2019 Embryo grade, but not developmental stage, is related to embryonic sex in superovulated beef cows Embryo grade, but not developmental stage, is related to embryonic sex in superovulated beef cows Caitlin Wiley,a Marianna Jahnke,a Patrick J. Gunn,b Tyler Dohlmana aVeterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, bDepartment of Animal Science, Iowa State University, Ames, IA Abstract Knowing calf sex before birth can have advantages for management and marketing. The objective was to detect relationships between embryonic sex and developmental stage and/or embryo quality at recovery. Mature, non-pregnant, non-lactating Angus and Simmental cows (n = 29) were superovulated using a 13-d, controlled internal drug release-based protocol following ultrasound-guided dominant follicle aspiration. Cows were artificially inseminated with frozen-thawed semen twice on d 6, 36 and 48 hours after controlled internal drug release-removal and PGF2α. Embryos were recovered by non-surgical flush 7 days after first insemination, with an average of nine transferrable quality embryos per donor. Embryos of quality grade 1-3 (n = 265) were biopsied, using a micromanipulator with a microsurgical blade to excise approximately 15 - 20% of the inner cell mass of each embryo. Embryo sex was determined with polymerase chain reaction on biopsies, including electrophoresis and analysis of gels to detect a male-specific Y-chromosome. Stage of embryo had no association with sex (p = 0.6). However, grade was associated with sex (p = 0.03), as proportionally more grade-1 embryos were male (51.2%), whereas a higher proportion of grade-2 embryos were females (75.2%). We concluded embryo quality at recovery was a better predictor of sex than developmental stage. Keywords: Embryo, embryo transfer, sex determination, superovulation Introduction Knowing sex of offspring prior to parturition can be economically beneficial. For instance, in the dairy industry, there is a strong interest in heifer calves as replacements, within the herd or for sale to other herds.1-3 Conversely, in some sectors of the beef industry, bulls are preferred for supplying feedlots with steers, whereas in other operations, heifers are desired for replacements.4 In addition to marketing benefits, fetal sex determination may assist in improving management at calving, as bull calves have higher rates of dystocia and mortality.5,6 In natural mating scenarios, there is a 51% probability of the conception resulting in a bull calf.3 For producers to influence sex of the offspring, sex-sorted semen, with ~90% accuracy, is the only validated approach to modify the sex ratio.3 Timing of insemination is a hypothetical method to influence fetal sex, but efficacy has not been consistent.7-9 To determine sex before calving, there are several options, with variable accuracy. Transrectal ultrasonography at 55 - 98 days after fertilization is one of the most common and least invasive methods to determine fetal sex.10,11 Other options for sex determination involve micromanipulation at the embryonic stage, including cytoplasmic analysis,12 male-specific antigens,13 and X-linked enzyme activity;14 however, these are not commonly used due to impracticality and inconsistent results. The most common and reliable method for embryonic sex determination is DNA analysis via polymerase chain reaction (PCR).15,16 Research has attempted to use developmental stage and/or embryo quality to predict embryonic sex. Several studies have been conducted with embryos produced by in vitro fertilization to determine if stage of embryo development at time of transfer has any association with fetal sex.7,8,17,18 The main findings were that male embryos developed faster than female embryos, if insemination occurred at the same time.7,17 However, timing of ovulation and fertilization is nearly impossible to predict in vivo, particularly in superovulated cattle that may ovulate over a 24-h interval. The objective was to determine if there is a relationship between embryonic stage and/or quality to embryonic sex from conventional in vivo-derived recoveries, as limited research has evaluated this relationship in conventional embryos. It Clinical Theriogenology • Volume 11 Number 1 • March 201953 was hypothesized there would be no significant difference in sex of embryos due to embryo stage or quality grade. Materials and Methods General This study was conducted at Iowa State University Lab Animal Research Station, Ames, IA and used embryos recovered from a superovulation study conducted at Iowa State University Zumwalt Station, Ames, Iowa in August 2016.19 All protocols and procedures used were approved by the Iowa State University Institutional Animal Care and Use Committee. Embryos were recovered from Angus and Simmental cows (n = 29) following a timed, 13-d superstimulation controlled internal drug release (Eazi-Breed™ CIDR®, Zoetis, Kalamazoo, MI)-based protocol initiated 1 day after trans-vaginal ultrasound guided dominant follicle ablation. Cows were artificially inseminated (AI) twice (12 hours apart) according to timed-AI schedule, 1.5 days after CIDR® removal and second prostaglandin F2α (PGF2α; Lutalyse®, Zoetis), concurrent with signs of estrus observed in all cows. Each insemination utilized one unit of frozen-thawed conventional semen sourced from a single bull collection, known to have high success rates in previous superstimulation research in our lab. Non-surgical embryo recovery was performed 7 days after initial timed-AI and embryos were evaluated according to International Embryo Technology Society standards by American Embryo Transfer Association certified personnel. Embryo biopsy Following evaluation and washing, embryos were placed in micro-drops of splitting medium (ViGro™ Splitting Plus Solution; Bioniche Animal Health, Athens, GA) used to immobilize embryos for micromanipulation. Biopsies were collected using a Bioniche Animal Health twinning system with a micromanipulator and an Olympus CKX41 microscope. A microsurgical blade excised the zona pellucida to allow removal of approximately 4 - 8 cells from the inner cell mass of the intact embryo contained in a micro-drop of splitting medium. Each biopsy was washed with ViGro™ Retrieval Supplement medium (Bioniche Animal Health) and immediately transferred to labeled micro centrifuge tube with 8 µl sterile water. Each tube was submerged in liquid nitrogen to ‘snap-freeze’ the samples and then were placed in racks at -18⁰C for approximately 1 month, until time allowed for PCR analysis. Embryo sexing was performed with a commercial PCR kit using primers specific to the Y- chromosome determinant according to the manufacturer’s instructions.20 Following electrophoresis, gels were placed on a UV trans-illuminator to determine Y-chromosome presence or absence. If the sample failed to produce distinguishable bands, results were not included in the dataset. Although 203 of the 265 embryos evaluated had distinguishable results, 62 biopsies were removed from the dataset due to inconclusive results. Statistical Analysis Data were analyzed in SAS 9.4 (SAS institute Inc., Cary, NC) using the GLIMMIX procedure for multivariate distribution. Embryo served as the experimental unit, with embryo grade, stage and flush group as fixed effects. Breed was included in the initial model as a covariate and subsequently removed due to a lack of significance (p > 0.10). Animal nested in group served as a random effect. Statistical significance was acknowledged at p ≤ 0.05. There were 62 embryo biopsies excluded from the dataset due to inconclusive PCR results. Results There was no difference in sex due to stage of embryo (p = 0.6; Table 1). However, sex differed in relation to quality grade (p = 0.03; Table 2). Specifically, there was a greater percentage of males from grade-1 versus grade-2 embryos (51.2 vs. 24.8%, respectively; p = 0.01). Clinical Theriogenology • Volume 11 Number 1 • March 2019 54 Discussion Sex determination before parturition is a growing sector of advanced reproductive technologies. As multiple ovulation embryo transfer (MOET) technology is widely used, sexing of embryos will continue to be a requested service and PCR has become the most relevant technology for early embryonic sex determination. Sexing of IVF derived embryos has advantages of monitoring maturation of oocytes, specific timing of fertilization, and development rates of embryos, all of which have been hypothesized to impact embryo sex.7,8,18,21 Several IVF studies reported correlations between embryonic development stage and sex. Previous work18 concluded male embryos develop at a faster rate than females. Early blastocysts (Stage 5) and blastocysts (Stage 6) had a greater proportion of males, whereas morulas (Stage 4) had a greater proportion of females. Previous work17 reported similar proportions of males developing to more advanced stages by d 8 after insemination. The reasoning for increased rate of development of males is unknown; however, it is hypothesized that the increased growth rate in males is associated with their gonadal sex differentiation.7,22 Furthermore, when the maturation state of oocytes was considered, early fertilization resulted in a higher ratio of females, whereas delayed fertilization produced a greater proportion of males.8,21 A potential explanation was that oocytes develop a mechanism allowing for improved processing of Y-bearing sperm during late maturation of metaphase II arrested oocytes. Furthermore, X- and Y-bearing sperm have different signaling messages and surface proteins, enabling oocytes early in maturation to process X-bearing sperm easier, whereas more mature oocytes process Y- bearing sperm more effectively later in estrus.21,23 However, the current study failed to identify a relationship between stage and embryonic sex. With superovulation protocols, a donor is expected to ovulate multiple ova during a 24 - 36 hour interval, in contrast to a single ovulation in non-stimulated donors.24 As such, the current study implemented two separate timed inseminations (12 hours apart) to have viable sperm at any point during this window of ovulation. Therefore, timing of fertilization relative to ovulation should have been evenly spaced and was not expected to alter the sex ratio, although exact timing of fertilization and rate of development were unknown. Embryo quality grade is an important component of embryo evaluation, as it can be used to predict viability after fresh transfer or cryopreservation. Quality grades take into account percentage of extruded cells, compactness of inner cell mass, shape, and color of cytoplasm.25 As the developmental stage of embryos increases with in vivo derived embryos, quality grades below a 1 are rarely appreciated, especially once embryos develop to at least Stage 6 (blastocyst). One reason for this is the blastocoel cavity expands and fills the perivitelline space, making it difficult to appreciate any extruded cells or debris, which eliminates the potential for downgrading embryo quality. Results from the current study, with in vivo-derived embryos, reflect these observations, with all Stage 6 and 7 embryos given quality grade 1. Meanwhile, quality grade 2 embryos in this study were at Stages 4 and 5. In a previous work,26 ovine IVF embryos were evaluated for embryonic sex based on rate of development at 207 hours after insemination. Within the advanced development stages (hatched, hatching, and expanded blastocysts), quality grades ranged from 1 - 4. These more advanced development stage with quality grades 1 - 3, tended to have a higher proportion of males (57.2%, p < 0.08).26 Despite advanced developmental stages, there were variations in quality that were associated with embryonic sex. However, quality grading of IVF embryos is not as well defined as conventional embryo grading, and IVF embryos are often graded harder due to increased cellular debris and extruded cells, especially in the more developed stages, increasing variations in quality grades with more advanced embryos.27 Our study had few grades 2 and 3 embryos (n = 29 and 19, respectively); therefore, further evaluation incorporating a larger dataset of in vivo derived embryos is warranted. Furthermore, due to the lack of variation in quality grade with more developed in vivo derived embryos as observed in this current study, it may be of value to evaluate the embryonic sex of embryo recoveries performed earlier, perhaps at d 6. These embryos would be less developed with potentially more variation in quality grade, thereby enhancing our ability to determine if quality grade is truly associated with sex determination. Clinical Theriogenology • Volume 11 Number 1 • March 201955 Conclusion There was a relationship between quality grade of in vivo-derived embryos and their genetic make-up, with a greater proportion of quality grade 2 embryos being heifers and a greater proportion of quality grade 1 embryos being bulls. However, there was no significant relationship between developmental stage at time of recovery and embryonic sex. Notwithstanding, limited observations of quality grades 2 and 3 embryos emphasized the need for further research to validate and extend the current findings. Acknowledgement This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Conflict of Interest None of the authors have any conflict of interest to declare. References 1. Kaimio I, Mikkola M, Lindeberg H, et al: Embryo production with sex-sorted semen in superovulated dairy heifers and cows. Theriogenology 2013;80:950-954. 2. Mikkola M, Andersson M, Taponen J: Transfer of cattle embryos produced with sex-sorted semen results in impaired pregnancy rate and increased male calf mortality. Theriogenology 2015;84:1118-1122. 3. Seidel GE: Economics of selecting for sex: the most important genetic trait. Theriogenology 2003;59:585-598. 4. Dahlen C, Larson J, Lamb GC: Impacts of Reproductive Technologies on Beef Production in the United States. In: GC Lamb, N DiLorenzo, editors. Current and Future Reproductive Technologies and World Food Production. Adv Exp Med Biol 752. New York, Springer; 2014. p. 97-114. 5. Morris CA, Bennett GL, Baker RL, et al: Birth weight, dystocia, and calf mortality in some New Zealand beef breeding herds. J Anim Sci 1986;62:327-343. 6. Dematawewa CMB, Berger PJ: Effect of dystocia on yield, fertility, and cow losses and an economic evaluation of dystocia scores for Holsteins. J Dairy Sci 1997;80:754-761. 7. Avery B, Madison V, Greve T: Sex and development in bovine in vitro fertilized embryos. Theriogenology 1991;35:953-963. 8. Gutierrez-Adan A, Perez-Garnelo S, Granados J, et al: Relationship between sex ratio and time of insemination according to both time of ovulation and maturational state of oocyte. Theriogenology 1999;51:397. 9. Rorie RW, Lester TD, Lindsey BR, et al: Effect of timing of artificial insemination on gender ratio in beef cattle. Theriogenology 1999;52:1035-1041. 10. Ali A: Effect of gestational age and fetal position on the possibility and accuracy of ultrasonographic fetal gender determination in dairy cattle. Reprod Domest Anim 2004;39:190-194. 11. Curran S, Ginther OJ: Ultrasonic determination of fetal gender in horses and cattle under farm conditions. Theriogenology 1991;36:809-814. 12. Wintenbergertorres S, Popescu PC: Transfer of cow blastocysts after sexing. Theriogenology 1980;14:309-318. 13. White KL, Anderson GB, Bondurant RH: Expression of a male specific factor on various stages of preimplantation bovine embryos. Biol Reprod 1987;37:867-873. 14. Williams TJ: A technique for sexing mouse embryos by a visual colorimetric assay of the x-linked enzyme, glucose-6- phosphate-dehydrogenase. Theriogenology 1986;25:733-739. 15. Thibier M, Nibart M: The sexing of bovine embryos in the field. Theriogenology 1995;43:71-80. 16. Shea BF: Determining the sex of bovine embryos using polymerase chain reaction results: A six-year retrospective study. Theriogenology 1999;51:841-854. 17. Xu KP, Yadav BR, King WA, et al: Sex related differences in developmental rates of bovine embryos produced and cultured in vitro. Mol Reprod Dev 1992;31:249-252. 18. Carvalho RV, DelCampo MR, Palasz AT, et al: Survival rates and sex ratio of bovine IVF embryos frozen at different developmental stages on day 7. Theriogenology 1996;45: 489-498. 19. Wiley C.E., Dohlman T.M., Jahnke M.M., et al: Effects of endogenous progesterone during ovarian follicle superstimulation on embryo quality and quantity in beef cows. Clinical Theriogenology 2018;10:427-434. 20. Herr C, Steel T, Lascelles A, et al: Instruction Manual: Splitting, Biopsy and Rapid Sexing of Cattle Embryos. edn., Pullmann, WA, AB Technology, Inc., 1995:58. 21. Dominko T, First NL: Relationship between the maturational state of oocytes at the time of insemination and sex ratio of subsequent early bovine embryos. Theriogenology 1997;47:1041-1050. 22. Mittwoch U: Sex differentiation in mammals and tempo of growth - probabilities vs switches. J Theor Biol 1989;137:445-455. 23. Moor RM, Osborn JC, Cran DG, et al: Selective effect of gonadotropins on cell coupling, nuclear maturation and protein synthesis in mammalian oocytes. J Embryol Exp Morphol 1981;61:347-365. Clinical Theriogenology • Volume 11 Number 1 • March 2019 56 24. Callesen H, Greve T, Hyttel P: Preovulatory endocrinology and oocyte maturation in superovulated cattle. Theriogenology 1986;25:71-86. 25. Wright RW, Ellington J: Morphological and physiological differences between in vivo and in vitro produced preimplantation embryos from livestock species. Theriogenology 1995;44:1167-1189. 26. Bernardi ML, Delouis C: Sex-related differences in the developmental rate of in-vitro matured in-vitro fertilized ovine embryos. Hum Reprod 1996;11:621-626. 27. Barfield J: Evaluation of in vitro-produced bovine embryos. Proceedings CETA/ACTE & AETA Joint Convention 2015. Table 1. Sex determination of bovine embryos, by developmental stage.1 Development stage 4 (n = 78) 5 (n = 99) 6 (n = 11) 7 (n = 15) p-value Female (%) 56.0 57.2 77.7 60.8 0.6 Male (%) 44.0 42.8 22.3 39.2 0.6 1Stage based on International Embryo Technology Society classification Table 2. Sex determination of bovine embryos, by embryo grade.1 Quality grade 1 (n = 155) 2 (n = 29) 3 (n = 19) p-value2 Females (%) 48.8a 75.2b 64.7ab 0.03 Males (%) 51.2a 24.8b 35.3ab 0.03 1 Quality grade based on International Embryo Technology Society classification (1 = Excellent or good, 2 = Fair, 3 = Poor). 2 p-values of the main effect of grade. a-c Within a row, LSMEANS without a common superscript differed (p ≤ 0.05). 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