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In Vitro Production of Sex Preselected Cattle Embryos Using a Monoclonal Antibody Raised Against Bull Sperm Epitopes T M.M.R

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In Vitro Production of Sex Preselected Cattle Embryos Using a Monoclonal Antibody Raised Against Bull Sperm Epitopes T M.M.R Animal Reproduction Science 205 (2019) 156–164 Contents lists available at ScienceDirect Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci In vitro production of sex preselected cattle embryos using a monoclonal antibody raised against bull sperm epitopes T M.M.R. Chowdhurya,e,1, Lianguang Xub,1, Rami Kongb,d,1, Bun-Young Parkd, Ayman Mesalamb,f, Myeong-Don Joob, Fahmida Afrina, Jong-In Jind, ⁎ Hyun-Tae Limb,c, Il-Keun Kongb,c, a Animal Genetic Resources Research Center, National Institute of Animal Science, RDA, Namwon 55717, Jeollabuk-do Province, Republic of Korea b Department of Animal Science, Division of Applied Life Science (BK21 Plus), Republic of Korea c Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Gyeongsangnam-do Province, Republic of Korea d GAST, Gyeongsang National University, Jinju 52828, Gyeongnam Province, Republic of Korea e Department of Physiology and Pharmacology, Faculty of Animal Science and Veterinary Medicine, Patuakhali Science and Technology University, Bangladesh f Department of Theriogenology, Faculty of Veterinary Medicine, Zagazig, 44519, Egypt ARTICLE INFO ABSTRACT Keywords: Sex preselection has always generated great interest among livestock producers. Among the WholeMom prevalent sperm sorting methods, there is much evidence that sex sorting has a negative effect on Sorted sperm sperm quality with an altered pattern of sperm motility, ultimately reducing the period of cell Genetic up-gradation viability. In this study, we have established a new approach for the preselected embryo pro- IVEP duction by using WholeMom®; a monoclonal antibody developed against bull sperm epitopes for simple and easy separation of X- and Y-sperm. There were no significant differences (P > 0.05) in the percentage of presumptive zygotes between the control and the X-sperm sorted group, but there was a difference in early cleaving embryos with there being 81.2 ± 1.4%, 78.3 ±1.0%,and 66.7 ± 1.1% for the control, X-sperm sorted, and Y-sperm sorted groups, respectively. Similarly, the percentage of embryos that developed to the blastocyst stage (Day 7) were also greater (P < 0.05) in the control and X-sperm sorted group compared with the Y-sperm sorted group being 34.8 ± 1.0%, 32.1 ± 0.8%, and 23.7 ± 1.0% in the control, X-sperm sorted, and Y-sperm sorted groups, respectively. Furthermore, B-SRY F2 and B-SRY R2 gene expression data indicated there was a detection accuracy of 81.0% for the female embryos and 72.5% for the male embryos produced in vitro. In conclusion, in cattle in vitro derived embryo production using pre-selected sexed semen and subsequent embryo transfer can facilitate the mass production of individuals that are genetically superior. 1. Introduction Currently, there is an increased worldwide demand for dairy and beef products which has resulted in greater attention on improving embryo production efficiency (Holden and Butler, 2018). The use of sex preselection along with use of other genomic, proteomicand ⁎ Corresponding author at: Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 660-701, Gyeongnam Province, Republic of Korea. E-mail address: [email protected] (I.-K. Kong). 1 These three authors equally contributed to this work. https://doi.org/10.1016/j.anireprosci.2018.11.006 Received 23 August 2018; Received in revised form 23 October 2018; Accepted 14 November 2018 Available online 16 November 2018 0378-4320/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). M.M.R. Chowdhury, et al. Animal Reproduction Science 205 (2019) 156–164 phenomics technologies, provide a platform for development of a promising breeding strategy to meet the increased demand for milk or meat (Chen et al., 2012) and also are promising as a means for sustainable production of elite dairy or beef cattle (Rath et al., 2012). Sex sorting technology would be the primary way of obtaining genetically elite cattle embryos with pre-selected sexes. For example, dairy cattle farmers prefer high yielding dairy cattle for milk production, while the elite bulls are selectively produced for their greater hereditary impact and economic importance (Chen et al., 2012). In the dairy industry, there is extra production of unwanted male calves. Male dairy calves increase the risk of dystocia as compared with heifer calves, and are a surplus by-product of breeding with conventional semen resulting in calves with relatively lesser economic value than heifer calves. Integrating sexed semen into the breeding programs can reduce the number of unwanted male dairy calves and reduce dystocia. Furthermore, provision of sexed semen could result in generation of herd replacements and additional heifers for herd expansion at a faster rate (Holden and Butler, 2018). Although there are various sperm sorting techniques none can be used that have a 100% accuracy. Furthermore, sex preselection before the formation of a conceptus is an ethically justified practice (Eftekhaari et al., 2015) whereby there is sorting of spermwithX or Y chromosomes prior to fertilization of the oocyte to generate either a male or a female offspring. Sex preselection could be accomplished before insemination by sorting of the sperm with a X or Y chromosome based on the distinctive characteristics of these two cell types. The success of separation of sperm with a X from those with a Y chromosome increased because of the desire of a significant number of commercial and registered dairy breeders to use in vitro fertilization (IVF) technologies to obtain elitefemales using preselected sires with superior genomic values (Garner et al., 2013). Nevertheless, there are some disadvantages with this being an economically expensive process. Some of the challenges with use of this process include a reduced life span of sex-sorted sperm, slowness of the process of sperm sorting per h (15 × 106 sperm/h of each sex), lesser sperm numbers used per insemination dose (2 × 106), and relatively lesser in vivo fertility compared to when sperm are used that has not been sex sorted. Furthermore, there needs to be expertise available for use of other technologies including artificial insemination (AI) (Garner et al., 2013). Numerous factors such as health issues including sex-linked disease, has been stimulated farmers to select traditional or up-to-date techniques in selecting the gender of the offspring. Several of these approaches are the use of the following methods: swim-upor washing of spermatozoa, percoll gradient sperm separation, use of glass wool column filter, albumin separation, microsort using FISH (Fluorescence in situ hybridization), free electrophoresis, pH adjustment, pre-implantation genetic diagnosis (PGD) and flowcyto- metry (Eftekhaari et al., 2015). Of these attempts, the only method proven to be commercially viable is flow cytometry or fluor- escence-activated cell sorting (Garner et al., 2013). Factors that should be taken into consideration during sorting process are: accuracy of sexing sperm, fertility when sexed sperm is used for breeding, genetic gain from use of sex-sorted sperm, and the economic value of sex-sorted sperm (Garner et al., 2013). During sorting, sorting pressure, speed, electrical deviation, and laser radiation, synergistically all lead to membrane alteration and pre-capacitation like changes in sperm that have been sex sorted, resulting in a reduced fertility when these sperm are used (Garner et al., 2013). Even with these limitations, production of sex-sorted semen usually followed by cryopreservation is being used commercially for cattle production. Development of technologies that would allow for the sorting rate to be more accurate without affecting sperm viability and fertility is still an evolving area of research. To overcome the existing limitations, the present studies were designed for sperm sorting based on the distinctive physiological differences between sperm with a X or Y chromosome and we hypothesized that these differences will be significant enoughtoenable successful separation of sperm with a X from those with a Y chromosome. WholeMom is a monoclonal antibody used to separate the sperm with a Y- from those with a X chromosome. WholeMom antibody binds with the plasma membrane of the heads of sperm with the Y chromosome. After treatment with WholeMom antibody for 20 to 25 min, most of Y-sperm heads are attached together by head- to-head agglutination and these sperm precipitated in the bottom. WholeMom antibodies do not bind to the sperm with the X chromosome. Thus, sperm with the X chromosome are freely motile with the capacity for transport to the site of oocyte fertilization in the female reproductive tract. We hypothesized that use of the WholeMom monoclonal antibodies would allow for the accurate sex selection of embryos without negatively affecting embryo development to the blastocyst stage. 2. Materials and methods 2.1. Reagents All chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise noted. 2.2. Animal ethics All of the methods and experimental procedures were conducted according to the approved (Approval ID: GAR-110502- X0017) guidelines and regulations by the institutional animal care and use committee (IACUC) of Gyeongsang National University, Republic of Korea. 2.3. Cumulus-oocyte complex (COC) recovery Ovaries from Korean native cows (Hanwoo) were collected after slaughter at the local slaughterhouse, placed in physiological saline (0.9% NaCl) at approximately 37.5 °C, and transported to the laboratory within 4 h of slaughter. After washing the ovaries with fresh Dulbecco’s phosphate buffered saline (D-PBS), COCs were retrieved as described (Chowdhury et al., 2017). COCs were re- covered from follicles (2–8 mm diameter) using an 18-G needle attached to a vacuum pump. Aspirated follicular fluid was expelled into dishes containing TL-HEPES medium (6.662 g/L sodium chloride, 0.238 g/L potassium chloride, 0.168 g/L sodium bicarbonate, 157 M.M.R.
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