Efficient one-step direct transfer to recipients of thawed bovine embryos cultured in vitro and frozen in chemically defined medium

Journal Pre-proof Efficient one-step direct transfer to recipients of thawed bovine embryos cultured in vitro and frozen in chemically defined medium Enrique Gómez, Susana Carrocera, David Martín, Juan José Pérez-Jánez, Javier Prendes, José Manuel Prendes, Alejandro Vázquez, Antonio Murillo, Isabel Gimeno, Marta Muñoz PII:

S0093-691X(20)30069-8

DOI:

https://doi.org/10.1016/j.theriogenology.2020.01.056

Reference:

THE 15353

To appear in:

Theriogenology

Received Date: 17 September 2019 Revised Date:

26 January 2020

Accepted Date: 28 January 2020

Please cite this article as: Gómez E, Carrocera S, Martín D, Pérez-Jánez JuanJosé, Prendes J, Prendes JoséManuel, Vázquez A, Murillo A, Gimeno I, Muñoz M, Efficient one-step direct transfer to recipients of thawed bovine embryos cultured in vitro and frozen in chemically defined medium, Theriogenology (2020), doi: https://doi.org/10.1016/j.theriogenology.2020.01.056. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

CREDIT AUTHOR STATEMENT Enrique Gómez: Conceptualization; Data curation; Formal analysis; Funding acquisition; Supervision; Methodology; Writing-original draft; Project administration Susana Carrocera: Investigation; Data curation; Resources David Martín: Investigation; Data curation; Editing; Resources Juan José Pérez-Jánez: Investigation; Validation Javier Prendes: Investigation José Manuel Prendes: Investigation Alejandro Vázquez: Data curation; Investigation; Resources; Writing - Review Antonio Murillo: Investigation; Writing - Review Isabel Gimeno: Investigation; Writing - Review Marta Muñoz: Conceptualizacion; Funding acquisition; Investigation; Methodology; Supervision; Writing - Review

REVISED 1

EFFICIENT ONE-STEP DIRECT TRANSFER TO RECIPIENTS OF THAWED BOVINE EMBRYOS

2

CULTURED IN VITRO AND FROZEN IN CHEMICALLY DEFINED MEDIUM

3 4

Enrique Gómez1*, Susana Carrocera1, David Martín1, Juan José Pérez-Jánez2, Javier Prendes2,

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José Manuel Prendes2, Alejandro Vázquez3, Antonio Murillo1,4, Isabel Gimeno1, Marta Muñoz1

6 7

1

8

2

9

Industrial de Roces 5, Gijón 33211, Spain.

Centro de Biotecnología Animal-SERIDA, Camino de Rioseco 1225, Gijón 33394, Spain. Cooperativa de Agricultores y Usuarios de Gijón, Carretera Carbonera 2230, Polígono

10

3

Asturian Biotechnology, Galeno, 2248, Polígono Industrial de Roces 5, Gijón 33211, Spain.

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4

Present address: Animal Production and Industrialization Research Unit, Engineering Faculty,

12

Universidad Nacional de Chimborazo, Riobamba EC060150, Ecuador.

13

* Corresponding Author: [email protected]

14 15

Abstract

16

Direct transfer (DT) of cryopreserved embryos to recipients facilitates on-farm application. We

17

analyzed a new freezing/thawing (F/T) procedure for in vitro produced (IVP) embryos,

18

integrating: 1) an ethylene-glycol based system; 2) a culture step without protein; and 3) a

19

synthetic protein substitute (CRYO3) in cryopreservation medium. IVP embryos from abattoir

20

ovaries were cultured in groups in BSA-containing synthetic oviduct fluid with or without 0.1%

21

fetal calf serum (FCS) until Day-6. Morulae and early blastocysts were subsequently cultured

22

without protein from Day-6 onwards. Day 7 and Day 8 expanded blastocysts (EXB) were

23

subjected to F/T or vitrification/warming (V/W). Thawed and warmed EXB were cultured in

24

vitro, and development rates, cell counts and dead cells were analyzed in surviving embryos.

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V/W improved survival over F/T (live and hatching rates at 2h, 24h and 48h) (P<0.0001), and

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FCS before Day 6 did not affect in vitro survival. After F/T, embryos had lower cell counts in the 1

REVISED 27

ICM, TE and total cells than after V/W. Day-7 embryos after F/T showed % apoptotic, %

28

pycnotic and % total dead cells higher (p<0.05) than their Day-8 counterparts, probably

29

because F/T reduced the numbers of ICM cells within Day-8 embryos. Thereafter, Day-7

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blastocysts were transferred to heifers in an experimental herd. There were no differences in

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birth rates with frozen (-FCS [n=40]: 45%; +FCS [n=14]: 28%), vitrified (-FCS [n=47]: 53%; +FCS

32

[n=11]: 36%) and fresh (-FCS [n=30]: 47%; +FCS [n=17]: 53%) embryos. However, frozen

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embryos produced with FCS showed 5/9 miscarriages after Day-40. Calves born from frozen

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(n=22), vitrified (n=29) and fresh (n=22) transfers did not differ in birth weight, gestation

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length and daily gain weight (P>0.10). Subsequently, transfer of frozen embryos (n=29) derived

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from oocytes collected from live, hormonally stimulated cows in experimental herd, led to

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pregnancy rates of 57% (heifers) and 40% (dry cows). with EXB on Day-62 Finally, embryos

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produced with BSA were transferred to cows in an on-field trial (frozen [n=80]; fresh [n=58]),

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with no differences in pregnancy rates (days 30-40). Pregnancy and birth rates could not be

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predicted from in vitro approaches. The new F/T system yields pregnancy and birth rates

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comparable to vitrified and fresh embryos without birth overweight. The absence of products

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of animal origin, defined chemical composition, and direct transfer entail sanitary,

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manufacturing and application advantages.

44 45

Keywords: Bovine, IVP embryo, Freezing, Vitrification, Pregnancy.

46 47

1. Introduction

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Cryopreservation is essential in assisted reproductive technology, as it allows embryo storage

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for very long periods, successful commercial worldwide exchanges, and it is necessary in case

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of recipient shortage or embryo surplus. Two main cryopreservation procedures are available

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for bovine embryos: freezing/thawing (F/T), and vitrification/warming (V/W). Therefore,

52

improving survival after F/T and V/W are major objectives in cattle embryo technology. 2

REVISED 53

F/T and V/W have each particular advantages and disadvantages. Thus, V/W is simple, with no

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expensive equipment required; cheap and, when embryos are few, no time-consuming.

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However, V/W requires well-trained skills, direct transfer is rather a challenge, and some

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vitrification procedures are not compliant with sanitary requirements [1]. On its side, F/T

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requires expensive equipment but allows faster management when embryos are many, and

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these techniques fulfill sanitary requirements. A desirable step that facilitates on-farm

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application of cryopreserved embryos is the direct transfer (DT) in straw. Within F/T, DT has

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greatly simplified post-thawing rehydration with in vivo embryos [2]. We believe that both

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techniques F/T and V/W should be available in the IVP laboratory to be used depending on

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particular circumstances of embryo production and commercial concerns.

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V/W techniques have been generally described to perform better than F/T with in vitro

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produced (IVP) embryos. However, this notion lies in comparisons between V/W and F/T

65

mainly based on in vitro experiments [3-11]. In contrast, experiments that incorporate embryo

66

transfer (ET) to compare F/T vs. V/W are scarce and constrained by embryo selection and

67

discard before ET, low numbers of ETs [12] or circumscribed to cohorts of micromanipulated

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embryos [13]. However, upon verification of any kind of embryo cryopreservation reduces

69

pregnancy and/or birth rates, it is surprising to confirm that several reports performed with

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high numbers of ETs with IVP embryos that analyzed F/T vs. fresh embryos do not find such

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reductions [14-16] or the decrease in pregnancy and birth rates for F/T embryos is not

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relevant, being in the survival range observed for V/W embryos vs. fresh embryos [17].

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Together with the above, cattle breeding industries use F/T technology with IVP embryos for

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commercial exchanges. This suggests that recent improvements led to F/T technology with

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efficacy levels comparable to V/W with IVP embryos. Such information, nevertheless, was not

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publicly available until a recent work from Sanches and co-workers [18]. These authors

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obtained high pregnancy rates (PR) with an F/T procedure with DT of IVP embryos. The

78

technique described was a modification of a classical ethylene-glycol (EG)-based technique [2], 3

REVISED 79

simple and performed on n=800 embryo transfer (ET) operations (fresh, V/W and F/T) in a

80

unique herd with parous recipients. Although non-significant, F/T pregnancy rates were above

81

those of V/W, both below the PR reported with fresh embryos. Collectively, the above reports

82

are not supportive of V/W performing better than F/T when IVP embryos undergo

83

development to term upon transfer to recipients.

84

In the present study, we designed and analyzed a new F/T procedure that integrates three

85

findings from recent studies. First, we modified the cryopreservation system from Sanches et

86

al. [18] with a single seeding (i.e., ice nucleation induction) step, and a different cryoprotectant

87

column distribution (a larger first column) in the straw. Second, we used a 24h embryo culture

88

step in synthetic oviduct fluid (SOF) medium without protein that has shown to yield embryos

89

with improved survival after vitrification and transfer, significant reduction in miscarriage and

90

higher birth rates [19, 20]. Third, we replaced the protein supplements contained in

91

cryoprotectant solutions, typically products of animal origin [18], with a synthetic substitute

92

(CRYO3) that has improved in vitro survival to freezing of bovine embryos over serum albumin

93

(BSA) [21]. To our knowledge, CRYO3 has not been used with bovine embryos transferred to

94

recipients.

95

The DT-F/T system procedure described herein was tested vs. a group of embryos submitted to

96

a V/W procedure that consistently has produced birth rates >45% in our laboratory [19, 20]

97

and with embryos transferred fresh. We analyzed in vitro survival, and, in blastocysts that

98

hatched after cryopreservation, inner cell mass (ICM) and trophectoderm (TE) cell distribution

99

and apoptosis incidence by a single technique. Thereafter, the long-time effects of the DT-F/T

100

were compared with V/W and fresh embryos after ET, by analyzing pregnancy and birth rates,

101

pregnancy length and weight of calves born. Ultimately, a demonstration trial using oocytes

102

collected from hormonally stimulated cows and DT-F/T embryos alone in the experimental

103

herd, and an embryo transfer trial carried out in cows within commercial farms (fresh vs. DT-

104

F/T embryos) confirmed the pregnancy rates obtained in experimental herd. 4

REVISED 105 106

2. Materials and methods

107

All experimental procedures were approved by the Animal Research Ethics Committee of

108

SERIDA (PROAE 26-2016; Resolución de 25 de Julio de 2016 de la Consejería de Medio Rural y

109

Recursos Naturales), following the European Community Directive 86/609/EC. All reagents

110

were purchased from SIGMA (Madrid, Spain) unless otherwise stated. General procedures for

111

in vitro embryo production have been described [22, 23]. The following are descriptions in

112

brief.

113 114

2.1. Oocyte collection and in vitro maturation (IVM)

115

Ovaries were collected from slaughtered cows in SERIDA (Matadero de Guarnizo, Cantabria,

116

Spain) and Asturian Biotechnology –Asturbiotech- (SEMAGI, Gijón, Spain). Ovaries were

117

transported to the laboratory in saline with penicillin 100 IU/mL and streptomycin sulfate 100

118

µg/mL and kept at 25 °C to 30 °C during collection and transportation.

119

Antral follicles (3-8 mm in diameter) were aspirated with an 18-g needle connected to a

120

syringe and transferred to holding medium (HM) (TCM199; Invitrogen, Barcelona, Spain), 25

121

mM HEPES and 0.4 mg/mL BSA). Good-quality oocytes with more than three layers of compact

122

cumulus cells and homogenous cytoplasm were selected for in vitro maturation (IVM). The

123

cumulus–oocyte complexes (COCs) were rinsed three times in HM. Selected COCs were

124

washed three times in maturation medium (MM) consisting of TCM199 NaHCO3 (2.2 mg/mL)

125

supplemented with 10% (v/v) FCS (F4135), 1.5 μg/mL of porcine FSH-LH (Stimufol; ULg FMV,

126

Liège, Belgium) and 1 μg/mL 17 β-estradiol. COCs were transferred (n=30-50) into each well of

127

a four-well dish (500 μL of IVM medium per well) and cultured for 22 to 24 hours at 38.7 °C, 5%

128

CO2 and high humidity.

129 130

2.2. In vitro fertilization (IVF) 5

REVISED 131

Commercial frozen sperm from Asturiana de los Valles (AV) bulls (n=2) and Holstein (n=3) bulls

132

with proven fertility were thawed and used for IVF (Day 0) in SERIDA, and n=12 AV bulls in

133

Asturbiotech. Motile sperm were obtained by a swim-up procedure. Thawed semen was added

134

to a tube with 1mL of pre-equilibrated Sperm-TALP (Tyrode’s albumin lactate pyruvate). After

135

1h incubation, the supernatant upper layer with motile sperm was recovered. Sperm was

136

centrifuged for 7 minutes at 200x g and the supernatant was aspirated. COCs were washed

137

twice in HM and placed in four-well culture dishes containing pre-equilibrated fertilization

138

medium (Fert-TALP) with heparin (10 μg/mL; Calbiochem, La Jolla, CA, USA). Spermatozoa

139

were added at a concentration of 2 x 106 cells/mL in 500 μL of medium per well, with a

140

maximum of 50 COCs. IVF was accomplished by incubating oocytes and sperm cells together

141

for 18 to 20 hours at 38.7 °C in a 5% CO2 atmosphere with saturated humidity.

142 143

2.3. In vitro culture (IVC)

144

Cumulus cells were detached using a vortex and fertilized oocytes were cultured in modified

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synthetic oviduct fluid (mSOF) containing amino acids (BME amino acids solution, 45 μL/mL

146

and MEM non-essential amino acids solution, 3.3 μL/mL), citrate (0.1 μg/mL), myo-inositol (0.5

147

μg/mL), and BSA (6 mg/mL) with 0.1% (v/v) FCS (SIGMA F4135) or without FCS [24]. In vitro

148

culture was carried out at 38.7 °C, 5% CO2, 5% O2, 90% N2, and saturated humidity. Embryos

149

were cultured in groups from Day-0 until Day-6 with BSA or with BSA+FCS. On Day-6 (143h PI),

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excellent and good quality (grade 1 and grade 2) morulae and early blastocysts were selected

151

and cultured either individually (SERIDA; 12 µL) or in groups (Asturbiotech; 20 embryos/50 µL)

152

in mSOF with 0.5 mg/mL polyvinyl-alcohol PVA (P8136, instead of BSA or BSA+FCS) under

153

mineral oil. Blastocyst development was monitored by optical microscopy (60X) on Day-7

154

(168h PI) and Day-8 (184h PI).

155 156

2.4. Embryo vitrification, warming and in vitro survival 6

REVISED 157

Vitrification procedures have been described in detail [7]. Briefly, expanded blastocysts were

158

vitrified in two-steps with fibreplugs (CryoLogic Vitrification Method; CVM). Procedures were

159

performed on a heated surface (41 °C) in a warm room (25 °C). Embryos were handled in a

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basic vitrification medium (VM: TCM 199-HEPES + 20% (v/v) FCS). Groups of one to five

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blastocysts were exposed to VM with 7.5% ethylene-glycol (EG, 102466-M), 7.5% DMSO

162

(D2650, vitrification solution-1) for 3 min, and then moved into a drop containing VM with

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16.5% EG, 16.5% DMSO and 0.5 M sucrose (vitrification solution-2; VS2). The time spent by the

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embryos in VS2 (including loading) was 20 to 25 sec. Samples were vitrified by touching the

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surface of a supercooled block placed in LN2 with a hook. Vitrified embryos held in fibreplugs

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were stored in closed straws in LN2 until warming. Embryos were warmed in one-step by

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directly immersing the fibreplug end in 800µL of 0.25 M sucrose in VM, where the embryo was

168

kept for 5 min and washed twice in VM and twice in mSOF containing 6 mg/mL BSA and 10%

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FCS before ET or in vitro culture. In vitro survival rates were analyzed by culturing Day-7 and

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Day-8 vitrified embryos in droplets of 25µL of mSOF containing 6 mg/mL BSA and 10% FCS.

171

Embryo survival was evaluated in terms of re-expansion and hatching rates at 24 and 48 h.

172 173

2.5. Embryo freezing and thawing

174

Procedures were performed on a heated surface (35 °C) in a warm room (25 °C). Expanded

175

blastocysts were washed three times in PBS+4g/L BSA either individually or in groups up to

176

eight embryos. Subsequently, embryos were briefly washed once and loaded in freezing

177

medium, containing PBS (P4417), 1.5M EG and 20% CRYO3 (# 5617, Stem Alpha, France) for 10

178

min. Embryos in freezing medium were aspirated in a French straw, loaded between 2 columns

179

with PBS + 0.75M EG + 20% CRYO3, and 2 further columns PBS + 0.75M EG + 20% CRYO3 in

180

turn separated by air (see Figure 1). The straw was closed with a plug in tight contact with a

181

column of PBS + 0.75M EG + 20% CRYO3; this column took up approximately half of the

182

available length of the straw. Subsequently, straws were loaded in a programmable freezer 7

REVISED 183

(Crysalis, Cryocontroller PTC-9500,) at -6°C for 2 min and seeded once with supercooled

184

forceps in the upper column adjacent to that contained the embryo. Straws remained for eight

185

further min at -6°C and were subsequently dehydrated at -0.5°C/min up to -35 °C. Ten to

186

fifteen minutes after reaching this temperature, the straws were stored in LN2 until use. For

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thawing, embryos were held for 10s on air, and then 30s at 35 °C in a water bath. The straws

188

were carefully dried with disposable wipes humidified with 70% ethanol. For in vitro culture,

189

the straws were emptied in Petri dishes, making all contents to converge in a single drop. No

190

later than 1 min, the embryos were picked-up, washed and cultured in droplets of 25µL of

191

mSOF containing 6 mg/mL BSA and 10% FCS. For DT to recipients, each thawed straw with a

192

single embryo was directly mounted in an ET catheter without previous shaking or mixing

193

contents.

194 195

2.6. CDX2 and TUNEL staining in blastocysts

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Embryos were evaluated by simultaneous assessment of apoptotic index (TUNEL staining) and

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ICM and TE differential cell counts with CDX2 immunostaining. Hatched blastocysts (grade 1

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and grade 2) surviving vitrification/warming and freezing/thawing were fixed in 4%

199

paraformaldehyde with 0.2 mg/mL PVA and then washed and stored in phosphate buffered

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saline (SIGMA P4417) with 0.2 mg/mL PVA (PBS-PVA; pH=7.4, 4°C) until use. Embryos were

201

permeabilized for 40 min at 37°C with sodium citrate (0.1M; pH=6.0; SIGMA C8532) containing

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PVA (0.2mg/mL) and Triton (1% v/v), and subsequently washed in PBS-PVA. Samples and

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positive controls were then submitted to TUNEL reaction according to the manufacturer's

204

instructions (In situ Cell Death Detection Kit with Fluorescein, 11684795910, Roche®,

205

Mannheim, BW, Germany), whereas negative controls were incubated in TUNEL mixture

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without transferase. Following two washes in PBS-PVA, immunohistochemical detection of

207

CDX2 was performed. After permeabilization (15 minutes 0.5% Triton-X-100 in PBS -PVA) and

208

washing 5 min in rinse buffer (RB, 0.1% Triton X-100 in PBS-PVA), samples were transferred to 8

REVISED 209

blocking solution (5% normal goat serum and 0.1% Triton X-100) for 2h at room temperature.

210

Subsequently, samples were incubated for 72h at 4°C with the primary anti-CDX2 antibody

211

(Abcam 15258) diluted 1:50 in blocking solution. After washing in RB (three times 5min), the

212

embryos were incubated with Alexa Fluor conjugated secondary antibody (Goat anti-mouse

213

Alexa Fluor®594 conjugate Thermofisher A-11032) for 1h and 15 min at RT. Finally, embryos

214

were washed three times in RB, counterstained with DAPI and mounted on a glass slide with

215

Vectashield-H1000 (Vector Labs, USA) under a coverslip.

216

Embryos were analyzed with a confocal microscope (ultra-spectral Leica TCS-SP8-AOBS; Leica

217

Microsystems, Mannheim, Germany). An excitation wavelength of 488 nm was selected for

218

detection of fluorescein-12-dUTP, 594 nm to excite Alexa-594, and 405 nm wavelength to

219

excite DAPI. Photomicrographs of serial optical sections were recorded every 1.5–2 μm vertical

220

step along the Z-axis of each embryo. CDX2 positive cells, total embryonic cells, and DNA-

221

fragmented nuclei were analyzed using software ImageJ (Confocal Uniovi ImageJ; version

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1.51). Nuclei with green fluorescence (FITC) were considered TUNEL positive (fragmented

223

DNA). Total healthy nuclei were distinguished from TUNEL positive-necrotic and TUNEL

224

positive-pycnotic cells by DAPI staining based on the presence of only blue fluorescence [25].

225

Nuclei with red fluorescence were considered CDX2 positive (trophectoderm cells). Positive

226

controls for TUNEL were carried out by treating embryos with 10 IU/mL of DNase I (Takara,

227

2215A). Negative controls for immunohistochemistry were carried out omitting the primary

228

antibody.

229 230

2.7. Embryo transfer, pregnancy diagnosis, and calf phenotyping

231

Embryos were transferred in controlled conditions (experimental herd in SERIDA) (2.7.1 and

232

2.7.2) and an on-field trial performed in commercial farms with embryos produced in

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Asturbiotech (2.7.3).

234

2.7.1. Experimental assay in the experimental herd (abattoir oocytes) 9

REVISED 235

The DT-F/T system procedure was compared with V/W and ET, using embryos produced in

236

culture with BSA alone or BSA+FCS.

237

Detailed procedures have been described [26]. Briefly, recipient heifers from Holstein, AV, and

238

their crosses were synchronized in estrus with an intra-vaginal progestagen device (PRID

239

Alpha; Ceva Salud Animal) for 10-11 days combined with a prostaglandin F2α analog (Dynolitic,

240

Pfizer, Leonvet, Spain) injected 48 h before progestagen removal. ETs were performed with

241

fresh, vitrified/warmed and frozen/thawed Day-7 embryos, which show pregnancy rates

242

higher than their Day-8 counterparts [17,22,27,28,29]. All embryos cryopreserved and

243

transferred were expanded blastocysts, while embryos transferred fresh were early

244

blastocysts, blastocysts, and expanded blastocysts. All mbryos selected for transfer developed

245

to more advanced stages in culture from Day-6 to Day-7. Before the transfer, vitrified embryos

246

were warmed and examined in their morphology; embryos with fragmented or degenerate

247

appearance were discarded. Frozen/thawed embryos were directly transferred in straw and

248

not examined. Fresh embryos were washed twice and mounted in straw in PBS+4 g/L BSA. On

249

Day 7 (225±1.5 h after progestagen removal; fixed time), blastocysts were non-surgically

250

transferred to recipients under epidural anesthesia. Blood plasma P4 was measured on Day 0

251

and Day 7 (before embryo transfer) in samples collected into ethylenediamine tetraacetic acid

252

(EDTA) vacuum tubes via coccygeal vein puncture. An enzyme-linked immunosorbent assay

253

(ELISA) test operating on a 0–40 ng/mL-1 scale (DRG Diagnostics) was used. The test was

254

sensitive starting from 0.5 ng/mL-1 and cross-reactivity from steroids other than P4 was less

255

than 1%. Intra and interassay coefficients of variation were 6% and 7% respectively. Recipients

256

selected for transfer showed either standing estrus or, in the absence of clear estrous signs, P4

257

fold changes Day-7/Day-0 >8 and Day-7 P4 values >3.5 ng/mL. A healthy corpus luteum was

258

detected in transferred recipients in one ovary by ultrasonography before ET. To allow

259

individual variation to express within an equilibrate design, ETs were performed in rounds (5 –

260

7 recipients per round), each round with embryos from a single bull (n=5 bulls) and embryos 10

REVISED 261

from the three treatments (i.e. V/W, F/T and fresh) whenever possible. Pregnancy was

262

diagnosed by ultrasonography on Day 40 and Day 62. Birth rates were monitored. Body

263

weights of the calf and the mother were measured at birth, as well as gestation length; the

264

average daily gain weight of the fetus was calculated as birth weight (Kg)/gestation length

265

(days). Recipients diagnosed as non-pregnant were re-used for transfer up to three times.

266

2.7.2 Experimental assay in the experimental herd (oocytes from hormonally stimulated

267

cows)

268

The DT-F/T system procedure was assayed in a demonstration assay with Holstein cattle using

269

oocytes collected from donor cows. Donors were stimulatedulated with FSH-LH (Pluset,

270

(Laboratorios Calier, Barcelona, Spain) in a 2 x 3-days treatment with decreasing doses. Oocyte

271

collection procedures were performed by Oocyte Puncture Ultrasonography (OPU) as

272

described by Hidalgo et al, [30]. IVM, IVF (n=3 Holstein bulls) and IVC proceeded as described

273

above, but embryo cultures from Day-0 to Day-6 were performed with BSA, and no FCS. In this

274

trial, both expanded and hatched blastocysts, from Day-7 and Day-8, were transferred frozen

275

and thawed to recipients synchronized on Day-7. Recipients were heifers (n=15 ETs) and

276

uniparous, non-lactating cows (n=14 ETs). Recipients diagnosed as non-pregnant were re-used

277

for transfer no more than once. Pregnancy was diagnosed by ultrasonography at Day-40 and

278

Day-62.

279

2.7.3 On field randomized trial

280

In commercial conditions, in contrast to embryos subjected to V/W, fresh and DT-F/T embryos

281

can be transferred without a need of equipment and embryo manipulation. By this easiness,

282

only Day-7 fresh and DT-F/T were compared. Embryos were produced with BSA and no FCS

283

until Day-6 and subsequently cultured in groups without protein.

284

Embryos were transferred in n=57 commercial farms by four veterinarians. Embryo transfer

285

was performed after estrus synchronization or natural estrus, upon farmer demand. All

286

recipients were subjected to artificial insemination (AI) on Day-0 prior to ET on Day-7, in a 11

REVISED 287

known strategy to treat certain types of infertility [31,32,33]. Selected recipients were mainly

288

cows, non-lactating or at the end of lactation, that underwent previous AI ≥3 times without

289

reaching pregnancy. A minor number of recipients were heifers and beef cows. Fresh embryos

290

were transported from the laboratory to farms in straws loaded in portable incubators

291

(Minitübe Iberica, Reus, Spain) at 38.5 °C in air for <40min. Frozen embryos were thawed on

292

farm. All transferred embryos were Day-7 expanding- to fully- expanded blastocysts.

293

Pregnancy was diagnosed by ultrasonography between gestational days 30 to 40.

294 295

2.8. Statistics

296

Data were analyzed using the Proc GLM module of SAS/STAT (version 9.2; SAS Institute Inc.,

297

Cary, NC). Continuous variables requiring normalization were log-transformed. In experiments

298

concerning embryo cryopreservation and CDX2 and TUNEL staining the fixed effects included

299

were culture up to Day-6 (i.e. BSA or FCS+BSA), embryonic stage on Day-6 (morula or

300

blastocyst), age of the embryo (Day-7 or Day-8), and cryopreservation procedure (V/W or F/T).

301

Replicate, bull and recipient breed were considered as random effects. Hatching time (24h or

302

48h) was analyzed as a fixed effect within CDX2 and TUNEL staining. Significant interactions

303

between major effects were analyzed and detected. For embryo transfer and pregnancy

304

viability (Day-40, Day-62, birth) the treatments included a group of fresh transferred embryos.

305

For pregnancy and birth rates in the experimental herd, the effects included were

306

cryopreservation treatment, culture up to Day-6 (i.e. SOF+BSA, with or without FCS), bull,

307

recipient breed and number of ET (1,2,3). For calf measurements, the effects included were

308

protein replacement up to Day-6, cryopreservation treatment, bull, calf sex, calf breed and

309

mother weight (for normalization purposes). Bull and recipient breed were considered as

310

random effects. Calf body measurements and birth weight also included embryonic sex. Least

311

squares means and their errors (±SEM) were estimated for each level of fixed effects with a

312

significant F-value. However, pregnancy and birth rates were expressed as mean percentages. 12

REVISED 313

The Ryan–Einot–Gabriel–Welsch Q-test was used to compare the raw means of the levels from

314

the fixed effects (P<0.05). Pregnancy rates in the demonstration trial compared Day-7 vs. Day-

315

8 embryos, while the on-farm trial was considered randomized and analyzed by the Chi-Square

316

test.

317 318

3. Results

319

3.1. In vitro survival to cryopreservation

320

Table 1 shows the post-cryopreservation development of embryos submitted to V/W or F/T.

321

Major effects revealed that V/W showed improved performance over F/T at all survival stages

322

analyzed (P<0.0001). In contrast, the presence of FCS and the Day-6 stage of the embryos

323

(morula, blastocyst or combinations of both) did not affect overall development at any stage.

324

As expected, Day-7 embryos showed higher survival rates in vitro than Day-8 embryos

325

(P<0.001).

326

Interactions between cryopreservation treatment, culture to Day-6 and age of embryos

327

cryopreserved are shown in Table 1. Within F/T, Day-7 embryos that were cultured until Day-6

328

in BSA appeared to show improved development over other F/T embryos and, interestingly,

329

did not show significant differences at any stage with the groups of V/W embryos. Within

330

embryos that underwent V/W, no clear interactions appeared, but embryos produced with

331

BSA (days 7 and 8) and Day-7 embryos produced with FCS+BSA showed 100% survival at 2h

332

after thawing.

333 334

3.2. Cell Counts (CDX2 trophectodermal-cell staining) and TUNEL study

335

Differential cell counts were performed simultaneously with the apoptosis study in embryos

336

that hatched after cryopreservation. Hatching time affected TE cell counts (Hatching at 24h:

337

84.0±6.4; hatching at 48h: 106.9±5.4; P<0.01; not shown in tables). Significant interactions are

338

shown in Table 2, where F/T reduced the number of cells counted in the ICM of hatched 13

REVISED 339

embryos derived from Day-8 blastocysts. Overall, embryos undergoing F/T showed lower cell

340

counts than vitrified embryos in the ICM, TE and total cells.

341

In the apoptosis study (Table 3), the only significant major effect was observed within

342

supplements in culture until Day-6. Thus, the presence of 0.1% FCS + 0.6% BSA in culture, with

343

regards to 0.6% BSA alone, increased the percentage of apoptotic cells (P=0.018; 5.22±1.07 vs.

344

9.24±1.33, respectively) and tended to increase the percentage of total dead cells (P=0.056;

345

11.82±1.69 vs. 16.70±2.12, respectively) (not shown in tables). Interactions in the TUNEL study

346

were observed only for F/T within Day-7 embryos, which showed % apoptotic, % pycnotic and

347

% total dead cells significantly higher (p<0.05) than their Day-8 counterparts. These

348

parameters did not differ for Day-7 vs. Day-8 embryos after V/W, although V/W did trigger

349

pycnotic and % total dead cells higher than Day-8, F/T embryos.

350 351

3.3. Embryo transfer and pregnancy monitoring

352

3.3.1. Experimental assay in the experimental herd (abbatoir oocytes)

353

Embryos (n=159) were transferred to recipients after F/T and compared with embryos

354

cryopreserved by V/W and a group of fresh embryos transferred as controls. All frozen

355

embryos that were thawed were transferred without examination (direct transfer), but two

356

vitrified embryos were discarded upon morphology examination at warming. Pregnancies were

357

monitored on Day-40, Day-62, and birth(Table 4). Neither major effects nor interaction effects

358

were observed (P>0.05) derived from cryopreservation treatments or culture to Day-6 with or

359

without FCS. Thus, pregnancy rates with embryos undergoing F/T were similar to embryos

360

transferred fresh and after V/W.

361

3.3.2 Demonstration assay in the experimental herd (oocytes from hormonally stimulated

362

donors)

363

Embryos (n=29) were transferred to recipients after F/T at the expanded and hatched

364

blastocyst stages (Table 5). Pregnancies on Day-62 were only found for expanded blastocysts 14

REVISED 365

(Day-7: 4/7 heifers [57%] and 4/10 cows [40%]; Day-8: 2/7 heifers [29%] and 0/1 cows).

366

Hatched blastocysts did not yield pregnancies after F/T (0/4). Pregnancy rates with Day-7

367

embryos tended to be higher than those in Day-8 embryos (P=0.10)

368

3.3.3. On-field trial

369

Only frozen embryos produced with BSA were transferred since 5/9 pregnancies of embryos

370

cultured with FCS+BSA were lost in the experimental herd after Day-40 (Table 6). Pregnancy

371

rates at days 30-40 did not differ for embryos transferred fresh (n=58) or after F/T (n=80).

372

3.4. Calf weight and gestation length

373

Results in Table 7 show that there were no differences in birth weight, gestation length and

374

average daily gain weight of the fetus between embryos transferred fresh, after F/T and after

375

V/W. Calves with bodyweight >50Kg at birth were 6/29, 3/22 and 1/22 after V/W, F/T, and

376

fresh embryo transfer, respectively (P>0.05; not shown in tables).

377 378

4. Discussion

379

Experiments of embryo transfer in cattle can be preceded by an analysis of embryonic viability

380

in vitro because of the high costs of recipient pregnancy management to term. Predicting

381

pregnancy from in vitro platforms is particularly interesting within IVP embryos, which typically

382

show lower pregnancy rates –mainly after cryopreservation-, higher gestational losses in the

383

early pregnancy and in the late fetal and perinatal periods, and more cases of morbidity and

384

dystocia than in vivo produced embryos [14, 34-38].

385

In this study, we analyzed the performance of a new system of embryo culture combined with

386

F/T under chemically defined conditions for IVP embryos. We previously observed that a short-

387

time culture without protein increases survival to V/W and reduces miscarriage, leading to

388

higher birth rates [19, 20]. Those beneficial effects were in part predicted from in vitro

389

experiments [19]. In contrast, in the present work, neither cell counts nor in vitro survival to

390

cryopreservation predicted that frozen/thawed embryos would be able to set pregnancies and 15

REVISED 391

reach birth at comparable rates to embryos transferred both fresh and after V/W. Once

392

compared with their counterparts that underwent V/W, F/T embryos were affected by

393

reduction in development rates at each in vitro endpoint analyzed (hatching and live embryo

394

rates, at 24h and 48h). Furthermore, after F/T, embryos showed lower cell numbers within the

395

ICM, TE and total cells. Last, our TUNEL study also showed that Day-7 embryos that survived

396

F/T showed similar proportions of dead cells that Day-8 embryos after V/W; both with the

397

highest levels of cell damage in this experiment. However, despite of these negative survival

398

traits observed in vitro with F/T, pregnancy rates, gestation length, birth rates, and calf birth

399

weight were not affected.

400

One reason for the discrepancy between in vitro and in vivo experiments could lie on

401

differences in chemical composition between the post-cryopreservation culture medium

402

(FCS+BSA containing) and cryopreservation media, as vitrification solutions contain FCS, but

403

FCS is absent from our chemically defined F/T medium. In contrast, within F/T under

404

chemically undefined conditions (i.e., BSA containing media), expansion and hatching rates

405

after F/T were not affected at 2h and 24h compared with V/W and only hatching rates

406

decreased 48h after culture [7]. Therefore, the presence of FCS in survival culture medium

407

could work providing a more stable environment for vitrified embryos (treated and vitrified

408

with FCS-containing solution) than for frozen embryos (treated and frozen without FCS-

409

containing solutions). This aspect remains unknown, but it should be considered in future

410

studies in light of the comparable results in pregnancy rates and calves obtained with

411

frozen/thawed embryos after ET in this study. However, defining appropriate, homogeneous

412

post-cryopreservation survival conditions in vitro can be more difficult than expected as, to our

413

knowledge, after embryo cryopreservation, successful post-culture requires serum, protein or

414

cell-coculture. Thus, the developmental competence of embryos cryopreserved in chemically

415

defined conditions would be generally underestimated and ET studies are imperative.

16

REVISED 416

Minimum numbers of cells per embryonic compartment (i.e. ICM and TE) are required to set

417

up pregnancy, although such threshold values are to be defined yet. Culture systems can

418

produce embryos with the same (TCM199 with cumulus cells; [38]), fewer (TCM199+FCS;

419

[39]), or more (SOF, with either BSA or FCS; [40]) cells than in vivo embryos. Thus, increased

420

ICM/TE cell ratio has been postulated to be more representative of in vivo embryos than cell

421

counts [39]. Reduced cell counts in embryos that underwent F/T over V/W have also been

422

reported [7,8]. In our previous studies, we found that an individual culture step without

423

protein produces highly viable embryos after V/W [19, 20], and now after F/T. Interestingly,

424

although the absence of protein reduces cell counts in the ICM and total cells, embryonic

425

viability to term improves significantly [19]. In this work, F/T led to embryos with fewer cells

426

but with the same pregnancy rates, gestation length and calf weight at birth than fresh and

427

V/W embryos. Again, caution is necessary to use cell counts as an embryonic viability

428

predictor.

429

Morphological techniques to estimate embryonic quality in vitro may include the evaluation of

430

lipid contents and apoptosis rates. TUNEL staining allows the evaluation of apoptotic rates in

431

embryonic cells [25, 41]. Excessive apoptosis, and generally cell death, are associated with

432

reduced embryonic viability [42,43,44]. Lipid stocks in cells of fresh embryos also interfere

433

with the ability to survive cryopreservation [24, 45], and lipid negatively affects the quality of

434

the blastocysts [46].

435

Interestingly, quality assays can be interrelated; as an example, the incidence of apoptosis in

436

cells of fresh embryos correlates better than lipid contents with embryo survival after

437

cryopreservation [45, 47]. In this study, we tested quality with embryos surviving

438

cryopreservation, by which analyzing lipid granule contents in fresh embryos was considered

439

unnecessary. We observed that neither FCS supplementation nor Day-6 embryonic stage (i.e.

440

morula or early blastocyst) increased apoptosis rates in hatched blastocyst after V/W. Similar

441

to our results, F/T reduced cell counts and increased apoptosis rates in ICM and TE as 17

REVISED 442

compared with V/W [7, 8]. However, among embryos subjected to F/T, the lower incidence in

443

the frequently less viable Day-8 embryos over Day-7 embryos can be in part an artifact related

444

to the reduced numbers of ICM cells in Day-8 surviving embryos. In the ICM the incidence of

445

cell death is known to be quite higher than the trophectoderm [25, 41], by which few cells in

446

the ICM minimizes the overall TUNEL impact on such embryos. We used both main culture

447

conditions (i.e. BSA with or without FCS) from Day-0 to Day-6 with all treatments tested, i.e.

448

F/T, V/W and fresh. In contrast, Sanches and co-workers (2016) froze only embryos cultured

449

with 0.5% BSA, while fresh and vitrified embryos were cultured with BSA + 2.5% FBS [18].

450

Although in our work we did not observe differences in pregnancy and birth rates with and

451

without FCS within the cryopreservation systems, we were concerned aboutthe numerically

452

high incidence of late miscarriage after F/T with FCS (5 miscarriages / 9 Day-40 pregnancies).

453

Therefore, we performed our demonstration and on field trials with F/T embryos produced

454

without FCS. Unlike our study, Sanches and co-workers (2016) found lower pregnancy and

455

birth rates upon F/T and V/W than with fresh embryos. Both studies differ also in terms of

456

animals used, with only heifers in our experimental study, heifers and cows in the

457

demonstration assay, and mainly cows in our on-field trial, vs. cows (n=804) in Sanches and

458

colleagues [18]. In our work, while all frozen embryos that were thawed were also transferred,

459

two vitrified embryos were discarded upon morphology examination at warming; the impact

460

of this event is difficult to calculate. Our work is consistent with previous studies, whereby

461

pregnancy rates within embryos produced with SOF+BSA did not differ between F/T and fresh

462

transfer [37] and between V/W and fresh transfer [35].

463

Certain types of infertility in high-producing and/or heat-stressed dairy cows can be treated

464

with ET [31,32,33], given that ET overcomes the development steps more affected by stress

465

(i.e., intrafollicular oocyte development, fertilization, and early cleavage stages) [48]. In these

466

cows, the transferred embryos yield more calves than a previous AI in the same cycle

467

[31,32,33]. Thus, in our study, we showed the proof of concept that DT-F/T is as efficient as 18

REVISED 468

fresh ET during early pregnancy within on-field experiments. Frozen Day-7 EXB from

469

hormonally stimulated cows also held pregnancy rates comparable to abattoir-derived

470

embryos. In this study, frozen hatched embryos did not lead to pregnancies, fueling the

471

controversy about the viability of hatched blastocysts after cryopreservation [17,49,50].

472

Viability of Day-7 EXB, however, was superior to Day-8 EXB, as described by other authors

473

[17,22,27,28,29].

474

Birth weight can be altered by cell numbers, culture conditions, lipid contents, developmental

475

kinetics, and cryopreservation [14,20,35,37,40,51]. Under our culture conditions, no

476

differences in birth weight and gestation length existed between cryopreservation techniques

477

and fresh embryos. However, caution is needed, since numbers of calves born are until now

478

limited, and several calves >50Kg could arise from embryos after V/W. Higher cell number and

479

birth weight have been reported in in vitro produced embryos cultured in SOF enriched with

480

protein in comparison with in vivo produced embryos ]. Generally, birth weight increases

481

within IVP over in vivo counterparts [14, 35, 40]. Although IVP embryos generally develop

482

through longer pregnancy periods [14, 37], gestation length may depend on bulls. Within

483

cryopreservation techniques, vitrification can [51] or cannot [35] increase birth weight and

484

perinatal alterations. Calves from IVP embryos that underwent V/W show altered birth weight

485

dependent on development kinetics and likely related to lipid contents [20]. Thus, a variety of

486

factors and complex interactions during in vitro embryo production might lead to long-term

487

alterations in offspring weight.

488 489

6. Conclusions

490

In this work, we described an efficient chemically defined F/T system for IVP embryos,

491

sustained by an embryo culture step without protein and replacement of BSA and/or serum by

492

a chemical supplement (i.e. CRYO3) in cryopreservation media. Pregnancy and birth rates

493

could not be predicted by in vitro survival to cryopreservation and cell counts, and apoptotic 19

REVISED 494

rates did not show differences between F/T and V/W. Interestingly, pregnancy rates on Day-62

495

reached 47% to 55% with DT-F/T embryos produced with BSA and no serum in the three assays

496

performed here. To our knowledge, no other chemically defined F/T system proved to be

497

successful with IVP embryos, yielding pregnancy and birth rates in heifers comparable to those

498

of V/W and fresh embryos, and similar pregnancy rates to fresh embryos in artificially

499

inseminated, repeat-breeding cows in commercial farms. Importantly, in cows, transfer of DT-

500

F/T embryos might help to counteract infertility associated to productive and perhaps thermal

501

stress [48], and calves born from DT-F/T embryos do not show birth overweight, a highly

502

undesirable trait on animal breeding. We suggest caution to interpret in vitro studies aiming to

503

predict embryo viability. We have provided new evidence of DT-F/T as an efficient

504

cryopreservation system for IVP embryos, with the sanitary advantage of the absence of

505

products of animal origin and total definition in its chemical composition.

506 507

Acknowledgments

508

ASEAVA and ASCOL, for the generous donation of frozen semen from Asturiana de los Valles

509

and Holstein bulls. Spanish Ministry of Economy and Competitiveness – MINECO-project

510

AGL2016-78597-R and AGL2016-81890-REDT. I. Gimeno is a FPI fellowship holder (MINECO,

511

grant BES-2017-082200). FEDER. Among the authors there are members of the COST Action

512

16119, In vitro 3-D total cell guidance and fitness (Cellfit).

513 514 515 516

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694 695

Figure Legends

696

Fig. 1. In-straw distribution of cryoprotectant solutions and embryo for freezing and direct

697

transfer. EG: ethylene-glycol. CRYO3: macromolecular replacement.

27

598

Table 1

599

In vitro development of bovine embryos that were cultured from Day-0 to Day-6 with 0.6%BSA or 0.6%BSA+0.1%FCS and individually, without protein

600

replacements, from Day-6 onwards, and subjected to freezing and thawing (F/T) or vitrification and warming (V/W). Live Treatment Culture to D6 Day

24h

N

2h

24h

48h

Hatching

48h Hatched

Hatching

Hatched

F/T

BSA

7

87

87.7±5.3xy

86.5±5.5xy

80.1±6.2x

31.8±6.7

21.1±6.3

F/T

BSA

8

50

69.0±7.3yz

67.7±7.4

68.0±8.5

7.5±9.1c

0.7±8.5a

43.1±9.1bc

34.9±9.1bc

F/T

FCS

7

87

78.1±6.4

79.5±6.5

71.6±7.4

28.2±8.0bc

12.8±7.5

53.4±7.9bc

46.4±7.9bc

F/T

FCS

8

37

53.0±6.7z

52.5±6.9z

45.2±7.8y

17.9±8.4

11.2±7.9

40.5±8.4c

30.0±8.4c

V/W

BSA

7

69

100.5±5.3x

101.5±5.5x

98.0±6.2x

48.8±6.7a

31.8±6.3

82.3±6.7a

77.5±6.7a

V/W

BSA

8

40

102.0±7.3x

98.2±7.4xy

98.3±8.5x

21.2±9.1

23.5±8.6

78.9±9.1a

70.7±9.1a

V/W

FCS

7

60

105.1±6.9x

106.4±7.1x

97.9±8.7a

92.7±8.7a

V/W

FCS

8

34

85.7±7.4xy

84.8±7.6xy

103.1±8.1x 53.9±8.7ab 39.2±8.1b 79.3±8.6x

67.3±6.7ab 56.9±6.7ab

63.7±9.2ab 47.9±9.2

32.6±9.3

13.9±8.7

21.8±4.2*

12.5±4.0* 50.0±4.3*

Main effects F/T

261 71.9±3.4*

71.3±3.5*

65.6±4.0*

41.2±4.4*

18

V/W

203 96.4±3.6

96.6±3.7

93.5±4.2

40.6±4.4

28.0±4.2

79.8±4.5

71.6±4.6

BSA

246 88.4±3.5

87.2±3.5

84.3±4.0

29.5±4.3

20.6±4.1

67.0±4.4

58.5±4.5

FCS

218 80.4±5.0

80.7±5.1

74.8±5.8

32.8±6.1

20.0±5.8

62.7±6.2

54.2±6.4

Blastocyst

147 88.7±6.0

91.3±6.2

85.7±7.0

37.4±5.1

25.2±4.9

70.3±5.3

58.4±5.4

Morula

131 85.3±6.1

84.4±6.2

81.3±7.1

29.5±5.6

21.4±5.3

58.5±5.7

54.7±5.9

Morula + Blastocyst

186 91.4±5.9

91.6±6.1

87.0±6.9

25.1±4.8

14.6±4.6

67.7±4.9

58.1±5.0

Stage on Day-6

601

*: P<0.0001; x,y: P<0.01; a,b,c: P<0.05

602

Data, expressed as LSM±SE, correspond to N=11 embryo production replicates and N=6 post-cryopreservation culture replicates.

603

Hatching: embryos with shed zona pellucida (ZP) but included in ZP

604

Hatched: embryos outside of ZP

19

605

Table 2

606

Differential cell counts by CDX2 immunostaining in Day-7 and Day-8 blastocysts that hatched in culture with SOF+10%FCS after vitrification/warming or

607

freezing/thawing Cryopreservation

Day

N

ICM

TE

Total

Freezing

7

55

29.9±2

92.3±4

122.2±5

Freezing

8

15

25.1±3a

89.3±8

114.4±10

Vitrification

7

63

34.9±1b

101.0±4

135.9±5

Vitrification

8

27

32.2±2b

97.1±6

129.3±7

27.9±2*

90.6±4

118.5±5*

33.4±1

99.1±4

132.5±4

0.0016

0.052

0.0099

Cumulative Freezing Vitrification P value

608

Data from N=8 post-cryopreservation culture replicates

609

ICM: Inner Cell mass; TE: Trophectoderm

610

a,b

: P<0.05

19

611

*: significant differences at P values shown.

20

Table 3 Percentage of TUNEL positive nuclei in Day-7 and Day-8 blastocysts that hatched in culture with SOF+10%FCS after vitrification/warming or freezing/thawing Cryopreservation Day

N

% Necrotic % Apoptotic % Pycnotic % Total Dead

Freezing

7

55 6.06±0.71

8.45±1.00a

1.62±0.32a

Freezing

8

15 4.75±1.26

4.75±1.79b

0.88±0.58b 10.39±2.86b

Vitrification

7

63 4.39±0.82

4.81±1.18

1.36±0.38a

10.56±1.89

Vitrification

8

27 5.51±0.90

8.14±1.28

2.01±0.41a

15.67±2.04a

16.13±1.60a

Embryos collected from N=8 post-cryopreservation culture replicates. a,b

: P<0.05

20

Table 4 Day-40 and Day-62 pregnancy rates after transfer in experimental herd of Day-7 vitrified/warmed, frozen/thawed and fresh embryos cultured from Day-0 to Day-6 in groups with either BSA (0.6%) or FCS (0.1%) + BSA (0.6%), and subsequently in individual culture without protein supplements (0.5 mg/mL PVA) from Day-6 to Day-7. Culture Treatment Freezing/Thawing

Day-0 to Day-6 ETs Day-40 Day-62 BSA

40

FCS+BSA

14

Vitrification/Warming BSA

Fresh

Development rates (%)

47

FCS+BSA

11

BSA FCS+BSA

22 (55) 22 (55) 9 (64)

Birth 18 (45)

8 (57)

4 (28)

29 (62) 28 (60)

25 (53)

5 (45)

5 (45)

4 (36)

30

19 (63) 17 (57)

14 (47)

17

11 (65) 10 (59)

8/15* (53)

No differences observed (P>0.05) * Within fresh ETs two recipients dead beyond Day-62 after ET (one pregnant, one open), and they were not used to calculate birth rates.

21

Table 5 Pregnancy rates (%) on Day-40 and Day-62 of one-step, frozen/thawed expanded and hatched blastocysts produced from oocytes collected from live superovulated cows by oocyte puncture ultrasonography and transferred on Day-7 to estrus synchronized recipient uniparous, nonlactating cows and heifers Blastocyst Recipient

Pregnancy (%)

Stage

Day

N

Day-40 Day-62

Heifer

Expanded

7

7

4 (57)

4 (57)

Heifer

Expanded

8

7

2 (29)

2 (29)

Heifer

Hatched

8

1

0

0

Cow

Expanded

7

10

5 (50)

4 (40)

Cow

Expanded

8

1

0

0

Cow

Hatched

7

1

0

0

Cow

Hatched

8

2

0

0

Cumulative Day-7 vs- Day-8: Day-40 (P=0.092); Day-62 (P=0.100)

Table 6 Pregnancy rates (%) in farms (field trial) after transfer of Day-7 frozen/thawed and fresh embryos cultured from Day-0 to Day-6 in groups with BSA (0.6%) and subsequently in individual culture without protein (0.5 mg/mL PVA) from Day-6 to Day-7. Treatment

ETs

N (%)

Freezing/Thawing 80

40 (50.0)

Fresh

30 (51.7)

58

No differences were observed (P>0.10). Pregnancy diagnosis made by ultrasonography on gestational days 30-40.

22

628

Table 7

629

Birth weight, gestation length and average daily gain weight of calves born from

630

frozen/thawed, vitrified/warmed and fresh embryos cultured from Day-0 to Day-6 in groups

631

and subsequently in individual culture without protein supplements (0.5 mg/mL PVA) from

632

Day-6 to Day-7. Birth weight (Kg) N Freezing/Thawing

Raw

Normalized Raw range

Gestation

Daily gain

length (days)

weight (g/day)

22 40.9±3.3

41.5±3.0

12.5-56.0

282.7±2.6

144.1±10.7

Vitrification/Warming 29 43.7±2.3

42.5±2.1

28.5-65.5

284.7±1.8

153.1±7.4

Fresh

43.3±2.1

28.0-76.5

283.2±2.4

154.5±9.8

22 44.0±3.0

633

Birth weight values are shown both raw and normalized by mother weight at birth as

634

covariate.

635

Data are expressed as LSM±SE. No significant differences (P>0.05).

23

FIGURE 1

Seeding point EG 0.75M+CRYO3

EG 1.5M+CRYO3 AIR

AIR

AIR

EG 0.75M+CRYO3 AIR

EMBRYO

ID PLUG

EG 0.75M+CRYO3

EG 0.75M+CRYO3

COTTON-PLUG

HIGHLIGHTS - Bovine embryos produced in vitro and frozen in chemically defined media lead to birth rates comparable to vitrified and fresh embryos - Pregnancy and birth rates are not predicted from in vitro experiments - Cryopreservation of embryos developed in a 24h single culture step without protein does not induce birth overweight