Application of sexed semen technology to in vitro embryo production in cattle

Theriogenology 65 (2006) 219 227 www.journals.elsevierhealth.com/periodicals/the Application of sexed semen technology to in vitro embryo production in cattle Matthew B. Wheeler a,b,d, *, Jack J. Rutledge c, Amy Fischer-Brown a,c, Tara VanEtten a, Samantha Malusky a, David J. Beebe d a Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA b Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA c Department of Animal Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA d Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA Abstract Use of sexed semen in conjunction with in vitro embryo production is a potentially efficient means of obtaining offspring of predetermined sex. For thousands of years, livestock owners have desired a methodology to predetermine the sex of offspring for their herds. The ability to sort individual sperm cells into viable X- and Y-chromosome-bearing fractions made producers sex selection dreams reality in the 1990s and now semen can be sexed with greater than 90% accuracy with use of a flow cytometric cell sorter. Several concerns regarding the implementation of sexed semen technology include the apparent lower fertility of sorted sperm, the lower survival of sorted sperm after cryopreservation and the reduced number of sperm that could be separated in a specified time period. These issues are discussed in this review. There are also a number of issues that appear to influence the success rates of using sexed semen to produce bovine embryos in vitro. These issues include reductions in fertilization rates, lower cleavage rates, blastocyst rates and pregnancy rates, partial capacitation of the sperm, dilute sperm samples and sire variation. These subjects are also addressed in this paper. Finally, we will describe a recent field trial in which female Holstein embryos produced using the combined technologies of sex-selected semen and microfluidics were transferred either as single or bilateral twin embryos into beef cattle recipients, demonstrating these technol- * Corresponding author at: Department of Animal Sciences, Room 368, University of Illinois, 1207 W. Gregory Dr., Urbana, IL 61801, USA. Tel.: +1 217 333 2239; fax: +1 217 333 8286. E-mail address: mbwheele@uiuc.edu (M.B. Wheeler). 0093-691X/$ see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.09.032

220 M.B. Wheeler et al. / Theriogenology 65 (2006) 219 227 ogies contributions to viable embryo production. The results indicate that large-scale transfer of in vitro produced, Holstein heifer embryos to beef recipients is a feasible production scheme. # 2005 Elsevier Inc. All rights reserved. Keywords: Bovine; In vitro production; Embryo; Sexed semen 1. Introduction For thousands of years, livestock owners have desired a methodology to predetermine the sex of offspring for their herds. For dairy cattle, this means heifer calves. According to the Livestock Reporter Network, a 2005 market report from Lancaster County, PA listed 90 120 lb. Number 1 Holstein heifer calves selling for $ 510 590, whereas 90 120 lb. Number 1 Holstein bull calves sold for $ 170 208. Considering the need for replacement heifers in their own herds, as well as the nearly three-fold increase in monetary value for heifer claves over bull calves, it is no wonder that dairy producers are interested in options that allow them to predetermine the sex of their calves. The ability to sort individual sperm cells into viable X- and Y-chromosome-bearing fractions made producers sex selection dream a reality in the 1990s [1 8]. Semen can be sexed with greater than 90% accuracy with use of a flow cytometric cell sorter [9]. There are, however, slight differences in the sexing accuracy between X-sorted sperm (87.8%) and Y-sorted sperm (92.1%) in calves born [9]. The economics [10,11], applications [12,13] and issues surrounding commercialization [14] of the sexed semen technology have also been recently reported. There have been a number of recent reviews on the topic of the use of sexed semen in cattle production [11,13,15 21] as well as studies that have evaluated the use of sex selected semen for AI [7,22 25] and in vitro production (IVP) of embryos [5,6,26 31]. This paper will focus on reviewing the use of sexed semen for IVP of embryos. A major concern with the implementation of sexed semen technology is that the fertility of sorted sperm is somewhat lower than that of control, unsorted sperm [23]. However, most of the early studies concerning the fertility of sexed semen were confounded by the insemination of animals with fewer sperm per dose than with normal unsorted control sperm [19]. When numbers of sorted sperm per dose were equivalent to those used with conventional AI, pregnancy rates for the sexed sperm have been on the order of 60 80% of those found with unsorted control sperm [23,32]. Another concern is that the survival of sorted sperm after cryopreservation is also decreased [33]. Schenk et al. [33] concluded after a series of experiments to optimize the cryopreservation of sexed sperm that the present methods used for flow cytometric sexing of sperm resulted in somewhat lower post-thaw motility and acrosomal integrity when compared to unsorted control sperm. They went on to report that this damage is negligible compared to the damage caused by routine cryopreservation and that the fertilizing capability of sorted sperm is adequate based on several straightforward laboratory analyses [33]. One of the early major drawbacks to implementing the extensive use of sexed semen technology was the number of sperm that could be separated in a specified time period.

M.B. Wheeler et al. / Theriogenology 65 (2006) 219 227 221 This situation seems to have been adequately addressed by the new generation of flow cytometers [34,16]. Modification of the orienting nozzle of the flow cytometer has allowed for the increase in sorting rate from about 0.35 million sperm/h to between 5 and 6 million sperm/h of each population [16]. This speed may be increased even more with future equipment developments. A number of companies worldwide now have licenses to produce and sell sexed semen. The primary market appears to be to individual bull owners and for AI studs. 2. IVP with sex selected semen One very appealing attribute of using flow-sorted sperm for IVP is that considerably fewer sperm are needed for IVF. Over the past dozen years, sexed semen has been used in several studies to produce embryos in vitro [5,6,26 31]. However, a number of issues appear to influence the success rates when sexed semen is used to produce bovine embryos in vitro. These issues include lower fertilization rates [6], lower cleavage rates [26], lower blastocyst rates [26,35], lower pregnancy rates [6], partial capacitation of the sperm [28], dilute sperm samples [28], and sire variation [27]. In a large in vitro study [26], the overall percentages of oocytes fertilized with sorted and unsorted frozen bovine sperm appear to be similar using current methods [26] although the cleavage rates for sorted-fresh sperm were lower (66% versus 76%) than for unsorted-fresh control sperm (P < 0.01) [26]. In this study, however, there were no differences in cleavage rates between sorted-frozen and unsorted-frozen sperm [26]. Further, the events that occur in the first cell cycle seem to have similar timing with both sorted and unsorted sperm [26]. One difference seen in the study was that, while the sorting process had a negligible influence on cleavage rates, there was a significant effect on the numbers of oocytes developing to blastocysts with both fresh and frozen sorted sperm. Further, an unexplained delay of about half to a full day in blastocyst development was observed [26]. These authors reported that the blastocyst rate for sorted-sperm was 70% that of the blastocyst rate produced by unsorted sperm [26]. These results were similar to what had been previously reported [35]. Recently, the percentage of embryos developing to blastocysts from oocytes derived from ovum pickup was also observed to be lower with sorted sperm when compared to unsorted sperm [31]. We have observed similar results in our group, where the blastocyst rate for all oocytes is approximately 30 40% with non-sorted semen and between 10% and 20% with sorted semen [36]. We have also observed a slight decrease in cleavage rates, but this was not significant which is similar to results of others [26]. In a subsequent study [27], somewhat different results were observed. This study examined bovine oocytes after fertilization with frozen thawed sperm from three bulls exposed to three sperm treatments: (1) sperm stained with Hoechst 33342 and sorted with flow cytometry; (2) sperm stained but not sorted; (3) sperm not stained or sorted (control). There was no difference in blastocyst percentages between the three sperm treatments but, in contrast to other results [35,26,30,31], there was a significant decrease in cleavage rates with stained-sorted (53.1%) and stained-unsorted sperm (59.9%) when compared to unstained-unsorted control sperm (69.9%). No differences were observed between X- and Y-chromosome-bearing sperm. In addition, the results indicated that there were significant

222 M.B. Wheeler et al. / Theriogenology 65 (2006) 219 227 differences in cleavage rate and blastocyst formation rate due to the use of different sires [27]. The authors speculated that, if additional oocytes had been included, results on blastocyst rates would have followed results for cleavage rates and significantly fewer blastocysts would have been produced [27]. Anotherconsiderationwhenspermaresortedbyflowcytometryisthatspermare subjected to an environment during the preparation for sorting and during the actual sorting that may affect capacitation differently than that experienced by sperm that have not been sorted [33].Spermarealsoinanextremely dilute solution after sorting and this condition, too, may influence capacitation. Because of these concerns, Lu and Seidel [28] hypothesized that sperm might become partially capacitated while waiting to be sorted or during the sorting process. To test this hypothesis, these investigators designed a series of studies to determine the optimal concentrations of heparin and sperm on cleavage rate and blastocyst development after fertilization of bovine oocytes with flow sorted sperm. There was an overall increase in sperm penetration when heparin concentration and sperm concentrations increased from 0 to 2 mg/ml and from 500,000 to 1.5 million sperm/ml, respectively [28]. However, no differences in sperm penetration were observed between 2 and 10 mg/ml of heparin or between sperm concentrations of 1.5 4.5 million sperm/ml. The same relationships were also observed regarding cleavage rates except the heparin effect was not different. Finally there was no significant effect of sperm or heparin concentration on blastocyst production nor was there a sperm concentration by heparin concentration interaction effect for any of the responses measured [28]. The controls (10 mg/ml heparin and 1.5 million sperm/ml) were similar to the sexed sperm treatments except that the percentage of polyspermy and percentage of blastocysts/oocyte were significantly higher (P < 0.05) in the controls than in any of the sexed sperm treatments. The study by Lu and Seidel [28] also examined sire effects and sire by treatment interactions: there were significant interactions for blastocyst percentage (heparin concentration x sire (P < 0.05), sperm concentration sire (P < 0.001)); percent sperm penetration (sperm concentration sire); percent two pronuclei (heparin sire (P < 0.01), sperm concentration sire (P < 0.03)). As shown previously [27,30], there was no significant decrease in blastocyst development between sexed and unsexed sperm. However, such and effect was observed when optimal concentrations of heparin and sperm were employed on a sire sire basis. Heparin and sperm concentrations are aspects that we likely will need to pay more attention to than we have in the past when selecting a sire for IVF if we are going to use sexed semen technology effectively and efficiently. 3. Large-scale field trial Recently, we evaluated a production scheme involving single and bilateral twin transfer of Holstein female embryos to beef cattle recipients [29]. The control IVF system has been consistent for our lab, with high cell number, inner cell mass allocations similar to those observed in vivo, appropriate elongation stage morphology, and most importantly, acceptable pregnancy rates and low birth weights of resulting calves. Modifications were made to the control culture system to test hypotheses relevant to specific projects. One

M.B. Wheeler et al. / Theriogenology 65 (2006) 219 227 223 Table 1 Single embryo transfer calving results [29] Treatment # Embryos transferred # Calves (%) Microfluidic 88 29 (33.0) White yolk 102 24 (23.5) Vortexed (control) 95 30 (31.6) Total 285 83 (29.1) modification was the supplementation of the culture medium (KSOMaaBSA; [37]) with 6% avian white yolk (WY). Another modification was the use of a microfluidic cumulus cell removal device. Cumulus removal with a microfluidic device has been shown to reduce the shear forces exerted on the embryo and improve rates of blastocyst development in vitro as compared with cumulus removal by vortex [38]. Further, the onset of transcription activity occurs earlier in vortexed embryos compared to microfluidically stripped embryos, suggesting a stress response to vortexing [38]. We were interested in examining the influence of white yolk supplementation to the culture medium and microfluidic cumulus removal on pregnancy and calving rates of in vitro produced bovine embryos fertilized with flow cytometrically sex-selected sperm. Holstein oocytes obtained from abattoir-derived ovaries were fertilized with the X- bearing fraction of gender-sorted Holstein semen. Cumulus cells were removed with aid of a vortex or microfluidic device (mfd). Half of the vortexed embryos were cultured in KSOMaaBSA (control) as were all mfd embryos. Remaining vortexed embryos were cultured in control medium with 6% avian white yolk (WY). Embryos were produced across five replicates. Control embryos were transferred as ipsilateral singles and bilateral twins; embryos for which cumulus cells were removed with a microchannel were transferred as ipsilateral singles. Pregnancy was diagnosed with ultrasound between 41 and 46 days of gestation and confirmed between 60 and 90 days of gestation. Effects of embryo production and recipient factors on fetal survival at both ultrasound events were analyzed using logistic regression. Survival was affected by embryo production replicate, but neither cumulus removal method nor culture medium was a significant factor. This is most likely due to inadequate numbers of animals. A Chi-square analysis was applied to survival rates at both ultrasound events and at calving (Tables 1 and 2). Approximately 95% of the calves were female. Transferring two embryos resulted in a higher calf yield per recipient than transferring single embryos (Fig. 1; P < 0.01), although individual embryo survival tended to be lower for those transferred as twins (Fig. 2; P < 0.1). The proportion of pregnant recipients was higher for Table 2 Twin embryo transfer calving results [29] Treatment # Embryos transferred # Calves (%) White yolk 180 44 (24.4) KSOMaaBSA 172 35 (20.3) Total 352 79 (22.4)

224 M.B. Wheeler et al. / Theriogenology 65 (2006) 219 227 Fig. 1. Offspring production per recipient [29]. twin versus single transfers at both ultrasound events (P < 0.05), but this difference was no longer significant at term (Fig. 3). It is noteworthy that five cases of hydroallantois were observed during the fifth month of gestation for one control twin, one WY single, and three WY twin transfers, originating from three embryo production replicates. Hasler et al. [20] reported approximately 1 case per 200 pregnancies following transfer of IVP embryos; the incidence in the current study exceeded 1 case per 100 pregnancies. Whether the increased incidence of hydroallantois is associated with the use of sex-selected semen is unknown, but is a question worthy of future research. These results demonstrate that cumulus cell removal with a microfluidic device and in vitro fertilization with sex-selected semen are viable components of a successful IVP system and that large-scale transfer of IVP Holstein heifer embryos to beef recipients is a feasible production scheme. Although twin transfers yielded more calves per recipient than single transfers, large gestational losses occurred. Efforts must be directed toward Fig. 2. Individual embryo survival [29].

M.B. Wheeler et al. / Theriogenology 65 (2006) 219 227 225 Fig. 3. Proportion of pregnant recipients irrespective of number of offspring [29]. understanding and eliminating these losses as well as in reducing the strikingly high incidence of hydrollantois. Acknowledgements The authors would like to thank all their colleagues at the University of Illinois (G. Barquero, S. Clark, C. Ferguson, D. Faulkner, F. Ireland, D. Kesler, S. Lane, and P. Lopes) and the University of Wisconsin (K. Haubert, N. Jensen, B. Lindsey, R. Monson, D. Northey, A. Reeder, K. Weigel, and H. Zeringue) who contributed to some part of the work described in this review. A portion of the work presented here was supported by USDA Multi-State Research Project W-1171 (to both Illinois and Wisconsin). References [1] Johnson LA. Sex preselection in swine: altered sex ratios in offspring following surgical insemination of flow sorted X- and Y-bearing sperm. Reprod Domest Anim 1991;26:309 14. [2] Johnson LA. Gender preselection in domestic animals using flow cytometrically sorted sperm. J Anim Sci 1992;70(Suppl 2):8 18. [3] Johnson LA. Isolation of X- and Y-bearing sperm for sex preselection. In: Charlton HH, editor. Oxford reviews of reproductive biology. Oxford: Oxford University Press; 1994. p. 303 26. [4] Johnson LA. Sex preselection by flow cytometric separation of X- and Y-chromosome-bearing sperm based on DNA difference: a review. Reprod Fertil Dev 1995;7:893 903. [5] Cran DG, Johnson LA, Miller NG, Cochrane D, Polge C. Production of bovine calves following separation of X- and Y-chromosome-bearing sperm and in vitro fertilization. Vet Rec 1993;132:40 1. [6] Cran DG, Johnson LA, Polge C. Sex preselection in cattle: a field trial. Vet Rec 1995;136:495 6. [7] Cran DG, McKelway WAC. King ME, Dolman DP, McEvoy TG, Broadbent PJ, et al. Production of lambs by low dose intrauterine insemination of flow cytometrically sorted and unsorted semen. Theriogenology 1997;47:267 [Abstract].

226 M.B. Wheeler et al. / Theriogenology 65 (2006) 219 227 [8] Abeydeera LR, Johnson LA, Welch GR, Wang WH, Boquest AC, Cantley TC, et al. Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry. Theriogenology 1998;50:981 8. [9] Tubman LM, Brink Z, Suh TK, Seidel Jr GE. Characteristics of calves produced with sperm sexed by flow cytometry/cell sorting. J Anim Sci 2004;82:1029 36. [10] Seidel Jr GE. Economics of selecting for sex: the most important genetic trait. Theriogenology 2003;59:585 98. [11] Seidel Jr GE. Sexing mammalian sperm intertwining of commerce, technology, and biology. Anim Reprod Sci 2003;79:145 56. [12] Hohenboken WD. Applications of sexed semen in cattle production. Theriogenology 1999;52:1421 33. [13] Weigel KA. Exploring the role of sexed semen in dairy production systems. J Dairy Sci 2004;87(Suppl E):E120 30. [14] Amann RP. Issues affecting commercialization of sexed sperm. Theriogenology 1999;52:1441 57. [15] Foote RH. Review: dairy cattle reproductive physiology research and management past progress and future prospects. J Dairy Sci 1996;79:980 90. [16] Johnson LA, Welch GR, Rens W. The Beltsville sperm sexing technology: high-speed sperm sorting gives improved sperm output for in vitro fertilization and AI. J Anim Sci 1999;77(Suppl 2):213 20. [17] Johnson LA. Sexing mammalian sperm for production of offspring: the state-of-the-art. Anim Reprod Sci 2000;60/61:93 107. [18] Garner DL. Sex-sorting mammalian sperm: concept to application in animals. J Androl 2001;22:519 26. [19] Seidel Jr GE, Garner DL. Current status of sexing mammalian spermatozoa. Reproduction 2002;124:733 43. [20] Hasler JF, Henderson WB, Hurtgen PJ, Jin ZQ, McCauley AD, Mower SA, et al. Production, freezing and transfer of bovine IVF embryos and subsequent calving results. Theriogenology 1995;43:141 52. [21] Hunter RHF. Advances in deep uterine insemination: a fruitful way forward to exploit new sperm technologies in cattle. Anim Reprod Sci 2003;79:157 70. [22] Morrell JM. Applications of flow cytometry to artificial insemination: a review. Vet Rec 1991;129:375 8. [23] Seidel Jr GE, Schenk JL, Herickhoff LA, Doyle SP, Brink Z, et al. Insemination of heifers with sexed sperm. Theriogenology 1999;52:1407 20. [24] Bodmer M, Janett F, Hassig M, Daas ND, Reichert P, Thun R. Fertility in heifers and cows after low dose insemination with sex-sorted and non-sorted sperm under field conditions. Theriogenology 2005;64:1647 55. [25] Schenk JL, Suh TK, Seidel Jr GE. Embryo production from superovulated cattle following insemination of sexed sperm. Theriogenology, doi:10.1016/j.theriogenology.2005.04.026. [26] Lu KH, Cran DG, Seidel Jr GE. In vitro fertilization with flow-cytometrically sorted bovine sperm. Theriogenology 1999;52:1393 405. [27] Zhang M, Lu KH, Seidel GE. Development of bovine embryos after in vitro fertilization of oocytes with flow cytometrically sorted, stained and unsorted sperm from different bulls. Theriogenology 2003;60:1657 63. [28] Lu KH, Seidel Jr GE. Effects of heparin and sperm concentration on cleavage rates of bovine oocytes inseminate with flow-cytometrically-sorted bovine sperm. Theriogenology 2004;62:819 30. [29] Fischer-Brown A, Barquero G, Clark S, Ferguson C, Faulkner D, Ireland F, et al. Twin vs. single transfer of IVP Holstein embryos to beef recipients. Reprod Fertil Dev 2005;17:230 [Abstract]. [30] Wilson RD, Fricke PM, Leibfried-Rutledge ML, Rutledge JJ, Penfield CM, Weigel KA. In vitro production of bovine embryos using sex-sorted sperm. Theriogenology, doi:10.1016/j.theriogenology.2005.07.007. [31] Wilson RD, Weigel KA, Fricke PM, Rutledge JJ, Leibfried-Rutledge ML, Matthews DL, et al. In vitro production of Holstein embryos using sex-sorted sperm and oocytes from selected cull cows. J Dairy Sci 2005;88:776 82. [32] Doyle SP, Seidel Jr GE, Schenk JL, Herickhoff LA, Cran D, Green RD. Artificial insemination of lactating Angus cows with sexed semen. In: Proceedings Western Section of the American Society of Animal Science, vol. 50; 1999. p. 203 5. [33] Schenk JL, Suh TK, Cran DG, Seidel Jr GE. Cryopreservation of flow-sorted bovine spermatozoa. Theriogenology 1999;52:1375 91. [34] Johnson LA, Welch GR. Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency. Theriogenology 1999;52:1323 41.

M.B. Wheeler et al. / Theriogenology 65 (2006) 219 227 227 [35] Merton JS, Haring RM, Stap J, Hoebe RA, Aten JA. Effect of flow cytometrically sorted frozen thawed semen on success rates of in vitro bovine embryo production. Theriogenology 1997;47:295 [Abstract]. [36] Monson RL, Fischer-Brown A. Personal communication; 2005 [37] Fischer-Brown AE, Lindsey BR, Ireland FA, Northey DL, Monson RL, Clark SG, et al. Embryonic disc development and subsequent viability of cattle embryos following culture in two media under two oxygen concentrations. Reprod Fertil Dev 2005;16:787 93. [38] Zeringue HC, Rutledge JJ, Beebe DJ. Early mammalian development depends on cumulus removal technique. Lab Chip 2004;5:86 90.