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Title: In vitro developmental competence of alpaca (Vicugnapacos) and llama (Lama glama) oocytes after parthenogeneticactivation
Author: Jaime Ruiz Leandra Landeo Jose Mendoza JorgeCorrea Mauricio Silva Marcelo H Ratto
PII: S0921-4488(15)30050-XDOI: http://dx.doi.org/doi:10.1016/j.smallrumres.2015.08.014Reference: RUMIN 5019
To appear in: Small Ruminant Research
Received date: 22-1-2015Revised date: 18-8-2015Accepted date: 19-8-2015
Please cite this article as: Ruiz, Jaime, Landeo, Leandra, Mendoza, Jose, Correa,Jorge, Silva, Mauricio, Ratto, Marcelo H, In vitro developmental competence of alpaca(Vicugna pacos) and llama (Lamaglama) oocytes after parthenogenetic activation.SmallRuminant Research http://dx.doi.org/10.1016/j.smallrumres.2015.08.014
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1 In vitro developmental competence of alpaca (Vicugna pacos) and llama (Lama glama) oocytes 1
after parthenogenetic activation 2
3
Jaime Ruiz1, Leandra Landeo1, José Mendoza1, Jorge Correa2, Mauricio Silva3, Marcelo H Ratto42* 4
5
1 Laboratorio de Biotecnologías Reproductivas, Facultad de Ciencias de Ingeniería, Universidad 6
Nacional de Huancavelica, Huancavelica, Perú. 7
2 Instituto de Ciencia Animal, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, 8
Valdivia, Chile. 9
3 Escuela de Medicina Veterinaria, Universidad Católica de Temuco, Temuco, Chile. 10
4 Ross University School of Veterinary Medicine, Basseterre, St. Kitts, W.I. 11
*Corresponding author. Email: mratto@rossvet.edu.kn 12
Research Highlights 13 14 Alpaca and llama oocytes can beactivated after a sequential incubation with Ionomycin and 6-15
DMAP 16 In vitro embryo development did not differ between species after oocyte chemical activation 17 Thisresults of the study could be applied to evaluate oocyte functionality after in vitro maturation or 18
cryopreservation procedures 19 20
Abstract 21
The study was designed to compare the cleavage and blastocysts rate of in vitro matured alpaca and 22
llama oocytes after chemical activation. Alpaca (n=90) and llama (n=85) ovaries were collected at a 23
local slaughterhouse and transported within 2-3 h to the laboratory. Cumulus Oocyte Complexes 24
(COCs) were aspirated from follicles 2-6 mm in diameter and classified according to the number of 25
cumulus cell layers and cytoplasm morphology. A total of 350 and 400 COCs were collected from the 26
alpaca and llama abattoir-derived ovaries, respectively (average, 3.8 vs 4.7 COCs per ovary, 27
respectively). Only category 1 and 2 COCs collected from alpaca (n=280) and llama (n=340) were in 28
vitro matured for 26-28 h in medium TCM 199 at 39ºC in an atmosphere of 5% CO2 in humidified 29
air. After in vitro maturation, oocytes were denuded of cumulus cells by vortex agitation, for 2 min in 30
mSOF-HEPES solution at 0.1% hyaluronidase. Mature (MII) alpaca (n=224) and llama (n=240) 31
2 oocytes were activated using 5 µM Ionomycin in SOF-HEPES supplemented with 1 mg/ml BSA at 32
room temperature for 4 min followed by incubation in mSOF-IVC supplemented with 3 mg/ml BSA, 2 33
mM 6-dimethylaminopurine (6-DMAP) and 12.5 μM cytochalasin B for 3 h at 39 ºC in an atmosphere 34
of 5% O2, 5% CO2 and 90% N2 in humidified air. Then, oocytes were transferred to 40 µl drops of 35
mSOF-IVC supplemented with 3 mg/ml BSA and cultured for 8 days at 39 ºC in an atmosphere of 5% 36
O2, 5% CO2 and 90% N2 in humidified air. A greater proportion of category 3 COCs was collected 37
from alpaca than llama ovaries; however, there were not significant differences in the remaining COCs 38
categories between species. A total of 224 and 240 alpaca and llama matured oocytes were chemically 39
activated, respectively. Cleavage (62.5 ± 2.7 vs 66.6 ± 5.2), morula (47.0 ± 2.0 vs 45.8 ± 1.4) and 40
blastocyst (22.5 ± 1.3 vs 18.7 ± 1.0) development rate did not differ between groups. In conclusion, 41
alpaca and llama oocytes can be effectively activated after a sequential incubation with 5 µM 42
Ionomycin and 2 mM 6-DMAP/12.5 μM cytochalasin B resulting in consistent in vitro embryo 43
development rates that could be used to assess oocyte viability/functionality after in vitro maturation or 44
cryopreservation. 45
Keywords: Alpaca, Llama, Oocytes, Chemical activation, Blastocyst. 46
47
1. Introduction 48
Parthenogenesis is a reproductive phenomenon occurring in many different lower animals (i.e. 49
insects, lizards, snakes, fishes and birds), in which an oocyte initiates its development to generate 50
offspring without the paternal contribution (Kharched and Birade, 2013). Although not natural to 51
mammals, parthenogenesis has been reported to occur spontaneously, to some extent, in several 52
species such as bovine (Lechniak et al., 1998), rat (Zernicka-Goetz, 1991), mice (Eppig et al., 2000; 53
Fedorushchenko et al., 1996) and camelids (Abdoon et al., 2007; Mesbah et al., 2004). 54
Parthenogenesis can also be artificially induced in mammals by physical, chemical and electrical 55
stimulation of the oocyte, processes that mimic the intracellular calcium oscillations induced by the 56
sperm during natural fertilization of the ova, and trigger the resumption and completion of meiosis. In 57
vitro production of parthenogenetic embryos has been reported in several species such as cows 58
3 (Dinnyés et al., 2000), buffalos (Gasparrini et al., 2004), goats (Ongeri et al., 2001), mice 59
(Krivokharchenko et al., 2003), pigs (Iwamoto et al., 2005), deer (Brahmasani et al., 2013), dromedary 60
(Wani, 2008; Khatir et al., 2009) and llamas and alpacas (Sansinena et al., 2003; Ruiz et al., 2013). 61
Oocyte chemical activation was considered, since its early development, as a useful method 62
that could play an important role in the development of other reproductive techniques such as intra 63
cytoplasmic sperm injection, cloning or in the evaluation of in vitro culture conditions for oocytes 64
(Kharched and Birade, 2013). Indeed, chemical activation has been used to assess the viability of 65
cryopreserved oocytes in several animal species such as pig and bovine (Wang et al., 1998; Dinnyes et 66
al., 2000), camelids (Ruiz et al., 2011; 2013) and humans (Imesch et al., 2013). 67
Also, chemical oocyte activation has been used in llamas and alpacas after Intra Cytoplasmic 68
Sperm Injection (ICSI; Sansinena et al., 2007; Conde et al., 2008), Somatic Cell Nuclear Transfer 69
(SCNT; Sansinena et al., 2003; Wani et al., 2010) or vitrification (Ruiz et al., 2013); however, the use 70
of these techniques could potentially alter the activation process and therefore hinder the correct 71
interpretation of results regarding to the outcomes of the oocyte activation protocol per se. 72
73
In previous llama studies (Sansinena et al., 2003; 2007) 36 to 63% of the oocytes were 74
activated using ionomycin and cycloheximide or ionomycin and the phosphorylation inhibitor 6-75
DMAP after SCNT or ICSI resulting in the development of 4-8 cells embryos; however, few oocytes 76
developed up to the morula stage and no blastocysts formation was recorded. On the contrary, the 77
activation of llama oocytes using ionomycin and 6-DMAP after ICSI resulted in a 36% of oocytes 78
(9/25) reaching the two cells stage, and from those 44% (4/9) developed into blastocysts (Conde et al., 79
2008). Similarly, the use of a sequential chemical activation protocol in alpacas consisting on 80
ionomycin and 6-DMAP resulted in about 5 to 17 % of blastocyst formation in control oocytes 81
compared to the 0 to 3 % of those that had been previously vitrified (Ruiz et al., 2013). It is difficult to 82
compare the results of the above studies with the present one since they have not been exclusively 83
designed to evaluate the efficiency of oocyte activation. 84
4
It has been documented that parthenogenetic activation depends on several 85
factors such as type of stimuli, age of the oocyte and animal species, for 86
instance protocols described for bovine may not be optimal for buffalo or 87
equine oocytes (Wani, 2008). Whether or not exists a differential response to chemical 88
activation protocols between alpacas and llamas oocytes is still unknown. 89
The present study was designed to compare the cleavage and blastocyst formation rate of in 90
vitro matured alpaca and llama oocytes after a chemical activation protocol based on ionomycin and 91
the phosphorylation inhibitor 6-DMAP. 92
93
2. Materials and methods 94
All chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, Mo, USA) unless 95
stated otherwise. 96
2.1. Collection and in vitro maturation of alpaca and llama Cumulus Oocyte Complexes (COCs) 97
Alpaca (n=90) and llama (n=85) ovaries were collected at a local slaughterhouse and 98
transported within 2-3 h to the laboratory of Reproductive Biotechnology of National University of 99
Huancavelica in Peru (3680 above sea level). Ovaries were washed in saline solution at 0.9%, follicles 100
(2-6 mm in diameter) were aspirated through a 21G needle connected to a 5 mL syringe and the 101
follicular contents were transferred into a 15 mL conical tube (Falcon) and allowed to settle for 20 m. 102
The sediment was aspirated with a Pasteur pipette and transferred into a 60 mm petri dish containing 103
mSOF-HEPES (Takahashi and First, 1992; Ruiz et al., 2011; 2013) for COCs identification and 104
evaluation. The COCs were evaluated using a stereomicroscope (10X) and classified as previously 105
described (Ratto et al., 2005). Briefly, COCs were classified according to the number of cumulus cell 106
layers and the morphology of cytoplasm as: category 1- COCs with > 5 layers of compact cumulus 107
cells and homogeneous cytoplasm; category 2- COCs with 2 to 5 compact layers of cumulus cells and 108
homogeneous cytoplasm; category 3- COCs with 1 to 2 layers of granulosa cells or partly denuded and 109
5 vacuolated cytoplasm; and category 4- denuded oocyte or oocytes with granular cytoplasm. Only 110
category 1 and 2 COCs were subjected to in vitro maturation. Groups of 8 to 12 alpaca (n=280) and 111
llama (n=340) oocytes were in vitro matured for 26-28 h (Ratto et al., 2005; 2007) in 50 µl drops of 112
maturation medium: TCM 199 (M2520) supplemented with sodium pyruvate (P5280) 0.2 mM, HEPES 113
(H3375) 25 mM, gentamicin sulphate (G3632) 50 µg/ml, FSH (F2293) 0.02 units/ml, estradiol - 17β 114
(E8875) 1 µg/ml and fetal calf serum (F6178) 10% (v/v) at 39ºC in an atmosphere of 5% CO2 in 115
humidified air. After in vitro maturation, oocytes were denuded by vortex agitation for 2 min in 116
mSOF-HEPES solution with hyaluronidase (H3506) at 0.1%, and washed at least twice in mSOF-117
HEPES. The presence of a polar body observed under stereomicroscopy (20X) was considered as a 118
valid indicator of oocyte nuclear maturation (metaphase II; Ruiz et al., 2011; 2013). 119
120
2.2. Parthenogenetic activation of oocytes and in vitro culture 121
Second metaphase alpaca (n=224) and llama (n=240) oocytes were activated according to the 122
procedure described by Wani (2008), with slight modifications (Ruiz et al., 2011; 2013). Briefly, 123
oocytes were exposed for 4 min to 5 µM Ionomycine in SOF-HEPES supplemented with 1 mg/ml 124
BSA (A7030) at room temperature followed by 3 h incubation in 80 μl drops of mSOF-IVC 125
supplemented with 3 mg/ml BSA, 2 mM 6-dimethylaminopurine (6-DMAP, D2629) and 12.5 μM 126
cytochalasin B (C6762) at 39 ºC in an atmosphere of 5% O2, 5% CO2 and 90% N2 in humidified air. 127
Then activated oocytes were transferred to 40 µl drops of mSOF-IVC supplemented with 3 mg/ml 128
BSA and in vitro cultured for 8 days at 39 ºC in an atmosphere of 5% O2, 5% CO2 and 90% N2 in 129
humidified air (Day 0: Oocyte activation). 130
131
2.3. Statistical analysis 132
Data were presented in proportion and mean % ± SEM. The proportion and the percentage of 133
oocyte category, cleavage, morula and blastocyst were analysed by Chi-square test and Student’s t- 134
6 test, respectively. The statistical power of the present study was 44 %. The level of statistical 135
significance was set at P < 0.05. 136
137
3. Results 138
A total of 350 and 400 COC were collected after follicle aspiration from 90 alpaca and 85 139
llama abattoir-derived ovaries (average, 3.8 vs 4.7 COCs per ovary, respectively). A higher proportion 140
(P < 0.03) of category 3 COCs were collected from alpaca than llama ovaries, however, there were no 141
significant differences (P = 0.1) in the remaining COCs categories between species (Table 1). A total 142
of 224 and 240 alpaca and llama matured oocytes were chemically activated, respectively. Cleavage, 143
morula and blastocysts formation rate did not differ (P = 0.8) between groups (Table 2; Figure 1). 144
145
4. Discussion 146
Based on the result of this study alpaca and llama oocytes develop to morula and blastocysts 147
stages after the administration of a chemical activation protocol based on a sequential treatment with 148
Ionomycin and 6-DMAP/ cytochalasin B. 149
The high rate of oocyte in vitro maturation obtained in the present study for alpaca and llama (80 and 150
85%, respectively) is similar to those described in previous studies (Ratto et al., 2005; 2007) and 151
greater that those reported by others (Sansinena et al., 2003; 2007; Conde et al., 2008). 152
Also, the percentage of cleavage, morula and blastocyst development rate obtained in the present 153
study were higher than those described in previous llama studies (Sansinena et al., 2003; Conde et al., 154
2008), where oocytes were chemically activated using ionomycin followed by cicloheximide instead 155
of 6-DMAP to inhibit protein synthesis. It has been documented in several species that the use of 6-156
DMAP exerts a higher inhibition of maturation promoting factor (Wang et al., 1998) resulting in early 157
pronuclei formation (Guo-Cheng et al., 2005). Indeed, lower cleavage and blastocyst rate in one of 158
these previous llama studies (Conde et al., 2008) could also be attributed to a longer (10 min) oocyte 159
exposure to ionomycin resulting in a reduced oocyte viability and further development. 160
7 The efficacy of parthenogenetic activation depends on several factors such 161
as type of stimuli, age of the oocyte and animal species. For instance, a high 162
rate of activation was observed in bovine oocytes after in vitro maturation for 163
30-40 vs 22 h followed by electric activation (Suzuki et al., 1999). Indeed, 164
in equine oocytes, the use of Ionomycin and 6-DMAP/ cytochalasin B did activate a great 165
number of oocytes after in vitro maturation for 42 h (Pimentel et al., 2002). In addition, oocytes of 166
some species (Bubalus bubalis) are more prone to parthenogenetic activation with ethanol, whereas a 167
low rate of activation has been described in dromedary using the same protocol (Wani, 2008). The 168
studies mentioned above suggest that animal species is an important factor that may influence the 169
response of oocytes to an activation protocol, therefore, considering that alpacas and llamas are 170
different species it is necessary to determine the effect of a specific protocol on oocyte developmental 171
competence after chemical activation. 172
There have been only 2 studies developed in alpaca oocyte activation (Ruiz et al., 2011; 2013) and the 173
present study is the first to describe a preliminary result from llama oocyte after chemical activation. 174
Previously, llama oocytes have been chemically activated (Sansinena et al., 2003; Conde et al., 2008) 175
with a similar protocol as the one used in this study, and blastocyst development ranged from 0 to 44 176
%; however, in those studies, oocytes were activated after Somatic Cell Nuclear Transfer (SCNT) or 177
Intra Cytoplasmic Sperm Injection (ICSI), both invasive procedures that may influence the oocyte 178
activation process, therefore caution should be taken to the interpretation of these results. The 179
proportion of blastocysts formation obtained in the present study was higher than that reported 180
previously (Ruiz et al., 2011; 2013). A plausible explanation could be attributed to an increased 181
selection pressure of oocyte quality before in vitro maturation procedure. On the other hand, morula 182
and blastocysts formation obtained in the present study (47.0 ± 2.0 and 45.8 ± 1.4, 35.8 ± 2.2 and 183
28.1 ± 1.1%, for alpaca and llama, respectively) were higher than those reported previously in 184
dromedary using a similar activation protocol (Wani, 2008). 185
8 In conclusion, alpaca and llama oocytes can be effectively activated after a sequential incubation with 186
5 µM Ionomycin and 2 mM 6-DMAP/12.5 μM cytochalasin B resulting in consistent high rates of in 187
vitro blastocyst development, that could be used to assess oocyte viability/functionality after in vitro 188
maturation or cryopreservation . 189
Conflict of interest 190
None conflict of interest statement 191 192
Acknowledgments 193
This work has been supported by Universidad Nacional de Huancavelica and the Socio-194
economic Development Fund Camisea (FOCAM) from Gobierno Regional de Huancavelica, 195
Perú. We thank the Belgian Technical Cooperation (BTC) in Peru and the Organization of 196
American States (OEA) that provided the financial support of Jaime Ruiz’s PhD program at 197
the Universidad Austral de Chile. 198
199
References 200 201 Abdoon, A.S.S., Kandil, O.M., Berisha, B., Kliem, H., Schams, D., 2007. Morphology of dromedary 202 camel oocytes and their ability to spontaneous and chemical parthenogenetic activation. Reprod. 203 Domest. Anim. 42, 88–93. 204 Brahmasani, S.R., Yelisetti, U.M. , Katari, V., Komjeti, S., Lakshmikantan, U., Mohanchandra, R.P., 205 Sisinthy, S. 2013. Developmental ability after parthenogenetic activation of in vitro matured oocytes 206 collected postmortem from deers. Small Rumin. Res. 113, 128-135. 207 Conde, P.A., Herrera, C., Trasorras, V.L., Giuliano, S., Director, A., Miragaya, M.H., Chaves, M.G., 208 Carchi, M.I., Stivale, D., Quintans, C., Agüero, A., Rutter, B., Pasqualini, S., 2008. In vitro production 209 of llama (Lama glama) embryos by IVF and ICSI with fresh semen. Anim. Reprod. Sci. 109, 298–308. 210 Dinnyes, A., Dai, Y., Jiang, S., Yang, X., 2000. High developmental rates of vitrified bovine oocytes 211 following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. 2000. 212 Biol. Reprod. 63, 513-518. 213 Eppig, J. J.,Wigglesworth, K., Hirao, Y., 2000. Metaphase I arrest and spontaneous parthenogenetic 214 activation of strain LTXBO oocytes: chimeric reaggregated ovaries establish primary lesion in 215 oocytes. Dev. Biol. 224, 60-68. 216 Fedorushchenko, A. N., Koval, T. I.U., Khamidov, D. K.H., 1996. The effect of a nerve growth factor 217 from different biological sources on the spontaneous maturation of mouse oocytes and on the 218 parthenogenetic activation of pronucleus formation. Tsitologiya 38, 1211-1216. 219 Gasparrini, B., Boccia, L., De Rosa, A., Di Palo, R., Campanile, G., Zicarelli, L., 2004. Chemical 220 activation of buffalo (Bubalus bubalis) oocytes by different methods: effects of aging on post-221 parthenogenetic development. Theriogenology 62, 1627 – 1637. 222 Guo-Cheng, L., Dong, H., Yan-Guanng, W., Zheng-Bin, H., Suo-Feng, M., Xin-Yong, L., Chong-Le, 223 C., Jing-He, T., 2005. Effects of duration, concentration and timing of ionomycin and 6-224
9 dimethylaminopurine (6-DMAP) treatment on activation of goat oocytes. Mol. Reprod. Dev. 71, 380-225 388. 226 Imesch, P., Scheiner, D., Xie, M., Fink, D., Macas, E., Dubey, R., Imthurn, B., 2013. Developmental 227 potential of human oocytes matured in vitro followed by vitrification and activation. J. Ovarian Res. 228 6:30. 229 Iwamoto, M., Onishi, A., Fuchimoto, D., Somfai, T., Takeda K., Tagami, A., Hanada, H., Noguchi, J., 230 Kaneko, H., Nagai, T. Kikuchi, K., 2005. Low oxygen tension during in vitro maturation of porcine 231 follicular oocytes improves parthenogenetic activation and subsequent development to the blastocyst 232 stage. Zygote 13, 335-345. 233 Khatir, H., Anouassi, A., Tibary, A., 2009. In vitro and in vivo developmental competence of 234 dromedary (Camelus dromedarius) oocytes following in vitro fertilization or parthenogenetic 235 activation. Anim. Reprod. Sci. 113, 212-219. 236 Kharched, S.D., Birade, H.S., 2013. Parthenogenesis and activation of mammalian oocytes for in vitro 237 embryo production: A review. Adv. Bios. Biotech. 4, 170-182. 238 Krivokharchenko, A., Popova, E., Zaitseva, L., Vilianovich, L., Ganten, D., Bader, M., 2003. 239 Development of parthenogenetic rat embryos. Biol. Reprod. 68, 829-836. 240 Lechniak, D., Cieslak, D., Sosnowski, J., 1998. Cytogenetic analysis of bovine parthenotes after 241 spontaneous activation in vitro. Theriogenology 49, 779-785. 242 Mesbah, S. F., Kafi, M., Nili, H., Nasr-Esfahani M. H., 2004. Spontaneous parthenogenesis and 243 development of camel (Camelus dromedarius) oocytes. Vet. Rec. 155, 498-500. 244 Ongeri, E.M., Bormann, C.L., Butler, R.E., Melican, D., Gavin. W.G., Echelard, Y., Krisher, R.L., 245 Behboodi, E., 2001. Development of goat embryos after in vitro fertilization and parthenogenetic 246 activation by different methods. Theriogenology 55, 1933-1945. 247 Pimentel, A.M., Bordignon, V., Smith, L.C., 2002. Effect of meiotic resumption delay on in vitro 248 maturation and parthenogenetic development of equine oocytes. Theriogenology 57: 735. 249 Ratto, M., Berland, M., Huanca, W., Singh, J., Adams, G., 2005. In vitro and in vivo maturation of 250 llama oocytes. Theriogenology 63, 2445-2457. 251 Ratto, M., Gómez, C., Berland, M., Adams, G., 2007. Effect of ovarian superstimulation on COC 252 collection and maturation in alpacas. Anim. Reprod. Sci. 97, 246-256. 253 Ruiz, J., Landeo, L., Artica, M., Ratto, M., Correa, J., 2011. Activación química de ovocitos de alpaca 254 vitrificados después de la maduración in vitro. Rev. Invest. Vet. Perú 22, 206–212. 255 Ruiz, J., Landeo, L., Mendoza, J., Artica, M., Correa, J.E., Silva, M., Miragaya, M., Ratto, M.H., 256 2013. Vitrification of in vitro mature alpaca oocyte: Effect of ethylene glycol concentration and time 257 of exposure in the equilibration and vitrification solutions. Anim. Reprod. Sci. 143, 72-78. 258 Sansinena, M., Taylor, S., Taylor, P., Denniston, R., Godke, R., 2003. Production of nuclear transfer 259 llama (Lama glama) embryos from in vitro matured llama oocytes. Cloning Stem Cells 5, 191-198. 260 Sansinena, M., Taylos, S., Taylor, P., Schmidt, E., Denniston, R., Godke, R., 2007. In vitro production 261 of llama (Lama glama) embryos by intracytoplasmatic sperm injection: Effect of chemical activation 262 treatments and culture conditions. Anim. Reprod. Sci. 99, 342-353. 263 Suzuki, H., Liu, L., Yang, X., 1999. Age-dependent development and surface ultrastructural changes 264 following electrical activation of bovine oocytes. Reprod. Fertil. Dev. 11:159–165. 265 Takahashi, Y., First, N.L., 1992. In vitro development of bovine one-cell embryos: influence of 266 glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology 37, 963-978. 267 Wani, N.A., 2008. Chemical activation of in vitro matured dromedary camel (Camelus dromedarius) 268 oocytes: Optimization of protocolos. Theriogenology 69, 591-602. 269 Wani, N.A., Wernery, U., Hassan, F.A.H., Wernery, R., Skidmore, J.A., 2010. Production of the First 270 Cloned Camel by Somatic Cell Nuclear Transfer. Biol. Reprod. 82, 373–379. 271 Wang, W., Macháty, Z., Abeydeera, L., Prather, R., Day, B., 1998. Parthenogenetic activation of pig 272 oocytes with calcium ionophore and the block to sperm penetration after activation. Biol. Reprod. 58, 273 1357-1366. 274 Zernicka-Goetz, M., 1991. Spontaneous and induced activation of rat oocytes. Mol. Reprod. Dev. 28, 275 169-176. 276 277
10 Figure 1. Representative photographs of in vitro mature alpaca and llama oocyte after a sequential 278
incubation with 5 µM Ionomycin and 2 mM 6-DMAP/12.5 μM cytochalasin B for 4 minutes and 3 279
hours, respectively followed by in vitro culture mSOF-IVC for 8 days (Day 0: Oocyte activation). 280
2 and 4 cell alpaca (A) and llama (B) embryos 2 days after chemical activation. Alpaca (C) and llama 281
(D) compact morulas 5 days after chemical activation (arrows). Alpaca (E) early blastocyst and llama 282
(F) expanded blastocyst 7 days after chemical activation. 283
284 285 Table 1.Cumulus Oocyte Complexes categories collected after follicular aspiration of follicles 286
between 3 to 6 mm in diameter, from alpaca and llama abattoir-derived ovaries (mean % ± SEM). 287
288
11 Species Ovaries
(n)
COC
(n)
Category 1*
Category 2*
Category 3
Category 4*
Alpaca
90 350
210/350
(60 ± 6.5)
70/350
(20 ± 6.7 )
53/350a
(15 ± 2.0 )
17/350
( 5 ± 1.7)
Llama 85 400
260/400
(65 ± 7.5 )
80/400
(20 ± 4.0)
40/400b
(10 ± 3.5)
20/400
(5 ± 2.2)
a vs bValues with different superscript within the column differ (P<0.03). 289
*No significant difference between species in any of the endpoints (P= 0.1). 290
There was no effect of replicates (n=4 replicates). 291
292
293
294
295
296
297
298
299
300
301
302
303
Table 2. Cleavage, morula and blastocyst development rateafter chemical activation of in vitro 304
matured alpaca and llama oocytes with ionomycin and 6-DMAP (mean % ± SEM). 305
306
Species Total
oocytes
Cleavage* Morula* Blastocysts/from
total Oocyte*
Blastocysts/from
Cleavage Oocyte*
Alpaca 224 140/224 105/224 50/224 50/140
12
(62.5 ± 2.7) (47.0 ± 2.0) (22.5 ± 1.3) (35.8 ± 2.2)
Llama 240 160/240
(66.7 ± 5.2)
110/240
(45.8 ± 1.4)
45/240
(18.7 ± 1.0)
45/160
(28.1 ± 1.1)
*No significant difference between species in any of the endpoints (P= 0.8). 307
There was no effect of replicates (n=4 replicates). 308
309