponded to those produced with FCS and ECS, which differed from embryos generated with SR (p < 0.05). The highest percentage of morulae and the lowest percentage of blastocysts were obtained with the SR supplement (p < 0.05). The oocytes cultured in FCS demonstrated a higher number of lipid droplets compared to those cultured in SR and ECS (p < 0.05). Less accumulation of lipids was observed in embryos supplemented with SR. The lowest and highest numbers of lipid droplets in oocytes corresponded to the Bi and Bt strain, respectively. The lowest amount of lipid droplets in embryos was observed in Bi (p < 0.05). In conclusion, supplementation of the in vitro development culture medium (synthetic oviduct fluid) with a synthetic substitute serum produced similar results in terms of embryo development compared to those obtained with FCS, but a decreased degree of lipid droplet accumulation was observed in the in vitro-cultured embryos.

Introduction In vitro fertilization (IVF) has been employed to produce a large amount of embryos for embryo transfer programmes due to an increase in the number of calves that can be obtained from genetically superior cows. This biotechnology includes oocyte maturation and fertilization as well as the in vitro culture of zygotes and embryos. Since the birth of the first yearling calf produced by in vitro fertilization, great advances in in vitro production (IVP) systems have been obtained, where approximately 40% of embryo (morulae and blastocyst) production can be achieved with respect to the initial number of maturated oocytes (Gordon 1994). Current culture systems have improved with regard to physiology, ultrastructure and embryo morphology (Gardner 2008). However, the quality of in vitroproduced embryos is lower than that of those produced in vivo (Crosier et al. 2001). Addition of foetal calf serum (FCS) to embryo culture media inhibits early embryonic stages of development, thus contributing to the blockade of the 4- to 8-cell embryonic development. However, after this stage, the addition of FCS stimulates both morulae and blastocysts development (Pinyopummintr and Bavister 1994; Thompson et al. 1998). Moreover, FCS appears to accelerate embryonic

development, which is manifested as an increase in the number of embryonic cells (Holm et al. 2002; FouladiNashta et al. 2005) and an improvement on both the embryonic production rate and hatching rate (Wang et al. 1997; G omez and Diez 2000). Yet, FCS is considered to be a variable and undefined compound (Gardner and Lane 1998) that produces great variability in the composition of embryonic media, and thus interferes with the reproducibility of the results. Some of the negative effects attributed to FCS include (i) an excessive production of lactate and the presence of dark and granulated cells into the ICM (Bavister et al. 1992; Krisher et al. 1999), (ii) an increase in the number of apoptotic cells (Byrne et al. 1999), (iii) a decrease in protein production (Kuran et al. 2001), (iv) a decrease in the ratio of ICM to trophoblast cells, and the number of union complexes between embryonic cells (Shamsuddin and Rodriguez-Martinez 1994), and (v) an abnormal accumulation of lipid droplets into the embryo cells (Dorland et al. 1994; Shamsuddin and RodriguezMartinez 1994; Thompson 1995; Abe et al. 1999a,b). Currently, IVP is commercially successful; nevertheless, the greatest challenge for the dissemination of this technology is the high sensitivity of these in vitroproduced embryos in response to cryopreservation (Mucci et al. 2006). This reduced cryotolerance is frequently associated with the accumulation of lipid droplets in the cytoplasm of the embryos (Abe et al. 2002; Rizos et al. 2002; De La Torre-Sanchez et al. 2006; Mucci et al. 2006; Barcelo-Fimbres and Seidel 2007). Some characteristics such as the loss of blastomeres and lack of embryonic development are indicators that these in vitro-produced embryos are highly sensitive to the cryopreservation process, which is reflected in the low conception rates compared to those obtained from in vivo-produced embryos (Sudano et al. 2011). The cause of the lipid accumulation in the in vitro-produced embryos is still unknown; however, many studies have attributed this effect to the use of foetal calf serum (Abe et al. 2002; Mucci et al. 2006; Barcelo-Fimbres and Seidel 2007), a common protein supplement in embryonic developmental cultures (Abe et al. 2002; Rizos et al. 2002; De La Torre-Sanchez et al. 2006; Mucci et al. 2006; Barcelo-Fimbres and Seidel 2007, 2007). In addition to its use as a protein source, FCS is also used as a source of energy, amino acids, vitamins, growth factors and heavy metal chelators; yet, it also contains undefined substances that have been associated with the presence of several alterations, for example large offspring syndrome (Lazzari et al. 2002). FCS has also been associated with numerous abnormalities, such as cell organelle modification (Abe et al. 2002), the © 2014 Blackwell Verlag GmbH

Lipid Droplets in Bovine Oocytes and Embryos

presence of degenerated mitochondria (Dorland et al. 1994), premature formation of the blastocyst and gene expression modifications (Rizos et al. 2002, 2003), in addition to pathogenic viruses (Barcelo-Fimbres and Seidel 2007). Because Zebu (Bos taurus indicus) represents 60% of the beef-producing cattle worldwide, there is a great demand for reliable cryopreservation procedures that contribute to an increase in the commercialization and exportation of these embryos. Zebu cattle are predominant in tropical regions due to its tolerance to heat stress and resistance to parasites compared to European breeds (Bos taurus taurus) (Meirelles et al. 1999; Hansen 2006). Heat stress is a particularly severe problem in cattle, with over 50% of the bovine population located in the tropics, which causes important economic losses in approximately 60% of dairy farms worldwide (Al-Katanani and Hansen 2001). Compared to European breeds, zebu cattle experiences a less severe reduction in feed intake (Seif et al. 1979), growth and milk rate and reproductive function (Rocha et al. 1998) in response to heat stress. Zebu embryos that are produced in vitro are usually more susceptible to cryodamage than Bos taurus embryos, which has been attributed to a greater quantity of lipids found in these embryos. In general terms, in vitro-produced bovine embryos are highly sensitive to cooling and conventional freezing protocols (Massip et al. 1995), which are mainly due to the extreme sensitivity of these embryos to slow cooling (0.5°C per minute), particularly at temperatures below 6°C (Mahmoudzadeh et al. 1994). This sensitivity appears to be related to the presence of large amounts of lipids in the cytoplasm of in vitro-produced bovine embryos (Leibo and Loskutoff 1993). Nevertheless, in vitro-produced Bos taurus embryos have higher survival rates compared to Bos indicus embryos when cryopreserved using a conventional freezing method. It has been reported that in vitro-produced Zebu embryos contain a greater number of mitochondria and consequently have more mitochondrial membranes than those from Bos taurus (Esper and Barbosa 1991). Thus, it has been suggested that a higher number of membranes result in a higher sensitivity of in vitro-produced Zebu embryos to cryopreservation. However, the reason why more lipids accumulate in the cytoplasm of both oocytes and embryos from Zebu cattle is not known, and few reports exist in the literature regarding this matter. The purpose of this study was to evaluate the content of lipid droplets (liposomes) in in vitro-cultured oocytes and embryos from three bovine strains that were supplemented with three different protein sources in order to determine whether it is possible to decrease the amount of lipids present in in vitro-produced embryos.

Materials and Methods Reagents, media and culture conditions Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA), unless otherwise stated. The in vitro maturation (IVM) medium consisted of TCM-199 (Gibco-BRL, NY, USA) supplemented with 0.2 mM sodium pyruvate, 25 lM sodium bicarbonate, © 2014 Blackwell Verlag GmbH

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0.5 lg/ml FSH (Pluset, Hertape-Calier, Brazil), 100 IU/ ml HCG (Intervet, Holland) and 1.0 lg/ml oestradiol. In vitro fertilization (IVF) medium consisted of Tyrode’s albumin, lactate, pyruvate (TALP) supplemented with 0.2 mM sodium pyruvate, 3 mg/ml fatty acid-free BSA, 25 mM sodium bicarbonate, 13 mM sodium lactate, 75 lg/ml kanamycin, 4 ll/ml PHE solution (2 mM penicillamine, 1 mM hypotaurine and 250 lM epinephrine) and 10 lg/ml heparin. In vitro culture (IVC) medium consisted of synthetic oviductal fluid (SOF) (Thompson 1995) supplemented with 0.2 mM L-glutamine, 0.34 mM sodium citrate, 2.8 mM myoinositol, 2% MEM essential amino acid solution, 0.2 mM sodium pyruvate, 75 lg/ml kanamycin, 5 mg/ml fraction V fatty acid-free BSA and 2.5% FCS and ECS. Animals In this study, ovaries from sexually mature Bos indicus (Bi), Bos taurus (Bt) and Bos indicus 9 Bos taurus (Bi 9 Bt) cows and heifers were used, independent of their reproductive condition, and were collected at a local slaughterhouse (Frigorifico y Empacadora de Tabasco) (Mexico), a commercial abattoir. Females were selected, prior to being sacrificed, based on visual inspection of their phenotypic characteristics, including the presence and size of the hump, ear length, belly button appearance, shape of the head and eyes, thickness of the legs, among other factors (Gasque Gomez 2008). Handling of ovaries The ovaries were removed approximately 10 min after sacrifice and were transported to the in vitro fertilization laboratory of the Brasuca Company (Tabasco, Mexico) within a 2-h time period; they were maintained at 35°C in physiological saline solution (NaCl 0.9% w/v in distilled water). Oocyte collection Oocytes were obtained from 2- to 8-mm-diameter follicles using the aspiration method with an 18-calibre hypodermic syringe. The follicular fluid obtained from each aspired follicle was placed into 50-ml conical tubes and left to settle for 15 min. The resulting pellet was placed in a Petri dish under a stereoscopic microscope, where the oocytes were located and morphologically evaluated (759) inside a laminar flow cabinet. For the maturation stage, only 1–3 graded oocytes were used according to the De Loos et al. (1989) classification. In vitro maturation (IVM) In vitro maturation was performed randomly using maturation culture medium (TCM-199), supplemented with three different protein sources: (i) 10% foetal calf serum (FCS), (ii) 10% oestrous cow serum (ECS) and (iii) 10% serum replacement (Gibco-Invitrogen). The culture conditions in the incubator for the in vitro fertilization (IVF) and the in vitro embryo development

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(IVD) were as follows: 38.5°C, 5% CO2 and saturated humidity. After the 24-h maturation period had elapsed, the oocytes were evaluated using a stereoscopic microscope (809) to quantify the number of oocytes in which cumulus cells showed expansion (expanded cumulus– oocytes complexes). This was the criterion to evaluate oocyte maturation. In vitro fertilization (IVF) The selected oocytes were washed in two drops of IVF culture medium (TALP) and subsequently transferred to new culture medium. IVF was performed in microdroplets made of modified Tyrode’s medium (90 ll) containing 3 mg per ml of BSA fatty acid-free, fraction V (Sigma, A8022), pyruvate (Sigma, P4562) and 50 mg per ml of gentamicin (Sigma G1264). The microdroplets were covered with mineral oil, and approximately 25 expanded oocytes were placed into each droplet. Next, semen for fertilization was thawed. Nellore bull’s semen was used for oocytes from Bi cows, European Swiss bull’s semen was used for the oocytes from Bt cows, and Brangus bull’s semen was used for the oocytes from Bi 9 Bt cows. The semen was thawed in a water bath at 35°C for 30 s, and sperm motility was assessed using a phase-contrast microscope. Subsequently, the semen was centrifuged at 751 9 g for 5 min in a 15-ml tube containing 2 ml of TL Hepes culture medium (Gibco-Invitrogen, USA). The supernatant was discarded and 2 ml of IVF culture medium supplemented with 0.003 g/ml of penicillamine and hypotaurine (to stimulate sperm motility) and 0.003 g/dl of heparin (to induce sperm capacitation) was added. Immediately thereafter, a second centrifugation was performed at 751 9 g for 5 min; the sperm pellet was resuspended in 30 ll culture medium for IVF. The sperm concentration was estimated using a haemocytometer, and the final concentration was adjusted to 1 9 106 sperm/ml. The oocytes and spermatozoa remained coincubated for 18 h at 38.5°C in 5% CO2 and a saturated humidity atmosphere. In vitro development (IVD) Once the fertilization time had elapsed, the switch of culture medium occurred depending on the supplement used: (i) SOF+FCS, (ii) SOF+ECS or (iii) SOF+SR. Oocytes were washed three times in 50 ll droplets of SOF covered with mineral oil in Petri dishes (35 9 10 mm). Twenty-five presumptive zygotes were placed into 100 ll of developmental medium, supplemented with both essential and non-essential amino acids and 0.1% penicillin/streptomycin (Thompson et al. 1992). The zygotes remained in this medium for 7 days, where the culture medium was replaced every 24 h. Next, the embryos were observed using a stereoscopic microscope (809) to determine the number of embryos that developed into morula and blastocyst stages according to the classification of Stringfellow and Seidel (1990). Morphological analysis In a different experiment, oocytes were processed as previously described (i.e. IVM, IVF, IVD). Some

oocytes (n = 67) were obtained after IVM and some embryos (blastocyst only n = 76) were obtained after IVD for microscopic analysis to detect the presence of saturated and unsaturated lipids, as well as to determine the quality of the embryos and oocytes produced with the three different treatments and cow strains. Selected oocytes and embryos were fixed by immersion in modified Karnovsky fixative (Karnovsky 1965), pH 7.4, with the addition of glutaraldehyde (2.5%) and paraformaldehyde (1%) for 60 min. Next, the embryos were washed in 0.1 M cacodylate buffer (pH 7.4), postfixed with 1% osmium tetroxide (OsO4) for 1 h (Zamboni and Merchant 1973), dehydrated by immersion in ethyl alcohol at different concentrations (70, 80, 90, 95 and 100%) for 20 min each and embedded into Epon resin (EPON 812, Pelco Int Mexico). At the end of this process, semi-fine sections (1 lm) were performed using an ultra-microtome LKB (Leica, Australia), stained with 0.5% toluidine blue and examined under bright field microscopy to establish the thickness of the subsequent cuts (fine). Next, fine cuts (50–70 nm) were performed, contrasted using uranyl acetate and lead citrate and mounted in cooper 100-mesh grids for Electronic Microscopy (Jeol, Japan). Image analysis To quantify the lipid droplets in the cytoplasm of the embryos and oocytes, toluidine blue metachromasia and OsO4 reaction were used and visualized under a light and electron microscope, respectively (Bozzola and Russell 1999); the oocytes were stripped once the maturation incubation period was completed. The levels of lipid extraction during the ethanol dehydration were dependent on the saturation degree of the lipids stored in the lipid droplets. Using a light microscope, poorly saturated lipids reacted strongly to OsO4, producing a black-maroonish colour; in contrast, the highly saturated lipids produced a greenish colour. As observed under an electron microscope, the droplets formed by unsaturated lipids were highly electron dense due to their reaction with OsO4 and because they resisted extraction during dehydration. For this procedure, each slide was analysed using a Zeiss Axioscope microscope, equipped with an AxioCamHRc camera (Zeiss, Germany). Images were stored using the AxioVision program (v. 4. 7. 2). Digital images of each sample were used to quantify lipid inclusions with the image analysis tools from ImageJ (version 1.46b, Wayne Rasband, NIH, USA). Statistical analysis The experimental design was a factorial 3 9 3 with six replicates; the factors were (1) three bovine strains and (2) three protein supplements. Data on embryo development were analysed using ANOVA, and when appropriate, the Tukey test was used for post hoc analyses. Analyses were performed using the SAS program (version 8 USA). Considering both cow strain and protein supplement, to compare the content of the cytoplasmic lipids in the oocytes and embryos, data on the number of lipid droplets were analysed using © 2014 Blackwell Verlag GmbH

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nonparametric tests (Kruskal–Wallis) with the Stats Soft program (v 5.0 UK). The level of significance was established at p < 0.05.

Results A total of 3921 oocytes were subjected to the embryo production process (i.e. maturation, fertilization, development): 1324 oocytes were treated with serum replacement (TCM199 + SR maturation; TALP + SR fertilization; SOF + SR development); 1294 oocytes were treated with FCS (TCM199 + FCS maturation; TALP + FCS fertilization; SOF + FCS development), and 1303 oocytes were treated with ECS (TCM199+ ECS maturation; TALP + ECS fertilization; SOF + ECS development). In vitro maturation and fertilization The proportion of matured and fertilized oocytes was no different between the three protein supplements; maturation and fertilization were approximately 93% and 98%, respectively (Table 1). Embryos produced with the three different protein supplements The percentage of embryos obtained from treatment with FCS (51.7%) and ECS (51.7%) was higher than that of the SR treatment (33.8%, p < 0.05), but did not differ between each other (Table 2). For embryo development, the percentage of morulae produced was different (p < 0.05) between the treatments: 18.1%, 14.7% and 12.3% for SR, FCS and ECS, respectively. The percentage of expanded blastocysts was also different (p < 0.05) between the treatments: the highest percentage corresponded with FCS (36.1%), followed by ECS (28.4%) and SR (22.2%) (Table 2). Embryos from three bovine strains There were differences (p < 0.05) in the proportion of embryos produced from the three strains; the highest percentage corresponded to Bi (50.8%), followed by Bi 9 Bt (42.2%) and lastly Bt (34.1%) (Table 3). In

Table 1. In vitro matured and fertilized bovine oocytes and cleaved embryos after the addition of three types of protein supplements Type of serum FCS ECS SR

% Matured oocytes (IVM/OPU)

% IVF (IVF/IVM)

% Embryos (IVD/IVF)

94.7ª (1225/1294) 93.2ª (1215/1303) 93.5ª (1238/1324)

98.9ª (1211/1225) 99.1ª (1204/1215) 98.0ª (1213/1238)

91.5a (1108/1211) 85.1b (1024/1204) 87.7b (1063/1213)

FCS, foetal calf serum; ECS, oestrous cow serum; SR, serum replacement; IVM, in vitro maturation; IVF, in vitro fertilization; IVD, in vitro development; OPU, ovum pickup. Different literals (a, b) in the same column represent statistical differences (p < 0.005).

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both Bt and Bi, the percentage of embryos supplemented with either FCS or ECS was higher than that with SR (p < 0.05). In contrast, in Bi 9 Bt, there were no differences between the supplements. In both Bi and Bi 9 Bt, the percentage of embryos supplemented with any of the supplements was higher than that of those from Bt (p < 0.05) (Table 4). Evaluation of in vitro matured oocytes using transmission electron microscopy Figure 1 shows an oocyte with cytoplasmic microvilli and cortical granules on its surface. The lipid droplets were of various diameters and variable density; however, two groups of lipid inclusions could be distinguished: (i) large droplets with moderate electron dense content and (ii) small droplets without electron density and with some particles inside (Fig. 1a, b and d). Both these types of lipid droplets were clearly distinguished from cytoplasmic vesicles and vacuoles because they lacked a plasma membrane. Yet, many of the small and some of the large droplets were surrounded by smooth endoplasmic reticulum (Fig. 1c and d). Mitochondria were found in complex packages that were associated with small-type lipid droplets and other cytoplasmic organelles, such as Golgi complex, endoplasmic reticulum, vacuoles and lysosomes (Figs 1a and 2a). The previously described packages were of irregular sizes and exhibited a distribution without a clear qualitative distinction between the three different bovine strains and protein supplements. Once the structure and distribution characteristics of the lipid droplets were established using electron microscopy, they were identified and quantified using a high-resolution optical microscope into semi-fine sections stained with toluidine blue. Some illustrative examples of the three bovine strains with the diverse protein supplements are shown in Figs 1b, 2b and 3. Quantification of lipid droplets Oocytes supplemented with FCS contained the highest number of lipid droplets (367), followed by those supplemented with ECS (243) and lastly those treated with SR (214); these values were significantly different from each other (p < 0.05). Regarding the embryos, the smallest number of lipid droplets corresponded to those supplemented with ECS (21), which were different (p < 0.05) from those supplemented with either FCS (74) or SR (83) (Table 5). With respect to the bovine strains, oocytes from Bi contained the lowest number (229) of total lipid droplets, followed by those from Bi 9 Bt (243) and next by those from Bt (318). These values were different (p < 0.05) from each other (Table 6). Importantly, embryos from Bi contained a smaller number (65) of lipid droplets than those from Bi 9 Bt (85) (p < 0.05); however, data on embryos from Bt are lacking. Further analysis with only data from Bi using the different protein supplements revealed that oocytes supplemented with FCS contained a higher number of lipid droplets than those supplemented with either ECS or SR (Fig. 4).

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Table 2. Bovine embryos produced in synthetic oviduct fluid (SOF) culture medium with the addition of different protein supplements % Blastocysts/Total embryos Type of serum FCS ECS SR

% Embryos Embryos/Oocytes

% Morulae Morulae/ Total embryos

Initial

Compact

Expanded

51.7a (573/1108) 51.7a (529/1024) 33.8b (360/1063)

14.7ª (84/573) 12.3b (65/529) 18.1c (65/360)

24.1a (138/573) 29.5b (156/529) 31.4b (113/360)

25.2a (207/573) 30.0b (150/529) 28.3c (80/360)

36.1a (207/573) 28.4b (150/529) 22.2c (80/360)

FCS, foetal calf serum; ECS, oestrous cow serum; SR, serum replacement. Different literals (a, b, c) in the same column represent statistic differences (p < 0.05).

Table 3. Bovine embryos of three different breeds cultured in synthetic oviduct fluid for 7 days % Blastocysts (Bl/IVD) Breed Bos taurus Bos indicus B.t 9 B.i

% Embryos (Embryos/IVD)

% Morulae (Morulae/IVD)

Initial

Compact

Expanded

34.1ª (299/869) 50.8b (599/1180) 42.2c (564/1146)

15.1a (45/299) 16.5b (99/599) 12.4c (70/564)

23.4ª (70/299) 28.3b (170/599) 31.4c (167/564)

22.7ª (68/299) 29.1b (174/599) 28.7ª (162/564)

38.8ª (116/299) 26.1b (156/599) 29.2c (165/564)

Different literals (a, b, c) in the same column represent statistical differences (p < 0.05).

Table 4. Number of embryos produced from oocytes of three bovine strains supplemented with three different protein sources: foetal calf serum (FCS), oestrus cow serum (ECS), serum replacement (SR) Bos taurus

Bos taurus 9 Bos indicus

Bos indicus

Supplement

M

B

Total (%)

M

B

Total (%)

M

B

Total (%)

FCS ECS SR

20a 10a 20a

104a 98a 47b

124 (30.4)ax 108 (28.7)ax 67 (16.9)bx

37a 45a 41a

212a 193a 71b

249 (83.8)ay 238 (84.1)ay 112 (38.8)by

32a 43a 42a

168a 140a 139a

200 (49.6)ay 183 (50.1)az 181 (47.9)ay

M, morulae; B, blastocysts; (%), (number of embryos (M+B)/number of fertilized oocytes)*100. Different letters in the same column (a, b) or the same row (x z) indicate significant differences (p < 0.05).

Discussion In this study, three different protein supplements (FCS, ECS and SR) were tested during in vitro oocyte maturation and fertilization, and in vitro embryonic development to identify the supplement that provided the best conditions for the production of viable embryos. There were no differences between protein supplements during any of the assessed stages: in vitro maturation, fertilization and development (percentages of maturated and fertilized oocytes, percentage of blastocysts). Furthermore, similar results have been previously published (Duque et al. 2003; Mucci et al. 2006; Moore et al. 2007) demonstrating that FCS can be substituted with similar results. However, there were differences between bovine strains in embryo production efficiency: oocytes from Bos taurus 9 Bos indicus produced the best results. This may be attributed to differences in the adaptability of the animals to tropical conditions (Nogueira 2004); thus, further studies are required on the subject.

Our results demonstrated that FCS can be substituted with a serum replacement, specifically SOF, with similar results and reducing FCS negative effects on the embryo quality, such as the abnormal accumulation of cytoplasmic lipid droplets (Dorland et al. 1994; Thompson 1995). Quantification of lipids in the oocyte and embryos has been performed using quantitative methods, such as gas chromatography, which is an expensive method that requires specific equipment, as well as groups of approximately 1000 cells (oocytes and embryos). In addition, lipids have been quantified via microfluorescence enzymatic trials, and although this procedure is easier, approximately 100 oocytes or embryos are still required (Barcelo-Fimbres and Seidel 2011). Given the less than ideal requirements of these procedures, cheaper and simpler tests are desired, and thus, light microscopy may be a good alternative. Currently, serum-derived synthetic products are frequently used in an attempt to establish a defined culture serum for both oocyte maturation and embryo © 2014 Blackwell Verlag GmbH

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(a)

(b)

(c)

(d)

Fig. 1. Light and electron micrographs of a Bos taurus matured oocyte supplemented with serum replacement: (a) electron microscopy displaying a part of the cortical cytoplasm with microvilli (mv), cortical granules (cg), large lipid droplets (L) and organelle packages formed by mitochondria (mt) and small lipid droplets (*). (b) Image of a toluidine blue-stained semi-fine section of the same oocyte shown in panel a. The white arrows indicate groups of large lipid droplets. Organelle packages with mitochondria and small lipid droplets (*) appear to be randomly distributed in the oocyte cytoplasm. The zona pellucida appears intact. c and d. Electron micrographs of higher resolution related to the oocyte in panel a. Mitochondria with scarce crests (mc) and endoplasmic reticulum cisterns (arrow heads) around lipid droplets (*) are evident. In the cytoplasm, surface microvilli (mv) and cortical granules (cg) can be appreciated. Bars: a, c and d = 1 lm, b = 50 lm

(a)

(b)

Fig. 2. Oocyte supplemented with oestrous cow serum: (a) electronic micrograph displaying part of an organelle package with abundant endoplasmic reticulum cisterns (er), mitochondria (mc), lysosomes (li) and lipid droplets; frequently, these droplets adhere to form droplets of a larger diameter (*). (b) Toluidine blue-stained semifine sections demonstrating many lipid droplets of a greenish colour (arrows). Bars: a = 1 lm; b = 50 lm

(a)

Fig. 3. Toluidine blue-stained semi-fine sections of bovine blastocysts treated with three different protein supplements: (a) blastocyst from Bos indicus supplemented with SR; (b) blastocyst from Bos taurus 9 Bos indicus supplemented with foetal calf serum. White arrows indicate lipid droplet groupings. zp, zona pellucida; mci, inner cell mass; bc, blastocoel; ct, mural trophoblast

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(b)

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EA Ordo~ nez-Leon, H Merchant, A Medrano, M Kjelland and S Romo

Table 5. Lipid droplets in in vitro matured bovine oocytes and embryos (blastocysts) in three different protein supplements Protein supplement (number of structures) FCS (58) ECS (39) SR (46)

Number of lipid droplets in oocytes

Number of lipid droplets in embryos

367  107a (n = 16) 243  39.5b (n = 31) 214  44.3c (n = 20)

74  26.9a (n = 42) 21  11.9b (n = 8) 83  23.2a (n = 26)

FCS, foetal calf serum; ECS, oestrous cow serum; SR, serum replacement. Values are means  SEM. Different literals (a, b, c) in columns differ significantly (p < 0.05).

Table 6. Lipid droplets in in vitro matured oocyte and embryos (blastocysts) from three bovine strains

Strain Bos taurus Bos indicus Bos indicus 9 Bos taurus

Number of lipid droplets in oocytes (n) 318  (n = 229  (n = 243  (n =

71.2a 21) 60.9c 25) 48.3b 25)

Number of lipid droplets in embryos (n) nd 65  20.3b (n = 71) 85  44.6a (n = 29)

nd, non-determined. Values are means  SEM. Values with different literals (a, b, c) in columns differ significantly (p < 0.05).

Fig. 4. Number of inner cytoplasmic lipid droplets in oocytes and embryos from three bovine strains cultured using three protein supplements

production, the presence of dark and granulated cells in the ICM (Bavister et al. 1992); Krisher et al. 1999), an increase in apoptotic cells (Byrne et al. 1999), minor protein synthesis (Kuran et al. 2001), decrease in the ICM/trophoblastic cell ratio as well as the number of union complexes between embryo cells (Shamsuddin and Rodriguez-Martinez 1994) and an abnormal accumulation of cytoplasmic lipid droplets (Shamsuddin and Rodriguez-Martinez 1994); Thompson 1995); Abe et al. 1999a,b). However, the underlying mechanism of why these lipids accumulate in the cytoplasm remains unknown; yet, it is believed that it may be a result of two processes: (i) the embryo is capable of absorbing lipids from the culture environment, particularly under conditions in which a rich lipid source (serum) exists, and (ii) it is postulated that this lipid accumulation is a result of the lack of mitochondrial capability to metabolize lipid complex via beta oxidation (De La Torre-Sanchez et al. 2006). In either case, lipids represent an obstacle for embryo survival; that is, embryos are more susceptible to oxidative damage and are very susceptible to cryopreservation. In the present work, oocytes supplemented with SR contained the lowest amount of lipid droplets, and similar results were obtained by Sudano et al. (2011). Sudano et al. eliminated the use of serum and observed a decrease in embryo lipid content, as well as an improvement in embryo quality. In contrast, Sagirkaya et al. (2007) found no significant differences when comparing SR to FCS, at 10% levels, during bovine oocyte maturation and fertilization. Several studies (Yamashita et al. 1999; Cho et al. 2001, 2002; Abe et al. 2002) have reported an improvement in embryonic cryosurvival when using BSA-supplemented and serum-free culture media, independent of the cryopreservation method employed. Furthermore, a lower amount of lipid droplets was observed in Bi compared to Bt oocytes, and thus, the proportion of embryos produced was higher in Bi than in Bt, which could be attributed to an enhanced Bi adaptability to the environment (Nogueira 2004). In conclusion, it is possible to supplement the embryodeveloping medium (SOF) with a synthetic serum substitute with similar results to those produced by the addition of FCS, but decreasing embryo lipid droplet accumulation is observed. This decrease in lipid accumulation may improve both the quality of bovine embryos to be cryopreserved and their cryosurvival, which would contribute to improve pregnancy rates with the use of frozen embryos. Acknowledgements

culture, thus minimizing the high variability obtained with the use of FCS. The SR used in this study is completely free of growth factors, cytosines, hormones, immunoglobulin and other macromolecules, and thus, it is ensured that the results are reproducible (Moore et al. 2007). FCS is currently the most commonly used product in in vitro embryo production; however, it also causes alterations in embryos subjected to this procedure (Sudano et al. 2011), such as excess lactate

Erika Alina Ordo~ nez-Leon was supported by the Mexican government (CONACYT); Brasuca Company S.A. and Frigorifico y Empacadora de Tabasco (Mexico) supported this study. This study was partially supported by CONACYT (Project 166012) to HM. We appreciate the assistance of Mr. Francisco R Gonzalez-Diaz from FES-Cuautitlan (UNAM) for the digital processing of images in this study.

Conflict of interest None of the authors have any conflict of interest to declare.

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Lipid Droplets in Bovine Oocytes and Embryos

Author contributions Erika A Ordo~ nez-Leon carried out most of the experiments, organized the data and participated in the writing of the article; Horacio Merchant designed and supervised the work on electron and optical microscopy and contributed to the writing of the article; Alfredo

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Submitted: 22 Aug 2013; Accepted: 18 Dec 2013 Author’s address (for correspondence): EA Ordo~ nez-Leon, Sindicato de Salubridad 115 3er piso Col Adolfo Lopez Mateos, Villahermosa, Tabasco, Mexico. E-mail: alina. [email protected].

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