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Biopsy of bovine embryos produced in vivo and in vitro does not affect pregnancy rates
Theriogenology 90 (2017) 25–31
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Biopsy of bovine embryos produced in vivo and in vitro does not affect pregnancy rates Regivaldo Vieira de Sousa a, b, Célia Regina da Silva Cardoso c, Guilberth Butzke c, Margot Alves Nunes Dode b, Rodolfo Rumpf d, Maurício Machaim Franco b, * a
School of Agriculture and Veterinary Medicine, University of Brasilia, Brasília, DF, Brazil Embrapa Genetic Resources and Biotechnology, Laboratory of Animal Reproduction, Brasília, DF, Brazil c Guilberth Serviços Veterinários S/A, São Paulo, SP, Brazil d Geneal Genética Animal–Análise, Pesquisa e Laboratório S/A, Uberaba, MG, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2016 Received in revised form 27 October 2016 Accepted 2 November 2016
Assisted reproductive techniques have significantly contributed to animal breeding programs. Similarly, genomics has provided important information and tools to improve the accuracy of selection. However, the greatest benefits of those tools can only be expected when they are combined, allowing animals to be selected accurately early in life. Therefore, obtaining DNA samples from embryos without compromising their viability is essential for the consolidation of preimplantation genomic selection. We aimed to evaluate the effect on the gestation rate of conducting a biopsy of in vivo (VV) and in vitro-produced (IVP) bovine embryos. The VV and IVP embryos were distributed into two groups: VV-B (biopsied embryos; n ¼ 380) and VV-C (intact embryos–controls; n ¼ 229) and IVP-B (biopsied embryos; n ¼ 91) and IVP-C (intact embryos–controls; n ¼ 227), respectively. After biopsy, embryos from both groups VV-B and IVP-B were cultured for an additional 3 hours before being transferred to synchronized recipients. To evaluate the quality of the DNA obtained in the biopsies, this was used to determine the sex of embryos by polymerase chain reaction. No effect (P > 0.05) of the biopsy was observed for any of the treatments, the pregnancy rate at D 60 post-transfer being similar for VV-B: 206/380 (54.21%) and VV-C: 128/229 (55.89%) and for IVP-B: 24/91 (26.37%) and IVP-C: 45/227 (19.82%). Also, no effect (P > 0.05) of the embryo’s stage of development was detected on percentage of pregnant recipients when in vitro embryos were transferred. From the biopsies analyzed, about 90% had the sex determined, confirming that DNA was there and it was efficiently amplified. The results indicated that biopsy does not affect the viability of IVV and IVP bovine embryos and can be used in commercial programs to associate assisted reproductive technologies with genomic selection. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: Bovine Embryo Biopsy Embryo viability Sex identification Genomic selection
1. Introduction The main focuses of genetic improvement of livestock are usually quantitative traits such as milk and meat * Corresponding author. Tel.: þ55 (61) 3448-4700; fax: þ55 (61) 33403624. E-mail address: [email protected] (M.M. Franco). 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2016.11.003
production. For many years, traditional genetic improvement, which relies on using the recorded phenotype of each animal together with the knowledge of its pedigree to estimate its breeding value, has been used to increase productivity. Although this technology has been very successful, leading to genetic gains in most farmed species (e.g., see Van Vleck et al. [1], Havenstein et al. [2]), there has long been an interest in using inherited genetic markers to
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accurately select animals for breeding and to speed up genetic progress [3]. Then, beginning with bovine genome sequencing in the mid 80s, a significant advance in genetic analysis and mapping technologies has occurred, leading to the development of a variety of genetic tools. One of them that has to be highlighted is the detection of tens of thousands of markers called single nucleotide polymorphisms (SNPs) [4], which resulted in a new method of selection called genomic selection (GS) [3]. With the GS method, animals can be selected for breeding on the basis of their genomic breeding values; that is, their genetic merit predicted by markers tagging the entire genome [5]. It is stated that an increase in genetic gain or income is from 60% to 120% compared with traditional methods of progeny testing [6] mostly due to the dramatic reduction in the costs of raising a large number of animals and selecting only a few as breeders. Although GS has dramatically changed traditional progeny testing schemes in cattle and other species, the largest increase in genetic gain could be achieved by shortening the generation interval. This can be done by associating GS with in vitro embryo production (IVP), in which an unborn animal’s genetic merit is predicted at the embryo stage, drastically reducing the generation interval [5,7]. Furthermore, IVP allows the production of large quantities of embryos, increasing selection intensity rapidly and leading to rapid genetic improvement. However, there are certain technical limitations as to how genomic screening of preimplantation embryos could be practical enough to be widely used. Issues such as performing embryo biopsies and obtaining sufficient DNA quality and quantity are the major concerns [5,8]. Regarding biopsy procedure, the bottleneck is how to manipulate the embryo without decreasing its viability. Embryo biopsy is not a new technique and has been widely used for many years in humans for selection against chromosomally or genetically abnormal embryos (for review see Harper & Sengupta [9]). Although it has been reported in several livestock species, including cattle, sheep, horses, and goats [10–15], it has mainly been used for sex determination [16,17]. In fact, for 20 years, embryo transfer teams have commonly used embryo biopsy technology for embryos produced in vivo (VV) combined with polymerase chain reaction (PCR)–based sex determination to limit the number of embryos to be transferred. Despite the invasiveness of embryo biopsy, the early embryos’ plasticity is such that the procedure does not seem to adversely affect the capacity of the VV-produced embryo to develop or to implant normally [18]. However, when the main objective is to associate GS and IVP, the viability of the biopsied embryo has to be maintained. Conversely, there is plenty of evidence indicating the various differences between VV and IVP embryos, which lead us to hypothesize that those produced in vitro are more fragile under micromanipulation and more sensitive to biopsy than the VV ones. In the majority of the studies using IVP embryos, the biopsy was performed at either 3, 4, or 5 days postinsemination, returning to culture to develop to the blastocyst stage, or at D7, not evaluating the pregnancy [10–12,19–28]. To our knowledge, there seems to be no
data available on the pregnancy rate of IVP bovine embryos that are biopsied at blastocyst stage, except for the studies reported by El-Sayed et al. [29] and Hoelke et al. [30]. However, in those studies, the biopsy was used for another purpose, and there was no concern with the pregnancy outcome itself; rather, the procedure was used as a tool to identify and categorize the biopsy to be analyzed. Therefore, we aimed to evaluate the effect on the pregnancy rate of carrying out a biopsy on D7 VV and in the IVP blastocyst. 2. Material and methods Unless otherwise indicated, chemicals were purchased from Sigma Aldrich (St Louis, MO, USA). The two experiments were conducted at different private companies. 2.1. In vivo embryo production (IVV embryos) To produce VV embryos, Holstein (22) and Jersey (8) donor females were subjected to superstimulatory protocols and artificial insemination. Donor cows were between 3 and 16 year old and heifers were between 13 and 16 month old. No animal showed fertility problems, and all animals were housed indoor at the time of embryo collections. Wave emergence was synchronized by administration of an intramuscular (im) injection of 2.0 mg of estradiol benzoate (Ric-BE, Syntex S.A., Buenos Aires, Argentina) and insertion of an intravaginal progesteronereleasing device with 1.9 g of progesterone (CIDR, Pfizer, Auckland, New Zealand) on Day 0. On Day 4, the superstimulatory treatment was initiated, using a total of 500 IU for Holstein and 375 IU for Jersey donors of FSH/LH (Pluset, Calier, Les Franqueses Del Vallès, Barcelona, Spain); the animals received applications twice daily in decreasing doses over a 4-day period. At the time of the sixth im injection of FSH, 25 mg of im PGF2a (Lutalyse, Pfizer) was used. Progesterone device was removed at the time of the last application of FSH. The ovulation was induced by using an IM injection of 25-mg lecirelina (Gestran, Tecnopec, São Paulo, Brazil). All females were artificially inseminated with frozen/thawed semen 12 and 24 hours after estrus manifestation. On Day 7 postinsemination, embryos were recovered by uterine flushing and were classified for quality and stage of development according to the IETS Manual [31]. Grades I and II of compacted morula (Mc), initial blastocysts (BI), blastocysts (BL), and expanded blastocysts (BE) were used for the experiment. Recovery rates were between 90 and 95%. The semen from each cow were collected 1 to 3 times. The rest time between collections was of 45 to 60 days. 2.2. In vitro embryo production (IVP embryos) Gyr (Bos Taurus indicus) heifers (29) that were between 2 and 7 year old were used as oocyte donors. Cumulus oocyte complexes (COCs) were obtained by ovum pick up, using an ultrasound device (Aloka SSD 500, Japan) coupled to a micro convex sector transducer at 7.5 MHz (Aloka, UST 9125, Japan). All of the follicles between 3 and 8 mm were punctured. The recovered COCs were selected under a stereomicroscope (ZeissdStemi SV6, Germany) and were
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transported to the laboratory. Only grade I and II COCs were used in the experiment. Selected COCs were washed and transferred to a 200-mL drop of maturation medium under mineral oil and incubated for 22 hours at 39 C with 5% CO2 in air. The maturation medium consisted of TCM–199, Earle’s salts (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 0.01 IU/mL of FSH, 0.01 IU/mL of LH 0.1 mg/mL of L-glutamine and antibiotics (amicacyn, 0.075 mg/mL, Sigma Chemical Co, St Louis, MO, USA). After maturation, COCs were transferred to a 100-mL drop of fertilization medium, which consisted of TALP [32] supplemented with penicillamine (2 mM), hypotaurine (1 mM), epinephrine (250 mM), and heparin (10 mg/mL). Motile spermatozoa were obtained using the Percoll (GE Healthcare, Piscataway, NJ, USA) gradient method using 1 mL of 90% and 1 mL of 45%, centrifuged at 700g for 15 minutes. Selected sperm were added into the fertilization drop at a final concentration of 1106 spermatozoa/mL. Sperm and oocytes were coincubated for 18 hours at 39 C with 5% CO2; the day of in vitro insemination was defined as Day 0. After coincubation, the presumptive IVP zygotes were washed and transferred to 200-mL drops of SOFaaci medium [33] supplemented with 2.77 mM of myo-inositol and 5% FBS and cultured at 39 C and 5% CO2 in the air for 7 days. Embryos were evaluated on Day 2 postinsemination for cleavage and Day 6 and 7 postinsemination for blastocyst rate. Day-7 embryos were classified according to the IETS Manual [31], and only the quality grade-I and II, BI, BL, and BX were used. 2.3. Embryo biopsy Biopsies were performed manually using an M&M micromanipulator (M&MdThe Micromanipulator Microscope Company, Escondido, CA, USA) and stainless steel blades at angles of 15 (Bio-Cut-Blades Feather, Feather Safety Razor Co, Chome Kita-Ku, Osaka, Japan). Embryos were micromanipulated on a 100 20 mm Petri dish (Corning, Tewksbury, MA, USA) containing 200 mL holding medium consisting of TCM–199, Hank’s salts supplemented with 10% FBS (both Invitrogen, Carlsbad, CA, USA) and antibiotic (amikacin; 0.075 mg/mL). The biopsied cells (10–20 blastomeres) were removed from the trophectoderm and were transferred to a lysing solution containing Proteinase K (Invitrogen, Carlsbad, CA, USA) 20 mg/mL, until sex determination. Biopsied embryos from both groups, VV and in vitro, were kept in holding medium for 3 hours before being transferred to the recipients.
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Brazil). The progesterone device was removed and an injection of 2 mL of cloprostenol was performed on Day 8, and on Day 9 estrus was observed. 2.5. Evaluation of DNA integrity by embryo sexing procedure To validate that the DNA from biopsies could be used for any genomic analysis, sex determination was performed by multiplex PCR using two pairs of primers. The first pair was specific to a region of the Y chromosome [34], whereas the second pair was specific to a bovine autosomal gene [35]. Bovine genomic DNA from a male and a female were used as a control. Polymerase chain reaction was performed by adding to each sample tube a PCR mix containing 20 nM of each pair of primers, 200 mM of each dNTP, 1 PCR buffer and 1U Platinum Taq DNA polymerase (Invitrogen) in a final volume of 30 mL. The PCR program used 40 cycles of 94 C for 20 seconds, 57 C for 30 seconds and 72 C for 30 seconds, followed by a final extension of 72 C for 30 minutes. Amplified PCR products were electrophoresed in 2.0% agarose gel stained with ethidium bromide (10 mg/mL) and visualized under UV illumination. When two amplicons were detected, the embryo was identified as male, whereas detection of one amplicon indicated a female embryo (Fig. 1). 2.6. Experimental design To evaluate the effect of biopsy of VV embryos on pregnancy rate, a total of 609 embryos were used in an experiment with eight replicates, in which each replica corresponded to a day of embryo recovered. For each replicate, selected embryos were randomly distributed into two groups: (a) in vivo biopsied (VV-B, n ¼ 380) and (b) in vivo control (VV-C, n ¼ 229). Both groups were composed of grade I and II embryos at Mc, BI, BL, and BX stage, and both were kept at room temperature for 3 hours in holding medium before transfer to the recipient.
2.4. In vivo and in vitro embryo transfer Embryos were transferred nonsurgically to the uterine horn ipsilateral to the CL of synchronized recipients that came into estrus 7 days before the transfer. Synchronization of the recipients was performed by administering an intramuscular injection of 2.0-mg estradiol benzoate (Ric-BE, Syntex S.A., Buenos Aires, Argentina) and inserting an intravaginal progesterone-releasing device with 1.9 g of progesterone (CRESTAR, MSD Animal Health, São Paulo, SP,
Fig. 1. Polymerase chain reaction results of bovine individual embryo biopsies, on 2.5% agarose (wt/vol) gel. Molecular marker (L); Bovine genomic DNA (CN, CM, and CAu); male embryos (M) and female embryos (F).
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Table 1 Pregnancy rate at 30 and 60 days of gestation and embryonic loss (EL) of biopsied (VV-B) and nonbiopsied (VV-C) bovine embryos produced in vivo. Group
VV-B VV-C
Embryos Total
Pregnancy 30 days, n (%)
Pregnancy 60 days, n (%)
EL
380 229
218 (57.36) 132 (57.64)
206 (54.21) 128 (55.89)
12 (5.50) 6 (4.45)
Data analyzed by chi-square (P > 0.05).
For the in vitro experiment, 318 IVP embryos were used. Similar to the VV group, embryos were selected and distributed into two groups: in vitro biopsied (IVP-B, n ¼ 91) and in vitro control (IVP-C, n ¼ 227), both composed of grade I and II embryos at BI, BL, and BX stage. In both groups, biopsied and intact embryos were kept for 3 hours, individually on SOF media at 39 C, 5% of CO2. After the incubation period, embryos were transferred to the recipients. All recipients were evaluated using an ultrasound with a 7.5-MHz linear probe at D30 and D60, for pregnancy, embryonic loss, and sexing. To confirm the good quality/integrity of the DNA collected in the biopsy, sex identification was performed on 271 biopsies from 380 VV-B and 91 biopsies from 91 IVP-B embryos. 2.7. Statistical analyses Conception rates and the effect of the embryonic stage on the pregnancy outcome after biopsy were carried out by the chi-square test. Differences were considered significant when P 0.05. 3. Results A total of 609 VV embryos were used to evaluate the effect of the biopsy on pregnancy. Biopsy had not affected embryo viability since pregnancy rate at 30 days was similar to the control embryos, which were not submitted to any micromanipulation procedure (Table 1). Similarly, no variation was observed between biopsied and intact embryos for embryonic loss evaluated by the pregnancy at Day 60 after transfer (Table 1). When embryos were biopsied at different stages of development, the pregnancy rate was higher for BI than for Mc (P < 0.05); however, it was similar for all the other stages (Table 2). Moreover, no impact of the embryo stage was detected on percentage of pregnant recipients when control embryos were transferred (Table 2).
To evaluate if biopsy affected the viability of IVP embryos, a total of 318 embryos were used. As reported in Table 3, the pregnancy of biopsy-derived blastocysts was not different from the nonbiopsy control group. When the results were evaluated, taking into account the stage of development, IVP embryos biopsied at BI, BL, and BX stage also had similar pregnancy rates (Table 4). From all the 362 biopsies evaluated, less than 10% were not suitable for identifying the sex of the embryos, either by the presence of a weak band or by the lack of amplification (Table 5). From a total of 243 VV-B embryos that had the sex identified, 168 (69.14%) were female and 75 (30.86%) were male. Regarding IVP-B embryos, from a total of 87 embryos that had the sex identified, 48 (55.17%) were female and 39 (44.83%) were male.
4. Discussion Genomic tools, such as single nucleotide polymorphism (SNP) chips, have led to a new method of selection called “genomic selection” in which dense SNP genotypes covering the genome are used to predict the breeding values. When GS is combined with reproductive technologies such as IVP and embryo biopsy, it can lead to substantial savings for breeders and to a dramatic increase in the rate of genetic progress. However, for these tools to become useful some limitations have to be overcome. The first, which is related to the ability of a micromanipulated embryo to result in a pregnancy, was the main focus of our study. To evaluate the effect on pregnancy rate of biopsy by microblade, we used embryos VV and IVP produced. As expected, we found no significant differences in pregnancy rates at 30 and 60 days when transferring biopsied or intact VV bovine embryos. Various authors have compared the viability of biopsied and nonbiopsied VV embryos and also shown that biopsy could be performed in bovine embryos without any variations in pregnancy rates [17,18,26,36]. These results indicate that despite the invasiveness of embryo biopsy, the early embryos’ plasticity is such that the procedure does not seem to adversely affect the capacity of the embryo to develop or to implant normally. Even so, there is evidence indicating the importance of the timing and method of biopsy in influencing the subsequent embryonic development and survival [18,37,38]. Therefore, we also investigated the effect of the developmental stage of the biopsied Day 7 embryos on the establishment of the pregnancy. Biopsied Mc, BI, BL, and BX had similar pregnancy to the control embryos at the same
Table 2 Effect of the stage of development of biopsied (VV-B) and nonbiopsied (VV-C) in vivo-produced bovine embryos on pregnancy rate at 30 days of gestation. Group
Pregnant recipients/N total (%) Mc
BI
BL
BX
VV-B VV-C
88/170 (51.76)a 86/157 (54.77)a
20/27 (74.07)b 9/17 (52.94)a
95/158 (60.12)a,b 32/49 (65.30)a
15/25 (60.00)a,b 5/6 (83.33)a
a,b
Data with different superscripts in the same line differ significantly (P < 0.05). Abbreviations: BI, initial blastocyst; BL, blastocyst; BX, expanded blastocysts; Mc, compacted morula.
R.V. de Sousa et al. / Theriogenology 90 (2017) 25–31 Table 3 Pregnancy rate at 60 days of gestation of biopsied (IVP-B) and nonbiopsied (IVP-C) bovine embryos produced in vitro. Group
Embryos
IVP-B IVP-C
N total
Pregnancy at 60 days, n (%)
91 227
24 (26.37) 45 (19.82)
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Table 5 Identification of embryo sex by PCR of biopsies from in vivo (VV-B) and in vitro (IVP-B) produced bovine embryos. Group
Sex identification by PCR (%) N
Identified
Nonidentified
VV-B IVP-B
271 91
243 (89.66) 87 (95.60)
28 (10.33) 4 (4.39)
Data analyzed by chi-square (P > 0.05).
Abbreviation: PCR, polymerase chain reaction.
stage. However, when the comparison was made within the group and between stages it was observed that biopsied Mc presented a lower pregnancy at D30 than the biopsied BI but was similar to BL or BX. Although there is a scarcity of literature regarding the comparison of morula with more advanced stages of development after transfer, it has been shown that bovine [39] and ovine [40] biopsied embryos at morula stage lead to decreased pregnancy rates compared with blastocysts. These findings are in agreement with our results, and they could easily be explained by the fact that the youngest embryos have fewer cells and, after manipulation, the number of surviving blastomeres may be much lower, affecting viability. However, our data showed that Mc had similar pregnancy outcome to BX and BE. We are not sure why the difference in pregnancy was only related to BI; it is possible that the low number of embryos at Mc stage affected the result. Genomic selection for domestic animals is becoming a useful tool, and its use will probably increase more and more in the near future. This is especially true if it is combined with IVP and biopsy technologies, because that association provides the most efficient way to obtain genetic gain more rapidly. Therefore, one of the most limiting factors is the viability of the IVP embryo after removal of the material necessary for the GS. The rationality for that is because it is generally accepted that embryos produced in vitro are morphologically, ultrastructurally, physiologically, transcriptionally, and metabolically different from their VV counterparts [41–45]. In summary, they are inferior in quality compared with those derived VV. Therefore, one can suppose that they would tolerate the stress of the biopsy less well. To test how IVP embryos respond to the biopsy, we evaluated the pregnancy rate. We found that no differences were observed between the biopsied and the control group, suggesting that removal of trophoblast cells did not affect embryo viability. It is important to
highlight that we cannot rule out the possibility that the similar pregnancy rate could be due to a mechanism of compensation from the biopsied group. In this group, the embryos had an open area on the zona pellucida due to micromanipulation, and this open area could have helped the hatching and increased the pregnancy as has been shown by Park, et al. [12]. Regarding the low-pregnancy rate observed for the IVP embryos, this was probably due to nutritional levels of the animals, resulting from the reduced food availability due to the early onset of the dry season. When we compared the pregnancy rates after transfer of bovine in vitro embryos biopsied at different stages of development, no differences were observed. The Mc stage was not included in the in vitro experiment because when embryos were removed from culture at D7 the majority were at blastocyst stage. These results suggested that even early blastocysts can be biopsied without affecting their viability. Furthermore, it is important to point out that the success of the technique is highly dependent on the ability and training of the operator, since very specific skills are needed. The second main limitation of using GS associated with IVP and biopsy techniques is the requirement of a minimum amount of good quality genomic DNA to perform the analysis. Although it was not our objective to evaluate the DNA present in the biopsy, we chose the microblade instead of aspiration or the needle method, because it has been shown that genetic material obtained using a microblade was of higher quality than that obtained by the other methods [18]. In addition, it has been indicated to be the most practical under field conditions and has been associated with good pregnancy outcome [18]. To evaluate the quality of DNA, we used the biopsy to determine the sex of embryos by PCR. From the biopsies analyzed, about 90% had the sex determined, suggesting that DNA was there and was correctly amplified. All the biopsies were done by the same technician, and the size had been previously determined in our laboratory as being between 10 and 20 cells. Using a different number of cells in biopsies for a genotyping investigation, Fisher et al. [7] found that a single-cell or three-cell sample failed to yield enough DNA for genotyping. In contrast, biopsies obtained from the trophoblast of a blastocyst with a microsurgical knife gave a call rate following wholegenome amplification and genotyping similar to that obtained with a 30- to 40-cell morula and 50-cell bisected blastocysts. A biopsy of eight to 10 cells obtained under laboratory conditions, using a microblade under a stereomicroscope gave a call rate of 90% [46]. Taking all this information together, it is very possible that our in vitro
Table 4 Effect of the stage of development of biopsied (IVP-B) and nonbiopsied (IVP-C) in vitro-produced bovine embryos on pregnancy rate at 60 days of gestation. Group
IVP-B IVP-C
Pregnant recipients/N total (%) BI
BL
BX
5/20 (25.00) 16/79 (20.51)
10/41 (24.39) 17/91 (18.68)
9/30 (30.00) 8/39 (20.51)
Data analyzed by chi-square (P > 0.05). Abbreviations: BI, initial blastocyst; BL, blastocyst; BX, expanded blastocysts.
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blastocyst biopsies would provide enough cells to enable reasonably robust whole-genome amplification and SNP genotyping. In conclusion, biopsy of bovine blastocysts produced VV and in vitro using microblade does not affect their viability and their ability to originate pregnancy at Day 60 of gestation. This method also provides a DNA of good quality that can be used for genetic analysis. Acknowledgments The authors thank Guilberth Serviços Veterinários S/C LTDA, Gênesis Biotecnologia Ltda/DF and Agropecuária Palma Ltda/DF for allowing them to use their embryos for the research, and Embrapa for financial support. Funding information: Grant sponsors: Embrapa Genetic Resources and Biotechnology MP1 01.13.06.001.04.00. References [1] Van Vleck LD, Westell RA, Schneider JC. Genetic change in milk yield estimated from simultaneous genetic evaluation of bulls and cows. J Dairy Sci 1986;69:2963–5. [2] Havenstein GB, Ferket PR, Scheideler SE, Larson BT. Growth, livability, and feed conversion of 1957 vs 1991 broilers when fed “typical” 1957 and 1991 broiler diets. Poult Sci 1994;73:1785–94. [3] Goddard ME, Hayes BJ, Meuwissen THE. Genomic selection in livestock populations. Genet Res 2010;92:413–21. [4] Kadarmideen HN, Mazzoni G, Watanabe YF, Strøbech L, Baruselli PS, Meirelles FV, et al. Genomic selection of in vitro produced and somatic cell nuclear transfer embryos for rapid genetic improvement in cattle production. Anim Reprod 2015;12: 389–96. [5] Ponsart C, Le Bourhis D, Knijn H, Fritz S, Guyader-Joly C, Otter T, et al. Reproductive technologies and genomic selection in dairy cattle. Reprod Fertil Dev 2013;26:12–21. [6] Pryce JE, Daetwyler HD. Designing dairy cattle breeding schemes under genomic selection: a review of international research. Anim Prod Sci 2012;52:107–14. [7] Fisher PJ, Hyndman DL, Bixley MJ, Oback FC, Popovic L, McGowan LT, et al. Brief communication: potential for genomic selection of bovine embryos. Soc Anim Prod 2012;72:156–8. [8] Kasinathan P, Wei H, Xiang T, Molina JA, Metzger J, Broek D, et al. Acceleration of genetic gain in cattle by reduction of generation interval. Sci Rep 2015;5:8674. [9] Harper JC, Sengupta SB. Preimplantation genetic diagnosis: state of the ART 2011. Hum Genet 2011;131:175–86. [10] Macháty Z, Páldi A, Csáki T, Varga Z, Kiss I, Bárándi Z, et al. Biopsy and sex determination by PCR of IVF bovine embryos. J Reprod Fertil 1993;98:467–70. [11] Chrenek P, Boulanger L, Heyman Y, Uhrin P, Laurincik J, Bulla J, et al. Sexing and multiple genotype analysis from a single cell of bovine embryo. Theriogenology 2001;55:1071–81. [12] Park JH, Lee JH, Choi KM, Joung SY, Kim JY, Chung GM, et al. Rapid sexing of preimplantation bovine embryo using consecutive and multiplex polymerase chain reaction (PCR) with biopsied single blastomere. Theriogenology 2001;55:1843–53. [13] Leoni G, Ledda S, Bogliolo L, Naitana S. Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embryonic genome analysis. J Reprod Fertil 2000;119:309–14. [14] El-Gayar M, Holtz W. Transfer of sexed caprine blastocysts freshly collected or derived from cultured morulae. Small Rumin Res 2005; 57:151–6. [15] Herrera C, Morikawa MI, Bello MB, von Meyeren M, Eusebio Centeno J, Dufourq P, et al. Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program. Theriogenology 2014;81:758–63. [16] Bondioli KR. Embryo sexing: a review of current techniques and their potential for commercial amdication in livestock production. J Anim Sci 1992;70:19–26. [17] Lopes RF, Forell F, Oliveira AT, Rodrigues JL. Splitting and biopsy for bovine embryo sexing under field conditions. Theriogenology 2001; 56:1383–92.
[18] Cenariu M, Pall E, Cernea C, Groza I. Evaluation of bovine embryo biopsy techniques according to their ability to preserve embryo viability. J Biomed Biotechnol 2012;2012:541384. [19] Agca Y, Monson RL, Northey DL, Peschel DE, Schaefer DM, Rutledge JJ. Normal calves from transfer of biopsied, sexed and vitrified IVP bovine embryos. Theriogenology 1998;50:129–45. [20] Lee JH, Park JH, Lee SH, Park CS, Jin DI. Sexing using single blastomere derived from IVF bovine embryos by fluorescence in situ hybridization (FISH). Theriogenology 2004;62:1452–8. [21] Tominaga K. Cryopreservation and sexing of in vivo- and in vitroproduced bovine embryos for their practical use. J Reprod Dev 2004; 50:29–38. [22] Yotsushima K, Sakaguchi M, Shimizu M, Okimura T, Izaike Y. Effects of fatty acid-free bovine serum albumin and fetal calf serum supplementing repair cultures on pre- and post-warm viability of biopsied bovine embryos produced in vitro. J Reprod Dev 2004;50: 471–6. [23] Pereira RM, Carvalhais I, Pimenta J, Baptista MC, Vasques MI, Horta AEM, et al. Biopsied and vitrified bovine embryos viability is improved by trans10, cis12 conjugated linoleic acid supplementation during in vitro embryo culture. Anim Reprod Sci 2008;106: 322–32. [24] Kubisch HM, Johnson KM. The effects of blastomere biopsy and oxygen tension on bovine embryo development, rate of apoptosis and interferon-s secretion. Reprod Domest Anim 2007;42:509–15. [25] Tominaga K, Iwaki F, Hochi S. Conventional freezing of in vitroproduced and biopsied bovine blastocysts in the presence of a low concentration of glycerol and sucrose. J Reprod Dev 2007;53:443–7. [26] Peippo J, Viitala S, Virta J, Räty M, Tammiranta N, Lamminen T, et al. Birth of correctly genotyped calves after multiplex marker detection from bovine embryo microblade biopsies. Mol Reprod Dev 2007;74: 1373–8. [27] Polisseni J, Sá WF, Guerra Mde O, Machado MA, Serapião RV, Carvalho BCd, et al. Post-biopsy bovine embryo viability and whole genome amplification in preimplantation genetic diagnosis. Fertil Steril 2010;93:783–8. [28] Shirazi A, Borjian S, Ahmadi E, Nazari H, Heidari B. The effect of biopsy during precompacted morula stage on post vitrification development of blastocyst derived bovine embryos. Avicenna J Med Biotechnol 2010;2:107–11. [29] El-Sayed A, Hoelker M, Rings F, Salilew D, Jennen D, Tholen E, et al. Large-scale transcriptional analysis of bovine embryo biopsies in relation to pregnancy success after transfer to recipients. Physiol Genomics 2006;28:84–96. [30] Hoelker M, Held E, Salilew-Wondim D, Schellander K, Tesfaye D. Molecular signatures of bovine embryo developmental competence. Reprod Fertil Dev 2013;26:22–36. [31] Robertson I, Nelson RE. Certification and identification of the embryo. In: Stringfellow DA, Seidel SM, editors. Manual of International embryo transfer Society. Third edition. Savoy, IL, USA: IETS; 1998. p. 103–34. [32] Parrish JJ, Krogenaes A, Susko-Parrish JL. Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development. Theriogenology 1995;44:859–69. [33] Holm P, Booth PJ, Schmidt MH, Greve T, Callesen H. High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. Theriogenology 1999;52:683–700. [34] Bondioli KR, Ellis SB, Pryor JH, Williams MW, Harpold MM. The use of male-specific chromosomal DNA fragments to determine the sex of bovine preimplantation embryos. Theriogenology 1989;31:95–104. [35] Ellis SB, Bondiolli KW, Williams ME, Pryor JH, Harpold MM. Sex determination of bovine embryos using male-specific DNA probes. Theriogenology 1988;29:242. [36] Carbonneau G, Morin N, Durocher J, Bousquet D. Viability of bovine ivf embryos biopsied with microsection or microaspiration technique for sexing. Theriogenology 1997;47:266. [37] Vajta G, Holm P, Greve T, Callesen H. Comparison of two manipulation methods to produce in vitro fertilized, biopsied and vitrified bovine embryos. Theriogenology 1997;47:501–9. [38] Shirazi A, Borjian S, Nazari H, Ahmadi E, Heidari B, Bahiraee A. Effects of timing on cell biopsy from pre-compacted morula stage bovine embryos on subsequent embryonic development. J Reprod Infertil 2010;11:25–32. [39] Lacaze S, Ponsart C, Humblot P. Influence of embryo stage on pregnancy rates following transfer of bovine biopsied embryos under on-farm conditions. Proceedings of the 25th AETE, Poznan, Poland: IETS; 2009. p. 208.
R.V. de Sousa et al. / Theriogenology 90 (2017) 25–31 [40] Naitana S, Loi P, Ledda S, Cappai P, Dattena M, Bogliolo L, et al. Effect of biopsy and vitrification on in vitro survival of ovine embryos at different stages of development. Theriogenology 1996;46:813–24. [41] Farin C, Farin P, Piedrahita J. Development of fetuses from in vitro– produced and cloned bovine embryos. J Anim Sci 2004;82:E53–62. [42] Crosier AE, Farin PW, Dykstra MJ, Alexander JE, Farin CE. Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro. Biol Reprod 2000;62:1459–65. [43] Crosier AE, Farin PW, Dykstra MJ, Alexander JE, Farin CE. Ultrastructural morphometry of bovine blastocysts produced in vivo or in vitro. Biol Reprod 2001;64:1375–85.
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[44] Lonergan P, Fair T, Corcoran D, Evans ACO. Effect of culture environment on gene expression and developmental characteristics in IVF-derived embryos. Theriogenology 2006;65: 137–52. [45] Machado GM, Ferreira AR, Pivato I, Fidelis A, Spricigo JF, Paulini F, et al. Post-hatching development of in vitro bovine embryos from day 7 to 14 in vivo versus in vitro. Mol Reprod Dev 2013;80:936–47. [46] Le Bourhis D, Mullaart E, Schrooten C, Fritz S, Coppieters W, Ponsart C. Breeding values concordance between embryos and corresponding calves. Reprod Fertil Dev 2011;24:180.
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Biopsy of bovine embryos produced in vivo and in vitro does not affect pregnancy rates Regivaldo Vieira de Sousa a, b, Célia Regina da Silva Cardoso c, Guilberth Butzke c, Margot Alves Nunes Dode b, Rodolfo Rumpf d, Maurício Machaim Franco b, * a
School of Agriculture and Veterinary Medicine, University of Brasilia, Brasília, DF, Brazil Embrapa Genetic Resources and Biotechnology, Laboratory of Animal Reproduction, Brasília, DF, Brazil c Guilberth Serviços Veterinários S/A, São Paulo, SP, Brazil d Geneal Genética Animal–Análise, Pesquisa e Laboratório S/A, Uberaba, MG, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2016 Received in revised form 27 October 2016 Accepted 2 November 2016
Assisted reproductive techniques have significantly contributed to animal breeding programs. Similarly, genomics has provided important information and tools to improve the accuracy of selection. However, the greatest benefits of those tools can only be expected when they are combined, allowing animals to be selected accurately early in life. Therefore, obtaining DNA samples from embryos without compromising their viability is essential for the consolidation of preimplantation genomic selection. We aimed to evaluate the effect on the gestation rate of conducting a biopsy of in vivo (VV) and in vitro-produced (IVP) bovine embryos. The VV and IVP embryos were distributed into two groups: VV-B (biopsied embryos; n ¼ 380) and VV-C (intact embryos–controls; n ¼ 229) and IVP-B (biopsied embryos; n ¼ 91) and IVP-C (intact embryos–controls; n ¼ 227), respectively. After biopsy, embryos from both groups VV-B and IVP-B were cultured for an additional 3 hours before being transferred to synchronized recipients. To evaluate the quality of the DNA obtained in the biopsies, this was used to determine the sex of embryos by polymerase chain reaction. No effect (P > 0.05) of the biopsy was observed for any of the treatments, the pregnancy rate at D 60 post-transfer being similar for VV-B: 206/380 (54.21%) and VV-C: 128/229 (55.89%) and for IVP-B: 24/91 (26.37%) and IVP-C: 45/227 (19.82%). Also, no effect (P > 0.05) of the embryo’s stage of development was detected on percentage of pregnant recipients when in vitro embryos were transferred. From the biopsies analyzed, about 90% had the sex determined, confirming that DNA was there and it was efficiently amplified. The results indicated that biopsy does not affect the viability of IVV and IVP bovine embryos and can be used in commercial programs to associate assisted reproductive technologies with genomic selection. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: Bovine Embryo Biopsy Embryo viability Sex identification Genomic selection
1. Introduction The main focuses of genetic improvement of livestock are usually quantitative traits such as milk and meat * Corresponding author. Tel.: þ55 (61) 3448-4700; fax: þ55 (61) 33403624. E-mail address: [email protected] (M.M. Franco). 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2016.11.003
production. For many years, traditional genetic improvement, which relies on using the recorded phenotype of each animal together with the knowledge of its pedigree to estimate its breeding value, has been used to increase productivity. Although this technology has been very successful, leading to genetic gains in most farmed species (e.g., see Van Vleck et al. [1], Havenstein et al. [2]), there has long been an interest in using inherited genetic markers to
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accurately select animals for breeding and to speed up genetic progress [3]. Then, beginning with bovine genome sequencing in the mid 80s, a significant advance in genetic analysis and mapping technologies has occurred, leading to the development of a variety of genetic tools. One of them that has to be highlighted is the detection of tens of thousands of markers called single nucleotide polymorphisms (SNPs) [4], which resulted in a new method of selection called genomic selection (GS) [3]. With the GS method, animals can be selected for breeding on the basis of their genomic breeding values; that is, their genetic merit predicted by markers tagging the entire genome [5]. It is stated that an increase in genetic gain or income is from 60% to 120% compared with traditional methods of progeny testing [6] mostly due to the dramatic reduction in the costs of raising a large number of animals and selecting only a few as breeders. Although GS has dramatically changed traditional progeny testing schemes in cattle and other species, the largest increase in genetic gain could be achieved by shortening the generation interval. This can be done by associating GS with in vitro embryo production (IVP), in which an unborn animal’s genetic merit is predicted at the embryo stage, drastically reducing the generation interval [5,7]. Furthermore, IVP allows the production of large quantities of embryos, increasing selection intensity rapidly and leading to rapid genetic improvement. However, there are certain technical limitations as to how genomic screening of preimplantation embryos could be practical enough to be widely used. Issues such as performing embryo biopsies and obtaining sufficient DNA quality and quantity are the major concerns [5,8]. Regarding biopsy procedure, the bottleneck is how to manipulate the embryo without decreasing its viability. Embryo biopsy is not a new technique and has been widely used for many years in humans for selection against chromosomally or genetically abnormal embryos (for review see Harper & Sengupta [9]). Although it has been reported in several livestock species, including cattle, sheep, horses, and goats [10–15], it has mainly been used for sex determination [16,17]. In fact, for 20 years, embryo transfer teams have commonly used embryo biopsy technology for embryos produced in vivo (VV) combined with polymerase chain reaction (PCR)–based sex determination to limit the number of embryos to be transferred. Despite the invasiveness of embryo biopsy, the early embryos’ plasticity is such that the procedure does not seem to adversely affect the capacity of the VV-produced embryo to develop or to implant normally [18]. However, when the main objective is to associate GS and IVP, the viability of the biopsied embryo has to be maintained. Conversely, there is plenty of evidence indicating the various differences between VV and IVP embryos, which lead us to hypothesize that those produced in vitro are more fragile under micromanipulation and more sensitive to biopsy than the VV ones. In the majority of the studies using IVP embryos, the biopsy was performed at either 3, 4, or 5 days postinsemination, returning to culture to develop to the blastocyst stage, or at D7, not evaluating the pregnancy [10–12,19–28]. To our knowledge, there seems to be no
data available on the pregnancy rate of IVP bovine embryos that are biopsied at blastocyst stage, except for the studies reported by El-Sayed et al. [29] and Hoelke et al. [30]. However, in those studies, the biopsy was used for another purpose, and there was no concern with the pregnancy outcome itself; rather, the procedure was used as a tool to identify and categorize the biopsy to be analyzed. Therefore, we aimed to evaluate the effect on the pregnancy rate of carrying out a biopsy on D7 VV and in the IVP blastocyst. 2. Material and methods Unless otherwise indicated, chemicals were purchased from Sigma Aldrich (St Louis, MO, USA). The two experiments were conducted at different private companies. 2.1. In vivo embryo production (IVV embryos) To produce VV embryos, Holstein (22) and Jersey (8) donor females were subjected to superstimulatory protocols and artificial insemination. Donor cows were between 3 and 16 year old and heifers were between 13 and 16 month old. No animal showed fertility problems, and all animals were housed indoor at the time of embryo collections. Wave emergence was synchronized by administration of an intramuscular (im) injection of 2.0 mg of estradiol benzoate (Ric-BE, Syntex S.A., Buenos Aires, Argentina) and insertion of an intravaginal progesteronereleasing device with 1.9 g of progesterone (CIDR, Pfizer, Auckland, New Zealand) on Day 0. On Day 4, the superstimulatory treatment was initiated, using a total of 500 IU for Holstein and 375 IU for Jersey donors of FSH/LH (Pluset, Calier, Les Franqueses Del Vallès, Barcelona, Spain); the animals received applications twice daily in decreasing doses over a 4-day period. At the time of the sixth im injection of FSH, 25 mg of im PGF2a (Lutalyse, Pfizer) was used. Progesterone device was removed at the time of the last application of FSH. The ovulation was induced by using an IM injection of 25-mg lecirelina (Gestran, Tecnopec, São Paulo, Brazil). All females were artificially inseminated with frozen/thawed semen 12 and 24 hours after estrus manifestation. On Day 7 postinsemination, embryos were recovered by uterine flushing and were classified for quality and stage of development according to the IETS Manual [31]. Grades I and II of compacted morula (Mc), initial blastocysts (BI), blastocysts (BL), and expanded blastocysts (BE) were used for the experiment. Recovery rates were between 90 and 95%. The semen from each cow were collected 1 to 3 times. The rest time between collections was of 45 to 60 days. 2.2. In vitro embryo production (IVP embryos) Gyr (Bos Taurus indicus) heifers (29) that were between 2 and 7 year old were used as oocyte donors. Cumulus oocyte complexes (COCs) were obtained by ovum pick up, using an ultrasound device (Aloka SSD 500, Japan) coupled to a micro convex sector transducer at 7.5 MHz (Aloka, UST 9125, Japan). All of the follicles between 3 and 8 mm were punctured. The recovered COCs were selected under a stereomicroscope (ZeissdStemi SV6, Germany) and were
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transported to the laboratory. Only grade I and II COCs were used in the experiment. Selected COCs were washed and transferred to a 200-mL drop of maturation medium under mineral oil and incubated for 22 hours at 39 C with 5% CO2 in air. The maturation medium consisted of TCM–199, Earle’s salts (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 0.01 IU/mL of FSH, 0.01 IU/mL of LH 0.1 mg/mL of L-glutamine and antibiotics (amicacyn, 0.075 mg/mL, Sigma Chemical Co, St Louis, MO, USA). After maturation, COCs were transferred to a 100-mL drop of fertilization medium, which consisted of TALP [32] supplemented with penicillamine (2 mM), hypotaurine (1 mM), epinephrine (250 mM), and heparin (10 mg/mL). Motile spermatozoa were obtained using the Percoll (GE Healthcare, Piscataway, NJ, USA) gradient method using 1 mL of 90% and 1 mL of 45%, centrifuged at 700g for 15 minutes. Selected sperm were added into the fertilization drop at a final concentration of 1106 spermatozoa/mL. Sperm and oocytes were coincubated for 18 hours at 39 C with 5% CO2; the day of in vitro insemination was defined as Day 0. After coincubation, the presumptive IVP zygotes were washed and transferred to 200-mL drops of SOFaaci medium [33] supplemented with 2.77 mM of myo-inositol and 5% FBS and cultured at 39 C and 5% CO2 in the air for 7 days. Embryos were evaluated on Day 2 postinsemination for cleavage and Day 6 and 7 postinsemination for blastocyst rate. Day-7 embryos were classified according to the IETS Manual [31], and only the quality grade-I and II, BI, BL, and BX were used. 2.3. Embryo biopsy Biopsies were performed manually using an M&M micromanipulator (M&MdThe Micromanipulator Microscope Company, Escondido, CA, USA) and stainless steel blades at angles of 15 (Bio-Cut-Blades Feather, Feather Safety Razor Co, Chome Kita-Ku, Osaka, Japan). Embryos were micromanipulated on a 100 20 mm Petri dish (Corning, Tewksbury, MA, USA) containing 200 mL holding medium consisting of TCM–199, Hank’s salts supplemented with 10% FBS (both Invitrogen, Carlsbad, CA, USA) and antibiotic (amikacin; 0.075 mg/mL). The biopsied cells (10–20 blastomeres) were removed from the trophectoderm and were transferred to a lysing solution containing Proteinase K (Invitrogen, Carlsbad, CA, USA) 20 mg/mL, until sex determination. Biopsied embryos from both groups, VV and in vitro, were kept in holding medium for 3 hours before being transferred to the recipients.
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Brazil). The progesterone device was removed and an injection of 2 mL of cloprostenol was performed on Day 8, and on Day 9 estrus was observed. 2.5. Evaluation of DNA integrity by embryo sexing procedure To validate that the DNA from biopsies could be used for any genomic analysis, sex determination was performed by multiplex PCR using two pairs of primers. The first pair was specific to a region of the Y chromosome [34], whereas the second pair was specific to a bovine autosomal gene [35]. Bovine genomic DNA from a male and a female were used as a control. Polymerase chain reaction was performed by adding to each sample tube a PCR mix containing 20 nM of each pair of primers, 200 mM of each dNTP, 1 PCR buffer and 1U Platinum Taq DNA polymerase (Invitrogen) in a final volume of 30 mL. The PCR program used 40 cycles of 94 C for 20 seconds, 57 C for 30 seconds and 72 C for 30 seconds, followed by a final extension of 72 C for 30 minutes. Amplified PCR products were electrophoresed in 2.0% agarose gel stained with ethidium bromide (10 mg/mL) and visualized under UV illumination. When two amplicons were detected, the embryo was identified as male, whereas detection of one amplicon indicated a female embryo (Fig. 1). 2.6. Experimental design To evaluate the effect of biopsy of VV embryos on pregnancy rate, a total of 609 embryos were used in an experiment with eight replicates, in which each replica corresponded to a day of embryo recovered. For each replicate, selected embryos were randomly distributed into two groups: (a) in vivo biopsied (VV-B, n ¼ 380) and (b) in vivo control (VV-C, n ¼ 229). Both groups were composed of grade I and II embryos at Mc, BI, BL, and BX stage, and both were kept at room temperature for 3 hours in holding medium before transfer to the recipient.
2.4. In vivo and in vitro embryo transfer Embryos were transferred nonsurgically to the uterine horn ipsilateral to the CL of synchronized recipients that came into estrus 7 days before the transfer. Synchronization of the recipients was performed by administering an intramuscular injection of 2.0-mg estradiol benzoate (Ric-BE, Syntex S.A., Buenos Aires, Argentina) and inserting an intravaginal progesterone-releasing device with 1.9 g of progesterone (CRESTAR, MSD Animal Health, São Paulo, SP,
Fig. 1. Polymerase chain reaction results of bovine individual embryo biopsies, on 2.5% agarose (wt/vol) gel. Molecular marker (L); Bovine genomic DNA (CN, CM, and CAu); male embryos (M) and female embryos (F).
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Table 1 Pregnancy rate at 30 and 60 days of gestation and embryonic loss (EL) of biopsied (VV-B) and nonbiopsied (VV-C) bovine embryos produced in vivo. Group
VV-B VV-C
Embryos Total
Pregnancy 30 days, n (%)
Pregnancy 60 days, n (%)
EL
380 229
218 (57.36) 132 (57.64)
206 (54.21) 128 (55.89)
12 (5.50) 6 (4.45)
Data analyzed by chi-square (P > 0.05).
For the in vitro experiment, 318 IVP embryos were used. Similar to the VV group, embryos were selected and distributed into two groups: in vitro biopsied (IVP-B, n ¼ 91) and in vitro control (IVP-C, n ¼ 227), both composed of grade I and II embryos at BI, BL, and BX stage. In both groups, biopsied and intact embryos were kept for 3 hours, individually on SOF media at 39 C, 5% of CO2. After the incubation period, embryos were transferred to the recipients. All recipients were evaluated using an ultrasound with a 7.5-MHz linear probe at D30 and D60, for pregnancy, embryonic loss, and sexing. To confirm the good quality/integrity of the DNA collected in the biopsy, sex identification was performed on 271 biopsies from 380 VV-B and 91 biopsies from 91 IVP-B embryos. 2.7. Statistical analyses Conception rates and the effect of the embryonic stage on the pregnancy outcome after biopsy were carried out by the chi-square test. Differences were considered significant when P 0.05. 3. Results A total of 609 VV embryos were used to evaluate the effect of the biopsy on pregnancy. Biopsy had not affected embryo viability since pregnancy rate at 30 days was similar to the control embryos, which were not submitted to any micromanipulation procedure (Table 1). Similarly, no variation was observed between biopsied and intact embryos for embryonic loss evaluated by the pregnancy at Day 60 after transfer (Table 1). When embryos were biopsied at different stages of development, the pregnancy rate was higher for BI than for Mc (P < 0.05); however, it was similar for all the other stages (Table 2). Moreover, no impact of the embryo stage was detected on percentage of pregnant recipients when control embryos were transferred (Table 2).
To evaluate if biopsy affected the viability of IVP embryos, a total of 318 embryos were used. As reported in Table 3, the pregnancy of biopsy-derived blastocysts was not different from the nonbiopsy control group. When the results were evaluated, taking into account the stage of development, IVP embryos biopsied at BI, BL, and BX stage also had similar pregnancy rates (Table 4). From all the 362 biopsies evaluated, less than 10% were not suitable for identifying the sex of the embryos, either by the presence of a weak band or by the lack of amplification (Table 5). From a total of 243 VV-B embryos that had the sex identified, 168 (69.14%) were female and 75 (30.86%) were male. Regarding IVP-B embryos, from a total of 87 embryos that had the sex identified, 48 (55.17%) were female and 39 (44.83%) were male.
4. Discussion Genomic tools, such as single nucleotide polymorphism (SNP) chips, have led to a new method of selection called “genomic selection” in which dense SNP genotypes covering the genome are used to predict the breeding values. When GS is combined with reproductive technologies such as IVP and embryo biopsy, it can lead to substantial savings for breeders and to a dramatic increase in the rate of genetic progress. However, for these tools to become useful some limitations have to be overcome. The first, which is related to the ability of a micromanipulated embryo to result in a pregnancy, was the main focus of our study. To evaluate the effect on pregnancy rate of biopsy by microblade, we used embryos VV and IVP produced. As expected, we found no significant differences in pregnancy rates at 30 and 60 days when transferring biopsied or intact VV bovine embryos. Various authors have compared the viability of biopsied and nonbiopsied VV embryos and also shown that biopsy could be performed in bovine embryos without any variations in pregnancy rates [17,18,26,36]. These results indicate that despite the invasiveness of embryo biopsy, the early embryos’ plasticity is such that the procedure does not seem to adversely affect the capacity of the embryo to develop or to implant normally. Even so, there is evidence indicating the importance of the timing and method of biopsy in influencing the subsequent embryonic development and survival [18,37,38]. Therefore, we also investigated the effect of the developmental stage of the biopsied Day 7 embryos on the establishment of the pregnancy. Biopsied Mc, BI, BL, and BX had similar pregnancy to the control embryos at the same
Table 2 Effect of the stage of development of biopsied (VV-B) and nonbiopsied (VV-C) in vivo-produced bovine embryos on pregnancy rate at 30 days of gestation. Group
Pregnant recipients/N total (%) Mc
BI
BL
BX
VV-B VV-C
88/170 (51.76)a 86/157 (54.77)a
20/27 (74.07)b 9/17 (52.94)a
95/158 (60.12)a,b 32/49 (65.30)a
15/25 (60.00)a,b 5/6 (83.33)a
a,b
Data with different superscripts in the same line differ significantly (P < 0.05). Abbreviations: BI, initial blastocyst; BL, blastocyst; BX, expanded blastocysts; Mc, compacted morula.
R.V. de Sousa et al. / Theriogenology 90 (2017) 25–31 Table 3 Pregnancy rate at 60 days of gestation of biopsied (IVP-B) and nonbiopsied (IVP-C) bovine embryos produced in vitro. Group
Embryos
IVP-B IVP-C
N total
Pregnancy at 60 days, n (%)
91 227
24 (26.37) 45 (19.82)
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Table 5 Identification of embryo sex by PCR of biopsies from in vivo (VV-B) and in vitro (IVP-B) produced bovine embryos. Group
Sex identification by PCR (%) N
Identified
Nonidentified
VV-B IVP-B
271 91
243 (89.66) 87 (95.60)
28 (10.33) 4 (4.39)
Data analyzed by chi-square (P > 0.05).
Abbreviation: PCR, polymerase chain reaction.
stage. However, when the comparison was made within the group and between stages it was observed that biopsied Mc presented a lower pregnancy at D30 than the biopsied BI but was similar to BL or BX. Although there is a scarcity of literature regarding the comparison of morula with more advanced stages of development after transfer, it has been shown that bovine [39] and ovine [40] biopsied embryos at morula stage lead to decreased pregnancy rates compared with blastocysts. These findings are in agreement with our results, and they could easily be explained by the fact that the youngest embryos have fewer cells and, after manipulation, the number of surviving blastomeres may be much lower, affecting viability. However, our data showed that Mc had similar pregnancy outcome to BX and BE. We are not sure why the difference in pregnancy was only related to BI; it is possible that the low number of embryos at Mc stage affected the result. Genomic selection for domestic animals is becoming a useful tool, and its use will probably increase more and more in the near future. This is especially true if it is combined with IVP and biopsy technologies, because that association provides the most efficient way to obtain genetic gain more rapidly. Therefore, one of the most limiting factors is the viability of the IVP embryo after removal of the material necessary for the GS. The rationality for that is because it is generally accepted that embryos produced in vitro are morphologically, ultrastructurally, physiologically, transcriptionally, and metabolically different from their VV counterparts [41–45]. In summary, they are inferior in quality compared with those derived VV. Therefore, one can suppose that they would tolerate the stress of the biopsy less well. To test how IVP embryos respond to the biopsy, we evaluated the pregnancy rate. We found that no differences were observed between the biopsied and the control group, suggesting that removal of trophoblast cells did not affect embryo viability. It is important to
highlight that we cannot rule out the possibility that the similar pregnancy rate could be due to a mechanism of compensation from the biopsied group. In this group, the embryos had an open area on the zona pellucida due to micromanipulation, and this open area could have helped the hatching and increased the pregnancy as has been shown by Park, et al. [12]. Regarding the low-pregnancy rate observed for the IVP embryos, this was probably due to nutritional levels of the animals, resulting from the reduced food availability due to the early onset of the dry season. When we compared the pregnancy rates after transfer of bovine in vitro embryos biopsied at different stages of development, no differences were observed. The Mc stage was not included in the in vitro experiment because when embryos were removed from culture at D7 the majority were at blastocyst stage. These results suggested that even early blastocysts can be biopsied without affecting their viability. Furthermore, it is important to point out that the success of the technique is highly dependent on the ability and training of the operator, since very specific skills are needed. The second main limitation of using GS associated with IVP and biopsy techniques is the requirement of a minimum amount of good quality genomic DNA to perform the analysis. Although it was not our objective to evaluate the DNA present in the biopsy, we chose the microblade instead of aspiration or the needle method, because it has been shown that genetic material obtained using a microblade was of higher quality than that obtained by the other methods [18]. In addition, it has been indicated to be the most practical under field conditions and has been associated with good pregnancy outcome [18]. To evaluate the quality of DNA, we used the biopsy to determine the sex of embryos by PCR. From the biopsies analyzed, about 90% had the sex determined, suggesting that DNA was there and was correctly amplified. All the biopsies were done by the same technician, and the size had been previously determined in our laboratory as being between 10 and 20 cells. Using a different number of cells in biopsies for a genotyping investigation, Fisher et al. [7] found that a single-cell or three-cell sample failed to yield enough DNA for genotyping. In contrast, biopsies obtained from the trophoblast of a blastocyst with a microsurgical knife gave a call rate following wholegenome amplification and genotyping similar to that obtained with a 30- to 40-cell morula and 50-cell bisected blastocysts. A biopsy of eight to 10 cells obtained under laboratory conditions, using a microblade under a stereomicroscope gave a call rate of 90% [46]. Taking all this information together, it is very possible that our in vitro
Table 4 Effect of the stage of development of biopsied (IVP-B) and nonbiopsied (IVP-C) in vitro-produced bovine embryos on pregnancy rate at 60 days of gestation. Group
IVP-B IVP-C
Pregnant recipients/N total (%) BI
BL
BX
5/20 (25.00) 16/79 (20.51)
10/41 (24.39) 17/91 (18.68)
9/30 (30.00) 8/39 (20.51)
Data analyzed by chi-square (P > 0.05). Abbreviations: BI, initial blastocyst; BL, blastocyst; BX, expanded blastocysts.
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blastocyst biopsies would provide enough cells to enable reasonably robust whole-genome amplification and SNP genotyping. In conclusion, biopsy of bovine blastocysts produced VV and in vitro using microblade does not affect their viability and their ability to originate pregnancy at Day 60 of gestation. This method also provides a DNA of good quality that can be used for genetic analysis. Acknowledgments The authors thank Guilberth Serviços Veterinários S/C LTDA, Gênesis Biotecnologia Ltda/DF and Agropecuária Palma Ltda/DF for allowing them to use their embryos for the research, and Embrapa for financial support. Funding information: Grant sponsors: Embrapa Genetic Resources and Biotechnology MP1 01.13.06.001.04.00. References [1] Van Vleck LD, Westell RA, Schneider JC. Genetic change in milk yield estimated from simultaneous genetic evaluation of bulls and cows. J Dairy Sci 1986;69:2963–5. [2] Havenstein GB, Ferket PR, Scheideler SE, Larson BT. Growth, livability, and feed conversion of 1957 vs 1991 broilers when fed “typical” 1957 and 1991 broiler diets. Poult Sci 1994;73:1785–94. [3] Goddard ME, Hayes BJ, Meuwissen THE. Genomic selection in livestock populations. Genet Res 2010;92:413–21. [4] Kadarmideen HN, Mazzoni G, Watanabe YF, Strøbech L, Baruselli PS, Meirelles FV, et al. Genomic selection of in vitro produced and somatic cell nuclear transfer embryos for rapid genetic improvement in cattle production. Anim Reprod 2015;12: 389–96. [5] Ponsart C, Le Bourhis D, Knijn H, Fritz S, Guyader-Joly C, Otter T, et al. Reproductive technologies and genomic selection in dairy cattle. Reprod Fertil Dev 2013;26:12–21. [6] Pryce JE, Daetwyler HD. Designing dairy cattle breeding schemes under genomic selection: a review of international research. Anim Prod Sci 2012;52:107–14. [7] Fisher PJ, Hyndman DL, Bixley MJ, Oback FC, Popovic L, McGowan LT, et al. Brief communication: potential for genomic selection of bovine embryos. Soc Anim Prod 2012;72:156–8. [8] Kasinathan P, Wei H, Xiang T, Molina JA, Metzger J, Broek D, et al. Acceleration of genetic gain in cattle by reduction of generation interval. Sci Rep 2015;5:8674. [9] Harper JC, Sengupta SB. Preimplantation genetic diagnosis: state of the ART 2011. Hum Genet 2011;131:175–86. [10] Macháty Z, Páldi A, Csáki T, Varga Z, Kiss I, Bárándi Z, et al. Biopsy and sex determination by PCR of IVF bovine embryos. J Reprod Fertil 1993;98:467–70. [11] Chrenek P, Boulanger L, Heyman Y, Uhrin P, Laurincik J, Bulla J, et al. Sexing and multiple genotype analysis from a single cell of bovine embryo. Theriogenology 2001;55:1071–81. [12] Park JH, Lee JH, Choi KM, Joung SY, Kim JY, Chung GM, et al. Rapid sexing of preimplantation bovine embryo using consecutive and multiplex polymerase chain reaction (PCR) with biopsied single blastomere. Theriogenology 2001;55:1843–53. [13] Leoni G, Ledda S, Bogliolo L, Naitana S. Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embryonic genome analysis. J Reprod Fertil 2000;119:309–14. [14] El-Gayar M, Holtz W. Transfer of sexed caprine blastocysts freshly collected or derived from cultured morulae. Small Rumin Res 2005; 57:151–6. [15] Herrera C, Morikawa MI, Bello MB, von Meyeren M, Eusebio Centeno J, Dufourq P, et al. Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program. Theriogenology 2014;81:758–63. [16] Bondioli KR. Embryo sexing: a review of current techniques and their potential for commercial amdication in livestock production. J Anim Sci 1992;70:19–26. [17] Lopes RF, Forell F, Oliveira AT, Rodrigues JL. Splitting and biopsy for bovine embryo sexing under field conditions. Theriogenology 2001; 56:1383–92.
[18] Cenariu M, Pall E, Cernea C, Groza I. Evaluation of bovine embryo biopsy techniques according to their ability to preserve embryo viability. J Biomed Biotechnol 2012;2012:541384. [19] Agca Y, Monson RL, Northey DL, Peschel DE, Schaefer DM, Rutledge JJ. Normal calves from transfer of biopsied, sexed and vitrified IVP bovine embryos. Theriogenology 1998;50:129–45. [20] Lee JH, Park JH, Lee SH, Park CS, Jin DI. Sexing using single blastomere derived from IVF bovine embryos by fluorescence in situ hybridization (FISH). Theriogenology 2004;62:1452–8. [21] Tominaga K. Cryopreservation and sexing of in vivo- and in vitroproduced bovine embryos for their practical use. J Reprod Dev 2004; 50:29–38. [22] Yotsushima K, Sakaguchi M, Shimizu M, Okimura T, Izaike Y. Effects of fatty acid-free bovine serum albumin and fetal calf serum supplementing repair cultures on pre- and post-warm viability of biopsied bovine embryos produced in vitro. J Reprod Dev 2004;50: 471–6. [23] Pereira RM, Carvalhais I, Pimenta J, Baptista MC, Vasques MI, Horta AEM, et al. Biopsied and vitrified bovine embryos viability is improved by trans10, cis12 conjugated linoleic acid supplementation during in vitro embryo culture. Anim Reprod Sci 2008;106: 322–32. [24] Kubisch HM, Johnson KM. The effects of blastomere biopsy and oxygen tension on bovine embryo development, rate of apoptosis and interferon-s secretion. Reprod Domest Anim 2007;42:509–15. [25] Tominaga K, Iwaki F, Hochi S. Conventional freezing of in vitroproduced and biopsied bovine blastocysts in the presence of a low concentration of glycerol and sucrose. J Reprod Dev 2007;53:443–7. [26] Peippo J, Viitala S, Virta J, Räty M, Tammiranta N, Lamminen T, et al. Birth of correctly genotyped calves after multiplex marker detection from bovine embryo microblade biopsies. Mol Reprod Dev 2007;74: 1373–8. [27] Polisseni J, Sá WF, Guerra Mde O, Machado MA, Serapião RV, Carvalho BCd, et al. Post-biopsy bovine embryo viability and whole genome amplification in preimplantation genetic diagnosis. Fertil Steril 2010;93:783–8. [28] Shirazi A, Borjian S, Ahmadi E, Nazari H, Heidari B. The effect of biopsy during precompacted morula stage on post vitrification development of blastocyst derived bovine embryos. Avicenna J Med Biotechnol 2010;2:107–11. [29] El-Sayed A, Hoelker M, Rings F, Salilew D, Jennen D, Tholen E, et al. Large-scale transcriptional analysis of bovine embryo biopsies in relation to pregnancy success after transfer to recipients. Physiol Genomics 2006;28:84–96. [30] Hoelker M, Held E, Salilew-Wondim D, Schellander K, Tesfaye D. Molecular signatures of bovine embryo developmental competence. Reprod Fertil Dev 2013;26:22–36. [31] Robertson I, Nelson RE. Certification and identification of the embryo. In: Stringfellow DA, Seidel SM, editors. Manual of International embryo transfer Society. Third edition. Savoy, IL, USA: IETS; 1998. p. 103–34. [32] Parrish JJ, Krogenaes A, Susko-Parrish JL. Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development. Theriogenology 1995;44:859–69. [33] Holm P, Booth PJ, Schmidt MH, Greve T, Callesen H. High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. Theriogenology 1999;52:683–700. [34] Bondioli KR, Ellis SB, Pryor JH, Williams MW, Harpold MM. The use of male-specific chromosomal DNA fragments to determine the sex of bovine preimplantation embryos. Theriogenology 1989;31:95–104. [35] Ellis SB, Bondiolli KW, Williams ME, Pryor JH, Harpold MM. Sex determination of bovine embryos using male-specific DNA probes. Theriogenology 1988;29:242. [36] Carbonneau G, Morin N, Durocher J, Bousquet D. Viability of bovine ivf embryos biopsied with microsection or microaspiration technique for sexing. Theriogenology 1997;47:266. [37] Vajta G, Holm P, Greve T, Callesen H. Comparison of two manipulation methods to produce in vitro fertilized, biopsied and vitrified bovine embryos. Theriogenology 1997;47:501–9. [38] Shirazi A, Borjian S, Nazari H, Ahmadi E, Heidari B, Bahiraee A. Effects of timing on cell biopsy from pre-compacted morula stage bovine embryos on subsequent embryonic development. J Reprod Infertil 2010;11:25–32. [39] Lacaze S, Ponsart C, Humblot P. Influence of embryo stage on pregnancy rates following transfer of bovine biopsied embryos under on-farm conditions. Proceedings of the 25th AETE, Poznan, Poland: IETS; 2009. p. 208.
R.V. de Sousa et al. / Theriogenology 90 (2017) 25–31 [40] Naitana S, Loi P, Ledda S, Cappai P, Dattena M, Bogliolo L, et al. Effect of biopsy and vitrification on in vitro survival of ovine embryos at different stages of development. Theriogenology 1996;46:813–24. [41] Farin C, Farin P, Piedrahita J. Development of fetuses from in vitro– produced and cloned bovine embryos. J Anim Sci 2004;82:E53–62. [42] Crosier AE, Farin PW, Dykstra MJ, Alexander JE, Farin CE. Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro. Biol Reprod 2000;62:1459–65. [43] Crosier AE, Farin PW, Dykstra MJ, Alexander JE, Farin CE. Ultrastructural morphometry of bovine blastocysts produced in vivo or in vitro. Biol Reprod 2001;64:1375–85.
31
[44] Lonergan P, Fair T, Corcoran D, Evans ACO. Effect of culture environment on gene expression and developmental characteristics in IVF-derived embryos. Theriogenology 2006;65: 137–52. [45] Machado GM, Ferreira AR, Pivato I, Fidelis A, Spricigo JF, Paulini F, et al. Post-hatching development of in vitro bovine embryos from day 7 to 14 in vivo versus in vitro. Mol Reprod Dev 2013;80:936–47. [46] Le Bourhis D, Mullaart E, Schrooten C, Fritz S, Coppieters W, Ponsart C. Breeding values concordance between embryos and corresponding calves. Reprod Fertil Dev 2011;24:180.