Improved in vitro development of OPU-derived bovine (Bos taurus) embryos by group culture with agarose-embedded helper embryos

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1 Available online at Theriogenology 74 (2010) Improved in vitro development of OPU-derived bovine (Bos taurus) embryos by group culture with agarose-embedded helper embryos E.M. Senatore a,j.xu b, M.V. Suárez Novoa c, G. Gong d,t.lin e, A. Bella f, J.F. Moreno g, M.E. Mannino a, X. Tian d, G.A. Presicce f,h, S.-C. Wu i, *, F. Du b, * a ARSIAL Centro Regionale per la Zootecnia, Rome, Italy b Renova Life Inc., College Park, USA c Universidad Lisandro Alvarado (UCLA), Barquisimeto, Venezuela d Department of Animal Science, University of Connecticut, Storrs, USA e Department of Animal Science, National Chung Hsing University, Taichung, Taiwan, ROC f Istituto Superiore di Sanità, Rome, Italy g Sexing Technologies, Navasota, USA h Consorzio di Ricerca e Sperimentazione per gli Allevatori (CRSA), Rome, Italy i Institute and Department of Animal Science and Technology, National Taiwan University, Taipei, Taiwan, ROC Received 11 November 2009; received in revised form 29 June 2010; accepted 29 June 2010 Abstract The average number of oocytes collected by ovum pick up (OPU) from Bos taurus cattle is 8 per live donor. The objective was to determine whether development of small numbers of cattle embryos (produced by OPU and IVF), was enhanced by including helper embryos, produced from abbatoir-derived oocytes and embedded in agarose. Oocytes were from abbatoir-derived ovaries (Experiments 1 and 2) or OPU of elite donors (Experiment 3). In Experiment 1, cleaved embryos (2 8 cells), were randomly allocated. Controls were groups of 1, 3, 5, 10, and 20 cleaved embryos cultured in 50 L serum-free SOF, whereas treatments were groups of 1, 3, and 5 embryos freely cultured along with helpers in groups of either 9, 7 or 5 embedded in agarose per droplet. Therefore, there were 10 cleaved embryos per droplet in combinations of 1 9, 3 7or5 5 (free helper), respectively. There was an increase in the progression to blastocyst for 1 5 embryos per droplet, compared to 10 and 20 ( % vs %, P 0.05). For the tested free embryos, those cultured with helpers had increased blastocyst development over their control counterparts ( % vs %, P 0.05). When the number of embryos per droplet was 10 or 20, blastocyst percentage was similar ( %, P 0.05). In Experiment 2, addition of an agarose chip to the culture medium did not significantly affect development to the blastocyst stage. In Experiment 3, after fertilizing OPU oocytes with sorted X-sperm, a group of three cleaved embryos were cultured in a droplet with either seven helpers (3 7) or alone (3 0). Blastocyst development of OPU oocytes in the 3 7 group was 37.1%, higher than that in the 3 0 group (11.8%, P 0.05). In conclusion, limited numbers of OPU/IVF oocytes had competent development when cultured with helpers (embedded in agarose to provide physical separation) Elsevier Inc. All rights reserved. Keywords: IVF; Ovum pick up; Embryo development; Agarose; Cattle 1. Introduction * Corresponding authors. addresses: scw01@ntu.edu.tw (S.-C. Wu); fuliangd@ renovalife.com (F. Du). Ultrasound-guided ovum pickup (OPU), in combination with IVF using conventional and/or sex sorted sperm, has enabled production of large numbers of X/$ see front matter 2010 Elsevier Inc. All rights reserved. doi: /j.theriogenology

2 1644 E.M. Senatore et al. / Theriogenology 74 (2010) embryos from live donor cattle of high genetic merit [1 5]. In that regard, IVP of embryos has greatly benefited from improvements in OPU technology [6]. The OPU procedure is semi-invasive, highly repeatable, and can be applied to animals in various reproductive states [7 10]. Moreover, accurate sperm sexing ( 90%) with cell sorters has facilitated production of replacement heifers, herd expansion, and genetic improvement [3,11,12]. The efficiency of retrieving cattle oocytes or cumulus-oocyte-complexes (COCs) via OPU is subject to many variables, including the genetic, physiological, and reproductive characteristics of the donors used [2,13]. Moreover, COCs retrieved from Bos taurus cattle have variable morphological and physiological quality, as well as competence for further development in vitro. In a recent study, the average number of COCs collected per OPU session was slightly 8 for Holstein cattle over a large number of donors [5]. Another study, however, reported a much lower average among donors when using Holstein and Danish Red and White breeds [14]. In order to preserve and maintain pedigree information for COCs from each donor, the development of oocytes and resulting embryos from each oocyte donor must be cultured separately. In circumstances where limited COCs are recovered from a specific donor, the number of presumptive zygotes, or cleaved embryos to be cultured together in a medium droplet may be well be suboptimal. Consequently, subsequent development is compromised, as cattle embryos develop better under group culture. In fact, several studies demonstrated a supportive and promoting effect on post-compacting embryonic development when more embryos were cultured within the same droplet or culture well [15,16]. Furthermore, the size of the culture droplet also affected embryo yield [17,18]. These effects are presumably due to embryotrophic factors, but neither the nature of these promoting factors nor the reason for their physiological effects has been elucidated. The general objective of the present study was to determine whether developmental competence (ability to reach the blastocyst stage after 7 d of in vitro culture) of a small number of OPU-derived cleaved embryos could have improved by the addition of helper embryos (helpers), produced from abbatoir-derived ovaries (the helpers were at a similar stage of development, but physically separated). Helpers were embedded into low-melting point agarose chips, and then cultured with embryos derived from IVF of oocytes recovered from an abbatoir (Experiments 1 and 2), or OPU/IVF-derived (Experiment 3) free floating embryos in the same culture droplet. In addition, in Experiment 3 the efficiency of the helper approach was tested by employing sorted X-sperm for fertilization of OPU-derived oocytes. Therefore, the objective was to determine the effect of helper embryos on preimplantational development of OPU-derived embryos produced by IVF with either conventional or sex-sorted sperm. 2. Materials and methods All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise noted. The COCs were matured at 39 C in 5% CO2 and humidified air, and fertilized oocytes were cultured in vitro at 39 C in 5% CO 2,5%O 2, and 90% N 2 in humidified air Oocytes collected from abbatoir-derived ovaries Bovine COCs for in vitro embryo production were recovered from abbatoir-derived ovaries (from Holstein cattle), brought to the laboratory within 2 3 h after recovery, and processed as previously described [3]. No tests were conducted to detect the presence of infectious agents in these ovaries. Recovery of COCs from antral follicles 2 8 mm in diameter was accomplished by vacuum pump aspiration (flow rate, ml/min). Oocytes were graded according to the following criteria: Grade A, 4 layers of cumulus cells; Grade B, 3 or 4 layers of cumulus cells; Grade C, 1 or 2 layers of cumulus cells; Grade D, denuded oocytes; and Grade E, oocytes with an expanded cumulus. To be consistent with usable COCs in Experiment 2 from OPU sessions, only COCs that were Grades A to C were selected for further processing. Selected oocytes were used in IVM, IVF, and group culture for Experiments 1 and 2, as described below Animals and oocytes collected by OPU Donors ranging from heifers to 6 8 y old pluriparous Holstein cows were used for this study, which was conducted in August at the University of Connecticut. They were stall fed and kept in a barn under controlled conditions. Twenty cattle were used for oocyte retrieval (four replicates). All procedures and protocols using live animals were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Connecticut. A portable Aloka 500 ultrasound unit equipped with a 5-MHz sector scanner vaginal probe (ALoka Co. Ltd, Tokyo, Japan), equipped with a 17-ga, 60-cm single lumen needle fitting a metallic needle guide were used

3 E.M. Senatore et al. / Theriogenology 74 (2010) for transrectal oocyte retrieval. Cattle were restrained in a squeeze chute and prepared for follicular aspiration as described [19]. The aspiration medium was phosphatebuffered saline (PBS) with 10 IU/mL heparin and 0.1% polyvinyl alcohol. The OPU was scheduled twice weekly (total of four replicates). Only COCs that were Grades A to C were used Maturation, fertilization and culture in vitro Embryos were produced as previously described [3]. Briefly, selected COCs were matured for 22 h in 75 L droplets of Medium 199 (Invitrogen, Carlsbad, CA, USA) containing Earle s salts, L-glutamine, 2.2 g/l sodium bicarbonate and 25 mm Hepes, supplemented with 10% (vol/vol) fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 0.5 g/ml ovine FSH (National Institute of Diabetes and Digestive and Kidney Disease, NIDDK, Los Angeles, CA, USA), 5.0 g/ml ovine LH (NIDDK), and 1.0 g/ml estradiol 17-. Droplets were covered with mineral oil and contained oocytes. Fertilization was accomplished by use of frozen/ thawed semen from bulls of known fertility and previously tested for IVF efficiency in our laboratory. In Experiments 1 and 2, sperm was subjected to a swim-up procedure for 1 h. Following centrifugation, the sperm pellet was resuspended to achieve a concentration of /ml. The final concentration of sperm was /ml in 50 L droplets of TALP fertilization medium supplemented with 10 g/ml heparin after adding both sperm and COCs. For culture, IVF droplets were covered with mineral oil, and sperm and COCs were co-incubated for h. Presumptive zygotes were stripped of enclosing cumulus cells by vortexing in a 0.1% hyaluronidase solution, and then moved into 50 L droplets of culture medium consisting of synthetic oviduct fluid (SOF) medium with 6 mg/ml BSA, essential and non essential amino acids, but no serum, under mineral oil (serum-free culture). Cultures were placed in a modulation chamber (Forma Scientific, Waltham, MA, USA), under a mixed gas atmosphere of CO 2 (5%), O 2 (5%) and balanced with N 2 (90%) for an additional h (total of h post IVF). In Experiment 3, for IVF of OPU oocytes with X-sorted sperm, Brackett and Oliphant (BO) medium [20] was used. Briefly, straws containing sexed semen at the concentration of /ml ( /ml per 0.25 ml straw) were thawed for 10 s in a 37 C water bath after 10 s of gentle shaking in air at room temperature. Sperm were washed in 8 ml of BO medium (supplemented with 3 mg/ml of BSA and 10 mm caffeine) and centrifuged at 1,500 g for 8 min. The sperm pellet was re-suspended and centrifuged once again, and re-suspended in BO washing medium (concentration, /ml). Matured COCs were rinsed in BO medium containing 6 mg/ml BSA and 10 g/ml heparin. Fertilization droplets (50 L) containing matured COCs were prepared in small (6 cm in diameter) Petri dishes. Processed semen was added (50 L) for a final droplet volume of 100 L under medical oil for a final sperm concentration of /ml, as described [3]. After sperm and COCs were co-incubated for 6 h, presumptive zygotes were moved into 50 L culture droplets in the serum-free medium described above, and cultured for 40 h (total 46 h post IVF) prior to adding helper embryos (embedded in agarose chips) to the culture Culture of cleaved embryos with agarose-embedded helper embryos A 1% solution of agarose, with low gelling and melting points (A-9414) was prepared in saline and sterilized by autoclaving. Solidified agarose was stored at 2 8 C prior to use. To embed embryos in agarose chips, sterile agarose was melted by warming it (65 C water bath) and maintaining it on a 39 C warming plate until inserting embryos. Cleaved embryos (2 8 cells) were selected and transferred into a Petri dish containing the melted agarose at a temperature between 35 and 39 C. Five, seven, or nine cleaved embryos were aspirated into a hand-made capillary along with agarose to form a sausage-like gel with a diameter of m (chip; Fig. 1), and then released into culture medium at C. When solidified, the agarose/embryo chips were transferred into 50 L droplets of SOF medium containing free embryos, and culture continued for an additional 5 d. The embedded embryos could easily be observed within the agarose chip; although diffusion could occur with the shared medium, they remained physically separated from the free embryos (Fig. 1) Experimental design Experiment 1. Minimum number of free embryos (IVF with conventional semen) to culture with agarose-embedded helpers The objective was to determine the minimum number of cleaved embryos that could be cultured in a single droplet without compromising their development to blastocysts. Abbatoir-derived oocytes underwent IVF with conventional semen. After h, cleaved embryos (1, 3, 5, 10, or 20 per group), were cultured in

4 1646 E.M. Senatore et al. / Theriogenology 74 (2010) Fig. 1. In vitro development of fertilized bovine embryos cultured in groups with agarose embedded embryos. (A), Embryos (2 8 celled stage) produced from abbatoir-derived oocytes were embedded in 1% agarose chips h after IVF, indicated by black arrow. Embryos at 2 8 celled stage as either: 1 (A), abbatoir-derived, indicated by white arrow) or 3 (B), OPU-derived, indicated by white arrow) were cultured, freely and separately, in a 50 L culture droplet, together with either 9 or 7 embryos embedded in an agarose chip (total of 10 embryos per droplet). After an additional 5 d of culture at 39 C in 5% CO 2,5%O 2 and 90% N 2 humidified air, the embryos (OPU-derived) developed into expanded blastocysts (C), white arrow), at a rate similar to that of the controls (D), in which a group of ten embryos was freely cultured in the same size droplet (50 L). Bar 140 m. a50 L medium droplet for an additional 5 d under the environmental conditions described above. The optimal number to maximize blastocyst yield was determined to be 10. Therefore, cleaved embryos (2 8 celled) in groups of either 1, 3, or 5 (to approximate the numbers likely available from OPU/IVF), were cultured together with either 9, 7, or 5 helpers (total of 10 embryos/droplet). The helpers were embedded in agarose chips (Fig. 1) Experiment 2. Effect of agarose on development of embryos in vitro The objective was to determine whether the addition of agarose affected blastocyst development. The design wasa2 2 factorial, with the number of embryos per group (3 vs. 10), and effect of agarose (with versus without). Cleaved embryos were derived from IVF with conventional semen and matured oocytes were retrieved from abbatoir-derived ovaries. The culture conditions were as described in Experiment Experiment 3. Developmental potential of OPU/IVF-derived embryos (X-sorted sperm) when group cultured with agarose embedded helper embryos Oocytes were collected by OPU, and fertilized in vitro, by standard procedures described above, using X-sorted sperm. Oocytes collected from abbatoir-derived ovaries, designated to produce helpers, underwent the same IVM/IVF on the same time schedule as that used for OPU/sexed-IVF embryos. Cleaved (2 8 cells) embryos (n 7) were embedded in each agarose chip. To test whether group culture could promote blastocyst development of OPU/sexed-IVF embryos, 46 h post IVF, groups of three cleaved embryos were cultured either with or without an agarose chip containing seven helpers (3 7 vs. 3 0) in 50 L culture medium droplets for an additional 5 d. Therefore, the total number of embryos per culture droplet was either 10 (3 OPU 7 helpers) or three (3 OPU 0 helpers) Statistical analyses The blastocyst development rate was determined for free embryos, and compared with the embedded helpers. Rates of blastocyst development of free floating 2 8 celled embryos in Experiment 1, and cleavage and subsequent development of OPU/IVF embryos in Experiment 2, were determined. For comparisons to Experiment 1, the rate of blastocyst development of OPU/ IVF embryos was calculated, along with the number of cleaved embryos (2 8 cells). Numbers of blastocysts were compared using the Student s t-test for unpaired

5 E.M. Senatore et al. / Theriogenology 74 (2010) Table 1 Development of small numbers of IVP bovine embryos cultured in groups with agarose-embedded helper embryos (Experiment 1). Treatment Control Agarose-embedded culture* No. CE per droplet No. replicates Total No. CE % of blastocyst (Mean SEM) a a a b b b b b CE cleaved embryos (2 8 cells). * Cleaved embryos in groups of either 1, 3, or 5 were cultured with either 9, 7, or 5 helper embryos embedded in agarose chips, respectively, in droplets containing 50 L culture medium. The total number of embryos was 10 per droplet. a,b Within a row, means without a common superscript differ (P 0.05). data, or by the Mann-Whitney test. The difference between proportions were evaluated by the chi-square test or, when appropriate, a Fisher s exact test. All statistical analyses were performed using STATA software version 9.2 (Stata Corporation, College Station, TX, USA). For all analyses, P 0.05 was considered significant. 3. Results 3.1. Experiment 1 The total number of oocytes used for IVM and IVF in Experiment 1 was 3,460; h after IVF, 72.1% oocytes were at the 2 8 cell stage. Cleaved embryos (n 2,388) were randomly allocated into a control or a treatment group. Controls consisted of groups of 1 (n 62), 3 (n 81), 5 (n 135), 10 (n 390), or 20 (n 480) freely floating cleaved embryos in 50 L droplets of culture medium (Table 1). In this experiment, morula development was not examined (due to the large numbers of embryos). Blastocyst development rates were 6.6, 11.1, and 24.4% in groups of 1, 3, or 5 embryos per droplet, respectively. However, even the best development achieved (with 5 embryos per droplet) was still significantly lower than that of the group with 10 embryos per droplet (24.4 vs. 39.2%). However, increasing the number of embryos to 20 per droplet resulted in a blastocyst rate of 43.3%, not significantly greater than that achieved with 10 embryos per droplet. Treated groups consisted of 1 (n 68), 3 (n 87), or5(n 135; Fig. 1) freely floating cleaved embryos in 50 L droplets of culture medium, with the addition of9(n 612), 7 (n 203), or 5 (n 135) helpers embedded in agarose (Table 1). In these groups of 1 9, 3 7 and 5 5, the blastocyst development of free floating embryos was 49.5, 41.2, and 39.2%, respectively, not significantly different from that of the controls when 10 (Fig. 1D) or 20 embryos were cultured per droplet (Table 1). In these groups of 10, the blastocyst development rate of agarose-embedded embryos was 38.5, 42.3, and 43.5%, respectively (no significant difference among groups; Fig. 1C). Blastocyst development rate did not differ significantly between freefloating and embedded embryos Experiment 2 A total of 711 oocytes were fertilized with conventional semen; the cleavage rate h after IVF was %. Cleaved embryos were used to test the effect of agarose. In the group of 3 per droplet, development to morula and blastocyst stages was % and %, respectively, significantly lower than with 10 per droplet (morula %, blastocyst %; Table 2). The addition of an agarose chip had no significant effect on embryo development to the blastocyst stage Experiment 3 A total of 561 COCs (Grades A to C) were retrieved by OPU (20 donors and four replicates). The mean number of oocytes collected from each donor was (range 3 20). After IVF and culture for 40 h, and prior to group culture, cleavage rates of OPU (69.2%) and OPU-helpers (72.3%) were similar (Table 3). Cleaved embryos were randomly assigned to control (3 OPU 0 helpers) and treatment (3 OPU 7 helpers) groups. Following culture with agarose-embedded helpers, the overall development rate to the blastocyst stage was significantly higher in the OPU-helper group (3 7, 37.1%; Fig 1B, 1C) compared with OPU group (3 0, 11.8%; Table 3). When the blastocyst development efficiency of the cleaved embryos was compared, the OPU-helper group (3 7), at 51.3%, was significantly higher than the 17.1% of the OPU embryo group (3 0) alone. When calculating the advantage of culturing along with agarose embedded helpers, the yield for OPU embryos averaged 2.63 blastocysts/donor/session ( %), an increase of 0.83 blasto-

6 1648 E.M. Senatore et al. / Theriogenology 74 (2010) Table 2 Development of small number of IVP bovine embryos cultured in groups with the addition of agarose chips. Treatment* No. CE No. replicates % Developed to (Mean SEM) Agarose No. CE/droplet Morulae Blastocysts With a a Without a a With b b Without b b CE cleaved embryos (2 8 cells). * Cleaved embryos in groups of either 3 or 10 were cultured with or without addition of agarose chips in droplets containing 50 L culture medium. a,b Within a column, means without a common superscript differ (P 0.05). cysts/donor/session achieved over OPU embryos cultured alone ( %; P 0.05). 4. Discussion This study unambiguously demonstrated the benefit of helper embryos (produced from abbatoir-derived ovaries) in agarose chips on development of bovine embryos produced by OPU and IVF. Blastocyst development was significantly better among embryos cultured with supplemental agarose-embedded helpers, when compared to control embryos. The development of embryos embedded in agarose chips was similar to that in the free-floating group when the total number in the group was 10. The need to improve the efficiency of OPU/IVF to maximize its use in cattle of high genetic merit, particularly those with limited oocyte yields, and to further facilitate the development of small numbers of embryos, has been emphasized for more than a decade [16,21]. In the present study, the number of COCs retrieved by OPU varied greatly (range 3 20 per session). This variation has been reported in both Bos taurus and Bos indicus breeds [2,4,5,13,14]. Although treatment with FSH or ecg [2,10,22] increased the number of available COCs for aspiration, the average number of COCs remained variable and not much greater than numbers reported for untreated donors [2,4,10,22]. Evidence of a beneficial effect of group embryo culture was highlighted in earlier studies in mice [23,24], sheep [25], and cattle [26]. The rate of blastocyst development and total cell number increased when bovine embryos were cultured in groups [26]. This benefit to embryo production, as evidenced by increased development, was likely caused by various effects of the culture environment [3,15,27 29]. In that regard, embryotrophic compounds are secreted during development, and are at maximal concentrations just prior to implantation in cattle. Several of these factors have been investigated and are believed responsible for supporting and enhancing embryo development [30]. These so called embryotrophic compounds are implicated in maternal recognition of pregnancy, and can be detected in the medium of in vitro cultured blastocysts [28,31]. Typically, the addition of growth and embryotrophic factors into culture media can compensate for the adverse effects of relatively large volumes of media, or of small numbers of embryos being cultured. Together with droplet size and embryo density, Gopichandran and Leese [32] have recently shown, Table 3 Development of OPU/sexed IVP bovine embryos group-cultured with agarose-embeded helper embryos. Group No. oocytes No. replicates Agarose treatment No. total CE/droplet % Developed to (Mean SEM)* 2 8 Cell Morula BL BL/CE OPU a a a a OPU helper a b b b Helper, agarose embedded embryos; BL blastocyst; CE cleaved embryo (2 8 cells); OPU Ovum pickup. * After IVF and culture in vitro for 40 h, cleaved embryos derived from OPU/IVF in groups of 3 were cultured with either 0 or 7 helper embryos embedded in agarose chips, in droplets containing 50 L culture medium. The number of cultured embryos per droplet was either 3 (OPU group) or 10 (OPU helper). The cleavage to the 2-8 celled stage, and development to morulae and blastocysts, except for the values of BLs/2-8 celled, were calculated based on the total number oocytes collected by OPU and used for IVF. a,b Within a column, means without a common superscript differ (P 0.05).

7 E.M. Senatore et al. / Theriogenology 74 (2010) using a novel embryo culture technique, that the degree of physical proximity of cattle embryos to one another can influence the production of embryotrophic factors, and affect preimplantational development. Furthermore, in our case, only a small number of cleaved cattle embryos were cultured in a medium free of both other cells and serum. Reduced developmental competence has been reported, and was expected, when compared to that attained when culturing a larger number. In fact, blastocyst formation was influenced by the number of embryos cultured per droplet [17]. In other studies, while emphasizing the importance of the number of cleaved embryos cultured in a group versus cultured singly, no effect on embryo development was attributed to the culture volume, although a lower medium to embryo ratio increased blastocyst cell number [7,33]. Others reported that the well of the well (WOW) culture system had higher development of single (cloned and zona-free) bovine embryos into blastocyst stages in vitro [34,35]. Presumably, different culture systems will result in various rates of embryo development. In the present study, all groups of embryos were cultured in 50 L droplets; therefore, the effect of droplet size on embryo development was not considered. Possible explanations for the reduced developmental potential of cleaved embryos cultured singly may be related to substrate depletion, or to toxic build-up in micro-droplets [28,36]. This was more strikingly evident when individual early developing embryos, especially those obtained by OPU, were cultured with early stage embryos produced from abbatoir-derived ovaries. In the present study, blastocyst production was increased when the number of fertilized oocytes in culture was 10 per 50 L droplet. In our previous study, the group culture system was highly effective; a threshold number of 10 cleaved embryos per droplet sustained an optimal rate of late stage embryo development [37]. In the current study, we also tested the use of a greater number of cleaved embryos (i.e. 20), with the same size droplet and confirmed that the developmental progression to more advanced stages, i.e. blastocyst, was not significantly increased. Finally, based on satisfactory results obtained in the first two experiments, the last experiment was designed to investigate the efficiency of employing the same protocol, but using sex sorted X-sperm cells for IVF. The sorting parameters had been thoroughly tested prior and shown to give satisfactory embryo development rates [3]. It is widely reported though, that sex sorted semen does not perform as efficiently as unsorted semen, and that a reduced number of embryos are generated under in vitro conditions [3,29]. In the present study, the number of resulting cleaved sexed embryos was sufficient enable a comparison of control (3 0) and treatment (3 7) groups. The use of helpers embedded in agarose chips enhanced the development of embryos (derived by IVF using sexed semen) to blastocyst stage, so that their development reached a rate similar to that of IVF embryos with unsorted semen. Within the available literature, wide variation in the mean number of blastocysts per donor per OPU session has been reported, depending on breed and hormonal treatment [4,5,10]. In the present study, a significantly higher mean yield of blastocyst/donor/session was achieved when helpers were added to the culture droplet, when compared to a small number of embryos cultured without helpers. The physical separation between OPU- and abbatoir-derived embryos achieved by embedding the latter in agarose chips proved successful, once the correct temperature of agarose (at the time of embryo placement) was determined. This study represented an important contribution for the advancement of in vitro embryo production utilizing a novel culture approach in conjunction with OPU. As a practical application, considering the high rate of blastocyst development of single embryos when cultured with agarose-embedded helpers, it can be foreseen that each in vitro cleaved embryo produced from a donor could be successfully cultured individually with support from agarose-embedded embryos produced from abbatoir-derived ovaries. In conclusion, this study demonstrated the potential benefit of helper embryos embedded in agarose chips for promoting development of OPU-derived oocytes fertilized with either conventional or sexed sperm. Combined with sperm sorting, OPU, IVF, and embryo transfer, this novel culturing approach will facilitate the production of embryos from live animals of high genetic merit, and advance the breeding and expansion of elite herds. Acknowledgments The authors sincerely thank Mary-Margaret Cole of Kellogg Dairy Center (KDC), David Schreiber in the Department of Animal Science, and Stephen B. Treaster in the Department of Molecular and Cellular Biology, at University of Connecticut for the assistance. This study was supported by the Small Business Innovation Research program of the USDA Cooperative State Research, Education, and Extension Service (CSREES), Grant Number to Dr. Fuliang Du.

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