Accepted Manuscript A new direct transfer protocol for cryopreserved IVF embryos Bruno Valente Sanches, Paula Alvares Lunardelli, Juliana Hayashi Tannura, Bruna Lopes Cardoso, Marcos Henrique Colombo, Douglas Gaitkoski, Andrea Cristina Basso, Daniel Robert Arnold, Marcelo Marcondes Seneda PII:
S0093-691X(15)00667-6
DOI:
10.1016/j.theriogenology.2015.11.029
Reference:
THE 13435
To appear in:
Theriogenology
Received Date: 8 September 2015 Revised Date:
25 November 2015
Accepted Date: 30 November 2015
Please cite this article as: Sanches BV, Lunardelli PA, Tannura JH, Cardoso BL, Colombo MH, Gaitkoski D, Basso AC, Arnold DR, Seneda MM, A new direct transfer protocol for cryopreserved IVF embryos, Theriogenology (2016), doi: 10.1016/j.theriogenology.2015.11.029. 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 proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT REVISED A new direct transfer protocol for cryopreserved IVF embryos
Bruno Valente Sanches1; Paula Alvares Lunardelli2; Juliana Hayashi Tannura1; Bruna
Basso1; Daniel Robert Arnold1; Marcelo Marcondes Seneda3.
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Lopes Cardoso1; Marcos Henrique Colombo1; Douglas Gaitkoski1; Andrea Cristina
In Vitro Brasil S/A. SP 340 Road, km 166, ZIP code: 238, Mogi-Mirim – SP, Brazil.
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State University of Londrina (UEL), Laboratory of Animal Reproduction, DCV, CCA,
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Corresponding author:
Marcelo Marcondes Seneda:
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ZIP code: 10.011, Londrina – PR, Brazil.
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State University of Londrina (UEL), Laboratory of Animal Reproduction, DCV, CCA.
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Celso Garcia Cid Road, Pr 445, km 380. Londrina, PR, Brazil, zip-code: 86051-990 Phone: +55 43 3371-5622
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Email: [email protected]
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ACCEPTED MANUSCRIPT ABSTRACT
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The global demand for in vitro produced (IVP) embryos of determined sex has greatly
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increased over the last decade. Efficient protocols for the direct transfer of IVP embryos
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are lacking. This study aimed to compare the pregnancy rates for fresh, vitrified or
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frozen/directly transferred IVP dairy cow embryos. Oocytes (n = 3171) recovered by
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ovum pick-up (n = 112) from Girolando (Holstein-Gir) females (n = 36) were selected
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and submitted to in vitro maturation (IVM) for 24 hours at 38.5°C with 5% CO2 in air
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with saturated humidity. In vitro fertilization (IVF) was performed with the thawed,
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sexed semen from 5 Holstein bulls. After IVF, presumptive zygotes were denuded and
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cultured for seven days under the same IVM and IVF conditions of temperature and
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humidity, except with 5% CO2 and 5% O2. Grade I blastocysts were randomly assigned
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for either the transferred fresh, vitrified/thawing or frozen/directly embryo transfer into
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previously synchronized recipient females. Conception rates were analyzed by binomial
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logistic regression, and a probability level of P < 0.05 was considered significant. The
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conception rates were 51.35±1.87% (133/259) for the fresh embryos, 35.89±3.87%
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(84/234) for the vitrified embryos and 40.19±4.65% (125/311) for the frozen directly
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transferred embryos. These data demonstrate that in vitro produced embryos with sexed
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semen could be directly transferred into recipient cows with similar conception rates to
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vitrified embryos. The comparison showed that the use of frozen embryos in direct
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transfer provides easier logistics and a more practical approach for the transfer of IVP
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embryos on dairy farms.
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Keywords: Bovine, Pregnancy, Direct Transfer, IVP Embryos.
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1. INTRODUCTION
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The global demand for in vitro produced (IVP) embryos of determined sex has increased greatly over the last decade, primarily due to the higher effectiveness of
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genetic improvement in dairy herds [1]. However, cryopreserved IVP embryos are less
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resistant to injuries than their in vivo counterparts are, and this has hindered the
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international genetic trade and the management of embryo banks [2].
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The culture media in which the embryos are grown may directly influence their cryosensitivity [3]. Many efforts have been made to improve the production of the IVP
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industry, with a major focus on media supplementation. The addition of components
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such as fetal bovine serum (FBS) to the culture medium may increase blastocyst
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development rates [4], yet it may also cause controversial effects to the quality of the
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blastocysts and the establishment of pregnancy [5], thereby influencing the outcome of
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the cryopreservation.
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The cryopreservation technique predominantly used for IVP embryos is
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vitrification [6] because of its simplicity, speed and low cost. However, this technique
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uses high concentrations of cryoprotectants and requires a laboratory and trained
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personnel to evaluate the embryos before transfer [7], which restricts the use of this
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technique on a large scale. On the other hand, slow freezing of embryos for subsequent
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direct transfer, despite having slightly higher operating costs, is more practical because
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it eliminates evaluation before the transfer. In addition, lower cryoprotectant
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concentrations can be used, reducing toxicity to the embryos [8].
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Since the 1990s, direct transfer has been applied to simplify the post-thawing
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rehydration step of in vivo embryos, so that the direct transfer technique is more
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accessible [8]. No data exist in the literature to report the use of direct transfer of frozen
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IVP embryos. In addition, data on direct transfer of vitrified IVP embryos are scarce,
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and report on the utilization of small numbers of embryos, which could be the reason
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for the contradictory pregnancy results reported [9; 10].
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Thus, the aim of this study was to 1) determine the effect of serum on conception rates from in vitro culture and 2) compare the conception rates of IVP
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embryos that were transferred fresh, vitrified or frozen/directly transferred in a large
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commercial dairy herd.
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57 2. MATERIAL AND METHODS
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This study was performed in accordance with the Animal Experimentation
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Ethics Committee at the State University of Londrina based on Federal Law 11.794
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from October 8, 2008.
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MO, USA) unless otherwise specified.
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Reagents used in this study were purchased from Sigma-Aldrich (St. Louis,
2.1. Collection of oocytes and in vitro maturation (IVM)
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A total of 112 ovum pick-up (OPU) procedures were conducted with
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ultrasound guidance in 36 female Girolando donors (1/2 Gir and 1/2 Holstein) ages 2-8
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years old, with an average milk yield of 5,000 kg per lactation.
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Prior to OPU, females received a caudal epidural anesthesia (5 ml of 2%
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lidocaine; Anestésico L, Pearson, São Paulo, SP, Brazil). The rectum was emptied, and
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the vulva and perineal area were cleaned. Subsequently, the transducer was fitted in the
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intravaginal device (Pie Medical, The Netherlands) and placed in the vagina, while
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ovaries were manipulated and positioned per rectum for performance of the OPU.
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After the aspiration sessions, the oocytes were washed in TCM-199 (Gibco BRL, Grand Island, NY) buffered with HEPES, supplemented with 10% fetal bovine
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serum (FBS; Gibco BRL; Grand Island, NY), 0.20 mM sodium pyruvate and 83.4
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mg/mL amikacin (Biochimico Institute, Rio de Janeiro, Brazil). The oocytes were pre-
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selected and classified in a mobile laboratory at the farm. Oocytes were transported to
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the laboratory in 5-mL polystyrene Falcon tubes containing 400 µL of TCM-199
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medium supplemented with 10% FBS, 1 mg/mL FSH (Folltropin, Bioniche Animal
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Health, Belleville, ON, Canada), 50 mg/mL hCG (Profasi, Serono, São Paulo, Brazil)
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and estradiol (1 mg/mL), 2.2 µg of sodium pyruvate and 83.4 mg/mL amikacin, covered
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by 350 µL of mineral oil. In the laboratory, the oocytes were maintained in the same
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tube in which they were transported, with the IVM medium being maintained at 38.5°C
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with 5% CO2 in air for 24 hours from the moment of the OPU.
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2.2. In vitro fertilization (IVF)
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In vitro fertilization was performed with female sexed semen from five Holstein bulls. Semen was thawed (at 35°C for 30 sec) and washed twice by
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centrifugation (200 x g for 5 minutes) in 1 mL of TALP supplemented with 2.2 µg of
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pyruvate and 83.4 g/mL amikacin, buffered with 2.3 g/mL of HEPES. The semen
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concentration was adjusted to 2x106 mobile spermatozoa (sptz)/mL. An insemination
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dose of 10 µL (105 sptz) was added to each 50-uL drop of TALP-IVF (TALP
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supplemented with 10 µg/mL heparin and 160 µL of penicillamine, hypotaurine and
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epinephrine solution) under mineral oil. Later, 25-30 oocytes were added in each drop.
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Oocytes and sptz were co-incubated for 20-24 hours at 38.5°C in an incubator with 5%
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CO2 in air and saturated humidity.
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2.3. In vitro culture (IVC) of the embryos
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After IVF, presumptive zygotes were co-cultured (groups of 25 oocytes per drop) in the incubator (38.5°C with 5% CO2, 5% O2 and saturated humidity) with
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granulosa cells.
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Embryos were cultured in 100-µL drops of SOF [11] supplemented with 0.5% bovine serum albumin (BSA; frozen/direct transfer) or BSA + 2.5% FBS (fresh and
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vitrified) under mineral oil. The cleavage rate was evaluated on the third day of culture.
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“Feeding” was performed on the third and fifth day of IVC. On day seven of the culture
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period, Grade 1 expanded blastocysts were randomly assigned to the experimental
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treatments.
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2.4. Embryo Vitrification
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Embryos were cryopreserved following the vitrification protocol as previously described by SANCHES et al. [3]. Briefly, expanded blastocysts were exposed for 1
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minute in the equilibration solution (ES) of 10% ethylene glycol (EG) + 10% dimethyl
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sulfoxide (DMSO) in TCM-HEPES medium (25 mM HEPES) + 10% FBS and then for
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20 seconds in the vitrification solution (VS = 20% EG + 20% DMSO in TCM-HEPES +
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0.5 M sucrose). During the 20 seconds of exposure to VS, three to five embryos were
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place in a Cryotop® (Kitazato, Shizuoka, Japan) and immediately placed in liquid
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nitrogen (-196°C) as previously described by Kuwayama et al. [12]. Vitrified embryos
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were stored in liquid nitrogen until thawed for embryo transfer.
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2.5. Thawing of the vitrified embryos
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For thawing of the vitrified embryos, the Cryotops containing the embryos were exposed to air for four seconds and then dipped in the warming solution (TCM-
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HEPES + sucrose 0.3 M) at approximately 35°C. The vitrification solution was
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removed by two washes with 0.3 M sucrose and 0.15 M sucrose for 5 minutes each,
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before proceeding to the application of the maintenance medium TCM-HEPES [13; 14].
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2.6. Slow freezing of the embryos
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Embryos were cryopreserved by the slow freezing method previously described for in vivo embryos [7]. Briefly, blastocysts and expanded blastocysts were
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exposed to the freezing solution (FS), which consisted of 1.5 M EG at 35°C for 10
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minutes. Embryos were loaded into 0.25-mL straws, placed on a central column of a 1.5
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M solution of EG, surrounded by four columns of the thawing solution (TS; 0.75 M EG
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diluted in DPBS; Nutricell, Campinas, São Paulo, Brazil), and separated by air columns
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from each other (Figure 1). After being loaded, the straws were placed in a freezing
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machine (TK 1000®; Uberaba, Minas Gerais, Brazil) that had been previously
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stabilized at -6°C. Two minutes after being placed in the machine, crystallization
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("seeding") of the columns immediately above and below the embryo column was
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conducted. Embryos remained at -6°C for 10 minutes.
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The freezing curve was initiated, lowering the temperature 0.5°C per minute to
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-32°C. At the end of the freezing curve, the embryos were immersed directly in liquid
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nitrogen, where they were stored until being transferred into the recipients.
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2.7. Thawing of frozen embryos and Direct Transfer
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At the time of direct transfer, the frozen embryos were removed from the liquid nitrogen, exposed to the air at room temperature for 10 seconds and immersed in water
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at 35°C for 30 seconds. The straw was dried with paper towels and gently agitated to
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mix solutions inside the straw to initiate rehydration. Embryos were then transferred
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into the uterine horn of the recipients.
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2.8. Synchronization of recipients for embryo transfer and pregnancy diagnosis
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All recipients were in the 45-90 day interval after calving during the first trimester of lactation and located on the same farm under identical management
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conditions (food, water, and facilities). The synchronization protocol was as follows: on
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day 0, administration of 2 mg of estradiol benzoate (Sincrodiol®, Ouro Fino, Cravinhos,
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São Paulo, Brazil) and placement of an intravaginal implant (CIDR®, Pfizer, Hamilton,
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New Zealand); on day 7, administration of 25 mg of prostaglandin F2α (Lutalyse®,
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Pfeizer, Piracicaba, São Paulo, Brazil); on day 9, removal of CIDR and application of 1
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mg of estradiol cypionate (ECP®, Pfizer, Hamilton, New Zealand). On day 18, each
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recipient was subjected to a transrectal ovarian examination (Aloka SSD 500, 5
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MHz linear transducer, Tokyo, Japan) to confirm the presence and size of the
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CLs.
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After epidural anesthesia using 5 ml of 2% lidocaine (Anestésico L, Pearson,
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São Paulo, SP, Brazil), and after the rectum had been emptied and the vulva and
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perineal area cleaned, a single embryo was nonsurgically transferred into the ipsilateral
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horn of the CL of each recipient.
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Pregnancy diagnosis was carried out by ultrasonography on days 30 and 60.
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2.9. Experimental design
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Experiment I – Comparison of the conception rates of in the vitro embryos cultured in
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the presence or absence of Fetal Bovine Serum (FBS)
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This experiment was conducted to determine if the presence of FBS in the culture media affects conception rates. Oocytes were collected by OPU from the
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Girolando donors and submitted to in vitro fertilization with sexed semen from the same
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Holstein bull. Presumptive zygotes were randomly assigned to be cultured in 100-µL
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drops of SOF [10] supplemented with either 8 mg/mL of BSA (no FBS group) or 2.5%
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FBS + 8 mg/mL of BSA (FBS group) under mineral oil. In vitro culture and media
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replacement were conducted as described above. After seven days in culture, the
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embryos from both groups were freshly transferred into previously synchronized
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recipient cows.
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Experiment II - Comparison of the conception rates with the IVP embryos transferred
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fresh, vitrified or frozen/directly transferred
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After the OPU and in vitro maturation, fertilization and culture, as described
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above, Grade 1 expanded blastocysts were randomly assigned to groups for use as either
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freshly transferred, vitrified or frozen for a later direct transfer on day 7. Synchronized
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recipients were utilized for the fresh, vitrified and frozen/direct transferred embryos.
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2.10. Statistical Analysis
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The conception rates at 30 and 60 days of pregnancy were analyzed by binomial logistic regression with IBM SPSS Statistics software, version 22 [15]. For
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experiment I, the serum was considered a fixed variable. For experiment II, the embryo
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treatment (fresh, vitrified, or frozen), technician and bull were used as fixed variables.
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Significance was considered when P < 0.05.
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In Experiment I, 665 oocytes from 25 cows were matured, fertilized and randomly assigned to be cultured in media supplemented without (n = 362) or with (n =
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303) 2.5% FBS. No difference in blastocyst rates was detected between the two groups
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(P = 0.791). Conception rates were not different at day 30 or day 60, regardless of the
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absence or presence of FBS (P = 0.269; Table 1).
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Table 1. Embryo development of IVP embryos cultured in serum with or without 2.5%
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FBS supplementation
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Group
Oocytes (n)
Blastocysts n (%)
Transferred embryos (n)
Pregnancies n (%) Day 30
Day 60
With FBS
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84 (27.06±4.26%)a
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38 (46.34±6.22%)a
30 (36.59±6.10%)a
Without FBS
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88 (24.31±2.56%)a
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33 (37.50±5.65%)a
31 (35.23±5.70%)a
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a
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0.05).
Same letters in the same column indicate no significant difference between lines (P >
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In Experiment II, 112 OPUs were carried out in 36 cows. A total of 804 embryos were produced from 2,506 oocytes and randomly assigned to either be
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transferred fresh (n = 259), transferred after being vitrified/thawed-cultured (n = 234) or
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frozen/directly transferred (n = 311), and conception rates were compared after 30 and
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60 days of pregnancy (Table 2). The embryo transfer technician and bull did not affect
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the conception rates (P > 0.10). Conception rates of fresh embryos (51.35±1.87% and
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43.24±1.23%, days 30 and 60, respectively) were significantly different from both
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vitrified and frozen embryos on days 30 and 60 (Table 2). The conception rates of the
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vitrified embryos (35.89±3.87% and 31.19±4.01%, days 30 and 60, respectively) were
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not different (P > 0.10) from those of directly transferred embryos (40.19±4.65% and
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34.72±4.15%, days 30 and 60, respectively; Table 2).
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Table 2. Comparison of the conception rates at 30 and 60 days after transferring fresh,
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vitrified or directly transferring the frozen IVP embryos.
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Pregnancies at day 30 n (%)
Pregnancies at day 60 n (%)
Fresh
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133 (51.35±1.87%)a
112 (43.24±1.23%)a
Vitrified
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84 (35.89±3.87%)b
73 (31.19±4.01%)b
Frozen
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125 (40.19±4.65%)b
108 (34.72±4.15%)b
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Transferred embryos (n)
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a, b
Different letters in the same column indicate a significant difference (P < 0.05).
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4. DISCUSSION
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This study evaluated the conception rates of IVP embryos that had been transferred fresh, vitrified or frozen by a direct transfer protocol. To the best of our
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knowledge, this study is the first of its kind, particularly regarding the use of sexed
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semen in frozen IVP embryos subjected to the direct transfer in a commercial setting.
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In addition to the increasing efficiency of using female-sexed semen in IVP
embryos, thereby providing more female cows for the dairy industry [16], research on
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cryopreservation of these embryos and transfer methods has become more promising
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because of the recent feasibility and the experiments regarding conception. In that
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context, fetal bovine serum as a supplement to the culture media has previously been
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considered responsible for decreasing the survival rate of embryos [17]. However, in
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our experimental conditions, there was no difference in conception rates between the
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two groups, allowing us to conclude that FBS is dispensable in in vitro culture.
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The most widely used method for cryopreservation of IVP embryos is
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vitrification, primarily because of the speed and lower costs of freezing. Vitrification
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ensures that embryo transfer is a much more efficient technique that is no longer
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dependent on the availability of synchronized recipients. The disadvantage of
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vitrification is that a qualified person is required to reheat the embryos, which
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complicates its field application and large-scale use [18]. In addition, a sufficient
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number of recipients must be arranged according to the number of embryos that might
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survive cryopreservation.
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Another important aspect to be considered is the cryoprotectant used in the
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procedure. Cryopreservation protocols should prevent the formation of intracellular ice
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crystals and attempt to minimize toxic and osmotic stress to the cells during freezing
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[19]. Decreasing exposure time of the embryos to toxic cryoprotective agents such as
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glycerol and ethylene glycol (EG) [20] before freezing and after thawing could reduce
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the toxic effects and allow higher post-thaw embryo viability [21].
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The direct transfer method enables rehydration of embryonic cells after thawing. In the
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early 1990s, Voelkel and Hu [7] demonstrated that the use of EG as a cryoprotectant
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could be an alternative to the direct transfer of frozen-thawed embryos, with slightly
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lower conception rates than those achieved with fresh embryos [7;22], ensuring that the
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technique is the most accessible and practical for use in the field. In addition, the direct
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transfer method allows for more flexibility in the number of recipients available,
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whether from synchronized or naturally cycling.
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In this study, direct transfer was performed after the embryos were slowly frozen in EG to 0.75 Molar, arranged in four columns beside the embryo. In that
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manner, the water inflow into the embryonic cells could occur more slowly, maintaining
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the integrity of the cells. A similar strategy was presented by other researchers for
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freezing in vivo embryos, using lateral columns consisting of a solution called the
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"holding medium," composed of EG to 0.37 Molar [7]. In that work, the pregnancy rate
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was not different from that of the control group (50%). Our pregnancy rates were lower;
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however, it is important to consider that in vivo produced embryos are subjected to
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lower stress levels during development, and they frequently achieve higher pregnancy
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rates.
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In our study, conception rates on days 30 and 60 with fresh IVP embryos
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(51.35±1.87% and 43.24±1.23%, respectively) were higher (P > 0.05) than those
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obtained from vitrified IVP embryos (35.89±3.87% and 31.19±4.01%, respectively) and
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frozen directly transferred (40.19±4.65% and 34.72±4.15%, respectively) IVP embryos.
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These results are significantly higher than reported by LIM et al. [23] regarding
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transferred IVP embryos cultured in the absence of FBS and cryopreserved by slow
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freezing (22.9%). Additionally, these results were higher than reported by other
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researchers utilizing vitrified IVP embryos and the Open Pulled Straw Technique [6].
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The combination of the culture of the embryos in the absence of FBS and the strategy of
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loading in the straw might have determined the success of our protocol.
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The data from our study demonstrate that in vitro produced embryos with
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sexed semen after slow freezing and direct transfer utilizing the method we describe
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result in pregnancy rates as satisfactory as those achieved by vitrification, allowing
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greater efficiency and practicality in advanced genetic dairy farming.
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ACKNOWLEDGEMENT
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The authors appreciated the commitment of the employees of Vitro Brazil S/A and the Cape Verde Group - Fazenda Santa Luzia.
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[13] Vieira AD, Mezzalira A, Barbieri DP, Lehmkuhl RC, Rubin MIB, Vajta G. Calves
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born after open pulled straw vitrification of immature bovine oocytes. Cryobiology
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2002;45:91-4.
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[14] Mezzalira A, Mezzalira JC, Moraes AN, Thaler Neto A, Vieira AD, Barreta MH, et
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al. Vitrification of bovine IVP embryos: age of embryos and exposure time to
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cryoprotectant influence viability. Arch Vet Sci 2004;9:107-11.
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[15] IBM SPSS Statistics Version 22, IBM Inc, Armonk, NY.
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[16] Xu J, Guo Z, Su L, Nedambale TL, Zhang J, Schenk J, et al. Developmental
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potential of vitrified Holstein cattle embryos fertilized in vitro with sex-sorted sperm. J
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Dairy Sci 2006;89:2510–8.
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368 [17] Lonergan P, Rizos D, Gutierrez-Adán A, Moreira MB, Pintado B, De La Fuente J.
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Temporal divergence in the pattern of messenger RNA expression in bovine embryos
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cultured from the zygote to blastocyst stage in vitro or in vivo. Biol Reprod
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2003;69:1424–31.
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[18] Morató R, Mogas T. New device for the vitrification and in-straw warming of in
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vitro produced bovine embryos. Cryobiology 2014;68:288–93.
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[19] Campos-Chillon LF, Walker DJ, De La Torre-Sanchez JF, Seidel Jr GE. In vitro
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assessment of a direct transfer vitrification procedure for bovine embryos.
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Theriogenology 2006;65:1200-1214.
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[20] Dochi O, Yamamoto Y, Saga H, Yoshiba N, Kano N, Maeda J, et al. Direct
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transfer of bovine embryos frozen-thawed in the presence of propylene glycol or
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ethylene glycol under on-farm conditions in an integrated embryo transfer program.
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Theriogenology 1988;49:1051-8.
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[21] Sommerfield V, Niemann H. Cryopreservation of bovine in vitro produced
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embryos using ethylene glycol in controlled feezing or vitrification. Cryobiology
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1999;38:95-105
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[22] Leibo SP, Mapletoft ERJ. Direct transfer of cryopreserved cattle embryos in North
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America. In: Proceedings of the 17th Annual Convention of AETA. San Antonio, TX;
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1998, p. 91-8.
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393 [23] Lim KT, Lang G, Ko KH, Lee WW, Park HJ, Kim JJ, et al. Improved
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cryopreservation of bovine preimplantation embryos cutured in chemically defined
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médium. Anim reprod Sci 2008;103-239-48.
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Figure 1 - Loading scheme for frozen embryo direct transfer.
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ACCEPTED MANUSCRIPT • With a consistent number of pregnancies, we describe a method for direct transfer of frozen IVF embryos;
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• Directly transferred frozen embryos provided the same pregnancy rates as vitrified embryos, even though vitrifying is the most currently used method of cryopreservation for IVP embryos;
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• Since we combine direct transfer with sexed semen, we provide a useful option for genetic enhancing, mainly for dairy cattle.
S0093-691X(15)00667-6
DOI:
10.1016/j.theriogenology.2015.11.029
Reference:
THE 13435
To appear in:
Theriogenology
Received Date: 8 September 2015 Revised Date:
25 November 2015
Accepted Date: 30 November 2015
Please cite this article as: Sanches BV, Lunardelli PA, Tannura JH, Cardoso BL, Colombo MH, Gaitkoski D, Basso AC, Arnold DR, Seneda MM, A new direct transfer protocol for cryopreserved IVF embryos, Theriogenology (2016), doi: 10.1016/j.theriogenology.2015.11.029. 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 proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT REVISED A new direct transfer protocol for cryopreserved IVF embryos
Bruno Valente Sanches1; Paula Alvares Lunardelli2; Juliana Hayashi Tannura1; Bruna
Basso1; Daniel Robert Arnold1; Marcelo Marcondes Seneda3.
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Lopes Cardoso1; Marcos Henrique Colombo1; Douglas Gaitkoski1; Andrea Cristina
In Vitro Brasil S/A. SP 340 Road, km 166, ZIP code: 238, Mogi-Mirim – SP, Brazil.
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State University of Londrina (UEL), Laboratory of Animal Reproduction, DCV, CCA,
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Corresponding author:
Marcelo Marcondes Seneda:
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ZIP code: 10.011, Londrina – PR, Brazil.
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State University of Londrina (UEL), Laboratory of Animal Reproduction, DCV, CCA.
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Celso Garcia Cid Road, Pr 445, km 380. Londrina, PR, Brazil, zip-code: 86051-990 Phone: +55 43 3371-5622
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Email: [email protected]
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ACCEPTED MANUSCRIPT ABSTRACT
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The global demand for in vitro produced (IVP) embryos of determined sex has greatly
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increased over the last decade. Efficient protocols for the direct transfer of IVP embryos
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are lacking. This study aimed to compare the pregnancy rates for fresh, vitrified or
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frozen/directly transferred IVP dairy cow embryos. Oocytes (n = 3171) recovered by
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ovum pick-up (n = 112) from Girolando (Holstein-Gir) females (n = 36) were selected
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and submitted to in vitro maturation (IVM) for 24 hours at 38.5°C with 5% CO2 in air
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with saturated humidity. In vitro fertilization (IVF) was performed with the thawed,
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sexed semen from 5 Holstein bulls. After IVF, presumptive zygotes were denuded and
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cultured for seven days under the same IVM and IVF conditions of temperature and
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humidity, except with 5% CO2 and 5% O2. Grade I blastocysts were randomly assigned
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for either the transferred fresh, vitrified/thawing or frozen/directly embryo transfer into
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previously synchronized recipient females. Conception rates were analyzed by binomial
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logistic regression, and a probability level of P < 0.05 was considered significant. The
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conception rates were 51.35±1.87% (133/259) for the fresh embryos, 35.89±3.87%
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(84/234) for the vitrified embryos and 40.19±4.65% (125/311) for the frozen directly
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transferred embryos. These data demonstrate that in vitro produced embryos with sexed
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semen could be directly transferred into recipient cows with similar conception rates to
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vitrified embryos. The comparison showed that the use of frozen embryos in direct
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transfer provides easier logistics and a more practical approach for the transfer of IVP
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embryos on dairy farms.
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Keywords: Bovine, Pregnancy, Direct Transfer, IVP Embryos.
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1. INTRODUCTION
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The global demand for in vitro produced (IVP) embryos of determined sex has increased greatly over the last decade, primarily due to the higher effectiveness of
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genetic improvement in dairy herds [1]. However, cryopreserved IVP embryos are less
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resistant to injuries than their in vivo counterparts are, and this has hindered the
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international genetic trade and the management of embryo banks [2].
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The culture media in which the embryos are grown may directly influence their cryosensitivity [3]. Many efforts have been made to improve the production of the IVP
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industry, with a major focus on media supplementation. The addition of components
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such as fetal bovine serum (FBS) to the culture medium may increase blastocyst
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development rates [4], yet it may also cause controversial effects to the quality of the
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blastocysts and the establishment of pregnancy [5], thereby influencing the outcome of
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the cryopreservation.
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The cryopreservation technique predominantly used for IVP embryos is
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vitrification [6] because of its simplicity, speed and low cost. However, this technique
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uses high concentrations of cryoprotectants and requires a laboratory and trained
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personnel to evaluate the embryos before transfer [7], which restricts the use of this
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technique on a large scale. On the other hand, slow freezing of embryos for subsequent
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direct transfer, despite having slightly higher operating costs, is more practical because
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it eliminates evaluation before the transfer. In addition, lower cryoprotectant
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concentrations can be used, reducing toxicity to the embryos [8].
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Since the 1990s, direct transfer has been applied to simplify the post-thawing
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rehydration step of in vivo embryos, so that the direct transfer technique is more
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accessible [8]. No data exist in the literature to report the use of direct transfer of frozen
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IVP embryos. In addition, data on direct transfer of vitrified IVP embryos are scarce,
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and report on the utilization of small numbers of embryos, which could be the reason
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for the contradictory pregnancy results reported [9; 10].
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Thus, the aim of this study was to 1) determine the effect of serum on conception rates from in vitro culture and 2) compare the conception rates of IVP
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embryos that were transferred fresh, vitrified or frozen/directly transferred in a large
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commercial dairy herd.
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57 2. MATERIAL AND METHODS
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This study was performed in accordance with the Animal Experimentation
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Ethics Committee at the State University of Londrina based on Federal Law 11.794
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from October 8, 2008.
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MO, USA) unless otherwise specified.
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Reagents used in this study were purchased from Sigma-Aldrich (St. Louis,
2.1. Collection of oocytes and in vitro maturation (IVM)
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A total of 112 ovum pick-up (OPU) procedures were conducted with
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ultrasound guidance in 36 female Girolando donors (1/2 Gir and 1/2 Holstein) ages 2-8
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years old, with an average milk yield of 5,000 kg per lactation.
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Prior to OPU, females received a caudal epidural anesthesia (5 ml of 2%
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lidocaine; Anestésico L, Pearson, São Paulo, SP, Brazil). The rectum was emptied, and
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the vulva and perineal area were cleaned. Subsequently, the transducer was fitted in the
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intravaginal device (Pie Medical, The Netherlands) and placed in the vagina, while
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ovaries were manipulated and positioned per rectum for performance of the OPU.
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After the aspiration sessions, the oocytes were washed in TCM-199 (Gibco BRL, Grand Island, NY) buffered with HEPES, supplemented with 10% fetal bovine
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serum (FBS; Gibco BRL; Grand Island, NY), 0.20 mM sodium pyruvate and 83.4
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mg/mL amikacin (Biochimico Institute, Rio de Janeiro, Brazil). The oocytes were pre-
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selected and classified in a mobile laboratory at the farm. Oocytes were transported to
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the laboratory in 5-mL polystyrene Falcon tubes containing 400 µL of TCM-199
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medium supplemented with 10% FBS, 1 mg/mL FSH (Folltropin, Bioniche Animal
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Health, Belleville, ON, Canada), 50 mg/mL hCG (Profasi, Serono, São Paulo, Brazil)
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and estradiol (1 mg/mL), 2.2 µg of sodium pyruvate and 83.4 mg/mL amikacin, covered
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by 350 µL of mineral oil. In the laboratory, the oocytes were maintained in the same
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tube in which they were transported, with the IVM medium being maintained at 38.5°C
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with 5% CO2 in air for 24 hours from the moment of the OPU.
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2.2. In vitro fertilization (IVF)
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In vitro fertilization was performed with female sexed semen from five Holstein bulls. Semen was thawed (at 35°C for 30 sec) and washed twice by
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centrifugation (200 x g for 5 minutes) in 1 mL of TALP supplemented with 2.2 µg of
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pyruvate and 83.4 g/mL amikacin, buffered with 2.3 g/mL of HEPES. The semen
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concentration was adjusted to 2x106 mobile spermatozoa (sptz)/mL. An insemination
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dose of 10 µL (105 sptz) was added to each 50-uL drop of TALP-IVF (TALP
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supplemented with 10 µg/mL heparin and 160 µL of penicillamine, hypotaurine and
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epinephrine solution) under mineral oil. Later, 25-30 oocytes were added in each drop.
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Oocytes and sptz were co-incubated for 20-24 hours at 38.5°C in an incubator with 5%
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CO2 in air and saturated humidity.
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2.3. In vitro culture (IVC) of the embryos
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After IVF, presumptive zygotes were co-cultured (groups of 25 oocytes per drop) in the incubator (38.5°C with 5% CO2, 5% O2 and saturated humidity) with
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granulosa cells.
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Embryos were cultured in 100-µL drops of SOF [11] supplemented with 0.5% bovine serum albumin (BSA; frozen/direct transfer) or BSA + 2.5% FBS (fresh and
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vitrified) under mineral oil. The cleavage rate was evaluated on the third day of culture.
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“Feeding” was performed on the third and fifth day of IVC. On day seven of the culture
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period, Grade 1 expanded blastocysts were randomly assigned to the experimental
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treatments.
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2.4. Embryo Vitrification
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Embryos were cryopreserved following the vitrification protocol as previously described by SANCHES et al. [3]. Briefly, expanded blastocysts were exposed for 1
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minute in the equilibration solution (ES) of 10% ethylene glycol (EG) + 10% dimethyl
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sulfoxide (DMSO) in TCM-HEPES medium (25 mM HEPES) + 10% FBS and then for
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20 seconds in the vitrification solution (VS = 20% EG + 20% DMSO in TCM-HEPES +
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0.5 M sucrose). During the 20 seconds of exposure to VS, three to five embryos were
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place in a Cryotop® (Kitazato, Shizuoka, Japan) and immediately placed in liquid
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nitrogen (-196°C) as previously described by Kuwayama et al. [12]. Vitrified embryos
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were stored in liquid nitrogen until thawed for embryo transfer.
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2.5. Thawing of the vitrified embryos
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For thawing of the vitrified embryos, the Cryotops containing the embryos were exposed to air for four seconds and then dipped in the warming solution (TCM-
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HEPES + sucrose 0.3 M) at approximately 35°C. The vitrification solution was
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removed by two washes with 0.3 M sucrose and 0.15 M sucrose for 5 minutes each,
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before proceeding to the application of the maintenance medium TCM-HEPES [13; 14].
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2.6. Slow freezing of the embryos
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Embryos were cryopreserved by the slow freezing method previously described for in vivo embryos [7]. Briefly, blastocysts and expanded blastocysts were
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exposed to the freezing solution (FS), which consisted of 1.5 M EG at 35°C for 10
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minutes. Embryos were loaded into 0.25-mL straws, placed on a central column of a 1.5
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M solution of EG, surrounded by four columns of the thawing solution (TS; 0.75 M EG
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diluted in DPBS; Nutricell, Campinas, São Paulo, Brazil), and separated by air columns
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from each other (Figure 1). After being loaded, the straws were placed in a freezing
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machine (TK 1000®; Uberaba, Minas Gerais, Brazil) that had been previously
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stabilized at -6°C. Two minutes after being placed in the machine, crystallization
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("seeding") of the columns immediately above and below the embryo column was
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conducted. Embryos remained at -6°C for 10 minutes.
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The freezing curve was initiated, lowering the temperature 0.5°C per minute to
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-32°C. At the end of the freezing curve, the embryos were immersed directly in liquid
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nitrogen, where they were stored until being transferred into the recipients.
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2.7. Thawing of frozen embryos and Direct Transfer
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At the time of direct transfer, the frozen embryos were removed from the liquid nitrogen, exposed to the air at room temperature for 10 seconds and immersed in water
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at 35°C for 30 seconds. The straw was dried with paper towels and gently agitated to
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mix solutions inside the straw to initiate rehydration. Embryos were then transferred
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into the uterine horn of the recipients.
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2.8. Synchronization of recipients for embryo transfer and pregnancy diagnosis
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All recipients were in the 45-90 day interval after calving during the first trimester of lactation and located on the same farm under identical management
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conditions (food, water, and facilities). The synchronization protocol was as follows: on
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day 0, administration of 2 mg of estradiol benzoate (Sincrodiol®, Ouro Fino, Cravinhos,
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São Paulo, Brazil) and placement of an intravaginal implant (CIDR®, Pfizer, Hamilton,
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New Zealand); on day 7, administration of 25 mg of prostaglandin F2α (Lutalyse®,
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Pfeizer, Piracicaba, São Paulo, Brazil); on day 9, removal of CIDR and application of 1
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mg of estradiol cypionate (ECP®, Pfizer, Hamilton, New Zealand). On day 18, each
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recipient was subjected to a transrectal ovarian examination (Aloka SSD 500, 5
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MHz linear transducer, Tokyo, Japan) to confirm the presence and size of the
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CLs.
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After epidural anesthesia using 5 ml of 2% lidocaine (Anestésico L, Pearson,
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São Paulo, SP, Brazil), and after the rectum had been emptied and the vulva and
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perineal area cleaned, a single embryo was nonsurgically transferred into the ipsilateral
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horn of the CL of each recipient.
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Pregnancy diagnosis was carried out by ultrasonography on days 30 and 60.
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2.9. Experimental design
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Experiment I – Comparison of the conception rates of in the vitro embryos cultured in
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the presence or absence of Fetal Bovine Serum (FBS)
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This experiment was conducted to determine if the presence of FBS in the culture media affects conception rates. Oocytes were collected by OPU from the
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Girolando donors and submitted to in vitro fertilization with sexed semen from the same
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Holstein bull. Presumptive zygotes were randomly assigned to be cultured in 100-µL
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drops of SOF [10] supplemented with either 8 mg/mL of BSA (no FBS group) or 2.5%
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FBS + 8 mg/mL of BSA (FBS group) under mineral oil. In vitro culture and media
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replacement were conducted as described above. After seven days in culture, the
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embryos from both groups were freshly transferred into previously synchronized
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recipient cows.
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Experiment II - Comparison of the conception rates with the IVP embryos transferred
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fresh, vitrified or frozen/directly transferred
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After the OPU and in vitro maturation, fertilization and culture, as described
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above, Grade 1 expanded blastocysts were randomly assigned to groups for use as either
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freshly transferred, vitrified or frozen for a later direct transfer on day 7. Synchronized
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recipients were utilized for the fresh, vitrified and frozen/direct transferred embryos.
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2.10. Statistical Analysis
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The conception rates at 30 and 60 days of pregnancy were analyzed by binomial logistic regression with IBM SPSS Statistics software, version 22 [15]. For
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experiment I, the serum was considered a fixed variable. For experiment II, the embryo
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treatment (fresh, vitrified, or frozen), technician and bull were used as fixed variables.
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Significance was considered when P < 0.05.
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In Experiment I, 665 oocytes from 25 cows were matured, fertilized and randomly assigned to be cultured in media supplemented without (n = 362) or with (n =
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303) 2.5% FBS. No difference in blastocyst rates was detected between the two groups
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(P = 0.791). Conception rates were not different at day 30 or day 60, regardless of the
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absence or presence of FBS (P = 0.269; Table 1).
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Table 1. Embryo development of IVP embryos cultured in serum with or without 2.5%
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FBS supplementation
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Group
Oocytes (n)
Blastocysts n (%)
Transferred embryos (n)
Pregnancies n (%) Day 30
Day 60
With FBS
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84 (27.06±4.26%)a
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38 (46.34±6.22%)a
30 (36.59±6.10%)a
Without FBS
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88 (24.31±2.56%)a
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33 (37.50±5.65%)a
31 (35.23±5.70%)a
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a
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0.05).
Same letters in the same column indicate no significant difference between lines (P >
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In Experiment II, 112 OPUs were carried out in 36 cows. A total of 804 embryos were produced from 2,506 oocytes and randomly assigned to either be
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transferred fresh (n = 259), transferred after being vitrified/thawed-cultured (n = 234) or
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frozen/directly transferred (n = 311), and conception rates were compared after 30 and
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60 days of pregnancy (Table 2). The embryo transfer technician and bull did not affect
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the conception rates (P > 0.10). Conception rates of fresh embryos (51.35±1.87% and
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43.24±1.23%, days 30 and 60, respectively) were significantly different from both
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vitrified and frozen embryos on days 30 and 60 (Table 2). The conception rates of the
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vitrified embryos (35.89±3.87% and 31.19±4.01%, days 30 and 60, respectively) were
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not different (P > 0.10) from those of directly transferred embryos (40.19±4.65% and
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34.72±4.15%, days 30 and 60, respectively; Table 2).
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Table 2. Comparison of the conception rates at 30 and 60 days after transferring fresh,
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vitrified or directly transferring the frozen IVP embryos.
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Pregnancies at day 30 n (%)
Pregnancies at day 60 n (%)
Fresh
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133 (51.35±1.87%)a
112 (43.24±1.23%)a
Vitrified
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84 (35.89±3.87%)b
73 (31.19±4.01%)b
Frozen
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125 (40.19±4.65%)b
108 (34.72±4.15%)b
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Transferred embryos (n)
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a, b
Different letters in the same column indicate a significant difference (P < 0.05).
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4. DISCUSSION
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This study evaluated the conception rates of IVP embryos that had been transferred fresh, vitrified or frozen by a direct transfer protocol. To the best of our
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knowledge, this study is the first of its kind, particularly regarding the use of sexed
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semen in frozen IVP embryos subjected to the direct transfer in a commercial setting.
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In addition to the increasing efficiency of using female-sexed semen in IVP
embryos, thereby providing more female cows for the dairy industry [16], research on
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cryopreservation of these embryos and transfer methods has become more promising
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because of the recent feasibility and the experiments regarding conception. In that
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context, fetal bovine serum as a supplement to the culture media has previously been
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considered responsible for decreasing the survival rate of embryos [17]. However, in
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our experimental conditions, there was no difference in conception rates between the
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two groups, allowing us to conclude that FBS is dispensable in in vitro culture.
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The most widely used method for cryopreservation of IVP embryos is
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vitrification, primarily because of the speed and lower costs of freezing. Vitrification
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ensures that embryo transfer is a much more efficient technique that is no longer
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dependent on the availability of synchronized recipients. The disadvantage of
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vitrification is that a qualified person is required to reheat the embryos, which
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complicates its field application and large-scale use [18]. In addition, a sufficient
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number of recipients must be arranged according to the number of embryos that might
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survive cryopreservation.
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Another important aspect to be considered is the cryoprotectant used in the
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procedure. Cryopreservation protocols should prevent the formation of intracellular ice
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crystals and attempt to minimize toxic and osmotic stress to the cells during freezing
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[19]. Decreasing exposure time of the embryos to toxic cryoprotective agents such as
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glycerol and ethylene glycol (EG) [20] before freezing and after thawing could reduce
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the toxic effects and allow higher post-thaw embryo viability [21].
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The direct transfer method enables rehydration of embryonic cells after thawing. In the
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early 1990s, Voelkel and Hu [7] demonstrated that the use of EG as a cryoprotectant
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could be an alternative to the direct transfer of frozen-thawed embryos, with slightly
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lower conception rates than those achieved with fresh embryos [7;22], ensuring that the
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technique is the most accessible and practical for use in the field. In addition, the direct
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transfer method allows for more flexibility in the number of recipients available,
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whether from synchronized or naturally cycling.
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In this study, direct transfer was performed after the embryos were slowly frozen in EG to 0.75 Molar, arranged in four columns beside the embryo. In that
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manner, the water inflow into the embryonic cells could occur more slowly, maintaining
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the integrity of the cells. A similar strategy was presented by other researchers for
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freezing in vivo embryos, using lateral columns consisting of a solution called the
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"holding medium," composed of EG to 0.37 Molar [7]. In that work, the pregnancy rate
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was not different from that of the control group (50%). Our pregnancy rates were lower;
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however, it is important to consider that in vivo produced embryos are subjected to
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lower stress levels during development, and they frequently achieve higher pregnancy
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rates.
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In our study, conception rates on days 30 and 60 with fresh IVP embryos
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(51.35±1.87% and 43.24±1.23%, respectively) were higher (P > 0.05) than those
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obtained from vitrified IVP embryos (35.89±3.87% and 31.19±4.01%, respectively) and
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frozen directly transferred (40.19±4.65% and 34.72±4.15%, respectively) IVP embryos.
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These results are significantly higher than reported by LIM et al. [23] regarding
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transferred IVP embryos cultured in the absence of FBS and cryopreserved by slow
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freezing (22.9%). Additionally, these results were higher than reported by other
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researchers utilizing vitrified IVP embryos and the Open Pulled Straw Technique [6].
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The combination of the culture of the embryos in the absence of FBS and the strategy of
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loading in the straw might have determined the success of our protocol.
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The data from our study demonstrate that in vitro produced embryos with
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sexed semen after slow freezing and direct transfer utilizing the method we describe
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result in pregnancy rates as satisfactory as those achieved by vitrification, allowing
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greater efficiency and practicality in advanced genetic dairy farming.
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ACKNOWLEDGEMENT
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The authors appreciated the commitment of the employees of Vitro Brazil S/A and the Cape Verde Group - Fazenda Santa Luzia.
REFERENCES
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Figure 1 - Loading scheme for frozen embryo direct transfer.
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ACCEPTED MANUSCRIPT • With a consistent number of pregnancies, we describe a method for direct transfer of frozen IVF embryos;
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• Directly transferred frozen embryos provided the same pregnancy rates as vitrified embryos, even though vitrifying is the most currently used method of cryopreservation for IVP embryos;
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• Since we combine direct transfer with sexed semen, we provide a useful option for genetic enhancing, mainly for dairy cattle.
