Accepted Manuscript

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

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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