The sexing of bovine embryos in the field

doi:10.1016/0093-691X(94)00008-I

Theriogenology

Volume 43, Issue 1, January 1995, Pages 71-80

https://doi.org/10.1016/0093-691X(94)00008-I Get rights and content

Abstract

Bovine embryo sexing has been tested under field conditions in France by officially approved embryo transfer teams of some AI Cooperatives since 1990. The aim of the present review is to report on the technical and logistical aspects of these procedures in the cattle industry. For fresh embryos, DNA technology has been shown to be highly sensitive (one cell), efficient (95%) and accurate (98%). Pregnancy rates of sexed embryos were not significantly different from that of controls. For frozen embryos, the biopsy procedure resulted in reduced pregnancy rates. Another method, based upon cell aspiration, resulted in improved pregnancy rates after freezing but only 3 out of 4 embryos could be sex-determined. This is currently being investigated. Recommended health certification standards have been established which would provide for the safe movement of micromanipulated embryos internationally. From the embryo transfer industry point of view, embryo sexing requires thorough training and monitoring. To the farmers, this technology allows a clear return on their investment due to the higher degree of security in obtaining offspring of the desired sex from a given mating and the savings achieved through better management of recipients.

References (25)

KJ Betteridge et al.
Approaches to sex selection in farm animals
WCD Hare et al.
Sexing two-week old bovine embryos by chromosomal analysis prior to surgical transfer. Preliminary methods and results
Theriogenology
(1976)
CM Herr et al.
Micromanipulation of bovine embryos for sex determination
Theriogenology
(1991)
M Leonard et al.
Sexing bovine embryos using Y chromosome specific DNA
Theriogenology
(1987)
H Niemann
Cryopreservation of ova and embryos from livestock: current status and research needs
Theriogenology
(1991)
K Utsumi et al.
Sexing of bovine embryos by PCR using bovine SRY primer
Theriogenology
(1994)
TJ Williams
A technique for sexing mouse embryos by a visual colorimetric assay of the X-linked enzyme, glucose 6-phosphate dehydrogenase
Theriogenology
(1986)
CE Bishop et al.
Sondes d'ADN spécifique du génome mâle des ruminants, leur préparation et utilisation
K Bondioli
Embryo Sexing: A review of current techniques and their potential for commercial application in Livestock production
J Anim Sci
(1992)
M Courot et al.
Dynamique de la sélection, Bilans et perspectives. Technologie de la reproduction: Sexage, Fécondation In Vitro, Clonage, Cellules embryonnaires souches, Transgénèse

SB Ellis et al.

Nucleic acid probes for prenatal sexing. International application published under the Patent Cooperative Treaty (PCT) Word Intellectual Property Organization

Publication no. Wo 86/07095

(1993)

Y Heyman et al.

Birth of cloned calves after nuclear transfer from sexed embryos

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The aim of this study was to establish a platform for genomic selection of in vitro-fertilized (IVF) Gir embryos. Multiple displacement amplification (MDA)-based embryo biopsy samples were genotyped, and genomic estimated breeding values (GEBV) for milk yield (305MY) were calculated. The concordance of GEBV and accuracy between embryo biopsies and the respective liveborn were assessed. Imputation was performed using two panels (Z-Chip and Bovine HD, Illumina) based on a database of 73,110 lactating cow’s database and pedigree files from 147,131 animals. Biopsied embryos had similar pregnancy rates (39% vs 40%), pregnancy loss rates (18% vs 20%), and pregnancy length compared to Control embryos. After genotyping, low call rate means were detected for biopsy samples compared to the respective calf samples (0.80 vs 0.98). Imputation presented 0.83 (Z-Chip) and 0.96 (HD) accuracy (CORRanim). Embryo GEBV accuracy levels were higher in BovineHD imputation (0.82) than Z-Chip imputation (0.55) or no imputation (0.62), and the correlation between embryo/calf pairs’ accuracy was 0.85 for BovineHD imputation, 0.11 for Z-Chip imputation, and 0.02 for no imputation. GEVB estimates correlation between embryo/calf pairs was 0.87 for BovineHD imputation, 0.80 for Z-Chip imputation, and 0.41 before imputation. The call rate of embryo samples did not affect the correlation between embryo/calf pairs for accuracy and GEBV before and after BovineHD imputation. Embryos obtained on the same farm presented GEBV 305MY differences of up to 800 kg, emphasizing the expected impact of embryo genomic selection for the Gir breed.
Vitrification and in-straw warming do not affect pregnancy rates of biopsied bovine embryos
2022, Theriogenology
Citation Excerpt :
Each biopsy technique has advantages and disadvantages [37]. The damage of the zona pellucida seemed to be reduced when embryos are biopsied using the needle or the aspiration technique [38]. Cenariu et al. [39] showed that the needle biopsy (57.0%) yielded higher pregnancy rates after transfer of biopsied IVD embryos compared to the aspiration technique (43.0%) or the microblade technique (31.0%).
In the cattle industry, in vivo or in vitro embryo production combined with genotyping and cryopreservation technologies allows the selection and conservation of embryos carrying genes for desirable traits. This study aimed to assess the efficiency of a vitrification method suitable for in-straw warming of biopsied in vivo derived (IVD) bovine embryos. Three experiments were carried out using two methodologies: the Cryotop®, the gold standard vitrification and 3-step warming methodology, or the VitTrans, a vitrification/in-straw 1-step warming method that enables direct embryo transfer to the uterus. In experiment 1, intact and biopsied in vitro produced (IVP) day 7 expanded blastocysts were vitrified using the Cryotop® and warmed in 1- or 3-steps. No differences in survival rates were recorded at 24 h after warming for intact or biopsied IVP blastocysts irrespective of the warming procedure. In experiment 2, the effect of the time from trophectoderm (TE) biopsy to vitrification/in-straw warming on post-warming survival rate was assessed. No significant differences in survival were observed when blastocysts were vitrified/in-straw warmed immediately after biopsy or after 3 h in culture when compared to intact blastocysts. In experiment 3, IVD embryos were vitrified 3 h after biopsy using the Cryotop® or the VitTrans method and pregnancy rates were assessed at day 60 after transfer. Fresh, biopsied embryos served as control. Similar pregnancy rates were observed when IVD biopsied embryos were transferred fresh or vitrified/warmed by the Cryotop® or VitTrans method. No significant effect of the embryo quality or developmental stage was detected on the percentage of pregnant recipients when IVD biopsied embryos were transferred fresh or after vitrification. While fresh female IVD embryos produced significantly higher pregnancy rates than male embryos, there were no differences in pregnancy rates when male or female vitrified/warmed embryos were transferred. About 81% from the biopsies analyzed successfully determined the embryo sex, confirming that DNA was there, and it was efficiently amplified. To conclude, our findings indicate that both vitrification methodologies produced similar post-warming outcomes for both intact and biopsied IVP embryos. Besides, vitrification/in-straw warming of biopsied IVD bovine embryos did not affect the viability to originate pregnancy, being a useful option for their direct transfer in field conditions.
Comparison of Cell-Free Fetal DNA Plasma Content Used to Sex Determination Between Three Trimesters of Pregnancy in Torkaman Pregnant Mare
2020, Journal of Equine Veterinary Science
Citation Excerpt :
The decision to sell a pregnant mare with a fetus of the desired or undesired sex and culling broodmare is easier when fetal gender is known [5]. Several approaches have been established for genetic sex typing, such as karyotyping; H-Y antigen detection in the mouse, rat, pig, sheep, goat, cow, and horse [10]; X-linked enzymatic determination; DNA probes for bovine embryos or cells obtained by amniocentesis [11]; ultrasonic examination of bovine fetal structures [12]; and the polymerase chain reaction (PCR) for the sexing of bovine embryos in the field [13–15]. Karyotyping by amniocentesis has been used for sexing of equine and bovine embryos [16,17].
This investigation aimed to compare the cell-free fetal DNA (cffDNA) plasma present in three trimesters of pregnancy in Torkaman pregnant mare. Peripheral blood samples of 32 pregnant mares in three trimesters of pregnancy were collected in tubes containing ethylenediaminetetraacetic acid at three time points. Circulating cffDNA was extracted from 3 mL of maternal plasma. Using outer and inner primers, a conventional polymerase chain reaction was performed for the sex-determining region Y (SRY) gene present in the Y chromosome. Of the total 32 Torkaman pregnant mares, 24 were carrying male fetuses and eight were carrying female fetuses. In total, the accuracy of the test was 48.75%, 68.75%, and 75% in the first, second, and third trimesters of pregnancy, respectively. The sensitivities were 25%, 58.32%, and 66.66%, respectively, whereas their specificities were 100% in all trimesters. In conclusion, the SRY gene can permit the detection of equine fetal sex with good accuracy through cffDNA analysis in maternal plasma just in the third trimester of pregnancy, although specificity in all duration of pregnancy was 100%.
Laser assisted blastomere extrusion biopsy of in vitro produced cattle embryos—A potential high throughput, minimally invasive approach for sampling pre-morula and morula stage embryos
2020, Animal Reproduction Science
Citation Excerpt :
The micro-blade biopsy approach which is generally used for cattle embryo biopsy is invasive, with post biopsy losses resulting in 5–10 % reduction in pregnancy rate when applied to high quality IVD morula and blastocysts (Lopes et al., 2001; Hasler et al., 2002). Greater losses occur when this approach is used to biopsy IVP morula and early blastocyst-stage embryos (Bredbacka, 1998; Hasler et al., 2002; Oliveira et al., 2017), embryos in early developmental stages (pre-morula; Williams et al., 1984), and embryos that are to be cryopreserved (Thibier and Nibart, 1995; Bredbacka, 1998; Hasler et al., 2002; Cenariu et al., 2012). Previously, embryos produced using MOET procedures that were biopsied to identify sex of the embryo, could be transferred fresh avoiding the effects of cryopreservation (Thibier and Nibart, 1995; Bredbacka, 1998; Shea, 1999; Lopes et al., 2001; Hasler et al., 2002); however, if complex genotypic selection is required, cryopreservation will be a necessity (Seidel, 2010; Ponsart et al., 2013).
Whilst adoption of in vitro production (IVP) of cattle embryos and subsequent biopsy for genetic evaluation is increasing, biopsy techniques primarily used were developed to sample in vivo-produced blastocysts. This study was conducted to develop a laser-assisted blastomere extrusion approach for rapid and minimal-invasive biopsy of IVP cattle embryos at pre-morula to morula stages of development (Day 5 or 6 post-fertilisation). Embryo development into blastocysts was not compromised when ≤3 cells were collected by blastomere extrusion on Day 5 (44.4 ± 4.4 % and 34.3 ± 4.6 %) or Day 6 (58.0 ± 4.3 % and 57.5 ± 5.3 %) post-fertilisation compared with non-biopsied control embryos. Similarly, capacity to withstand cryopreservation was not different between embryos biopsied at Day 5 and 6 post-fertilisation and control-embryos (58.8 ± 6.0 %, 63.5 ± 5.6 %, and 56.0 ± 4.8 %, respectively). When more cells were collected from embryos at Day 6 post-fertilisation (≥8 compared to ≤3 cells), subsequent embryo development was not different (63.6 ± 6.1 % and 73.1 ± 6.2 %, respectively) nor was the capacity to withstand cryopreservation (67.9 ± 9.0 % and 62.5 ± 8.7 %, respectively). For biopsies on Day 6 post-fertilization, 95 % of samples produced a PCR product; however, when compared to the whole embryo PCR results, approximately 11 % of biopsy-samples classified as being from a male embryo were from female embryos (false positive), indicating DNA contamination between samples. In conclusion, results of this study indicate laser-assisted blastomere extrusion is a time efficient and minimally invasive approach to biopsy IVP morula and pre-morula cattle embryos to facilitate genetic analysis.
Embryo transfer and other assisted reproductive technologies
2018, Veterinary Reproduction & Obstetrics
Optimizing production of in vivo-matured oocytes from superstimulated Holstein cows for in vitro production of embryos using X-sorted sperm
2014, Journal of Dairy Science
Citation Excerpt :
However, in most cases, this requires specialized equipment such as a micromanipulator and related PCR machines (Hasler et al., 2002). Moreover, embryo biopsy requires skill, increases the time required for processing, and potentially reduces the viability of the embryo (Thibier and Nibart, 1995; Hasler et al., 2002; Hirayama et al., 2004). Currently, sexed sperm is commercially available and represents an attractive alternative to embryo sexing (Seidel, 2003; Garner and Seidel, 2008; DeJarnette et al., 2009).
The present study aimed to establish an efficient system for the production of female embryos from dairy cows by in vitro fertilization (IVF) using X-sorted sperm and in vivo-matured oocytes collected by ovum pick up (OPU). Nonlactating Holstein cows (n = 36) were administered a controlled intravaginal progesterone-releasing (controlled internal drug release) device (d 0), underwent dominant follicle ablation (DFA) or ovulation by administration of 100 μg of GnRH on d 5, and were superstimulated with FSH and PGF_2α, following standard procedures. Controlled internal drug release devices were removed on the evening of d 8 or on the morning of d 9, depending on the experiment. For LH surge induction, 200 μg of GnRH was administered on the morning of d 10 (0 h). In experiment 1, the peak (48.1%) of ovulating follicles was detected at 29 to 32 h after GnRH injection (0 h), and the range in the timing of the initiation of ovulation was less by timing from GnRH administration (30.0 ± 2.8 h) rather than by timing the onset of estrus (32.7 ± 4.7 h). Only 0.9% of total ovulated follicles were recorded before 26 h after GnRH injection. Therefore, OPU was carried out at 26 h and IVF occurred at 30 h after GnRH in experiments 2 and 3. In experiment 2, 83.3 ± 10.8% of oocytes with expanded cumulus cells had extruded the first polar body at 30 h after GnRH injection. The aim of experiment 3 was to compare the effect of either DFA or GnRH-induced LH surge before superstimulation on the efficiency of embryo production by IVF following superstimulation. Progesterone concentrations from d 10 to 12 in the DFA group were lower than those in the GnRH group. A greater proportion of recovered oocytes with expanded cumulus cells from ≥8-mm follicles was observed in the DFA group than in the GnRH group (95.9 and 77.4%, respectively). Blastocyst rates in the DFA and GnRH groups (58.0 and 52.8%, respectively) did not differ from those of oocytes collected from nonstimulated OPU and matured in vitro (49.9%). However, the proportion of high-quality blastocysts was higher in the DFA group compared with the GnRH group (54.9 vs. 21.5%). Our results demonstrate that high rates of good-quality blastocysts can be produced by IVF with X-sorted frozen sperm using in vivo-matured oocytes collected by OPU from cows after DFA and superstimulation combined with ovulation induction.

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The sexing of bovine embryos in the field

Abstract

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Sondes d'ADN spécifique du génome mâle des ruminants, leur préparation et utilisation

Embryo Sexing: A review of current techniques and their potential for commercial application in Livestock production

J Anim Sci

Dynamique de la sélection, Bilans et perspectives. Technologie de la reproduction: Sexage, Fécondation In Vitro, Clonage, Cellules embryonnaires souches, Transgénèse

Nucleic acid probes for prenatal sexing. International application published under the Patent Cooperative Treaty (PCT) Word Intellectual Property Organization

Publication no. Wo 86/07095

Birth of cloned calves after nuclear transfer from sexed embryos