Theriogenology 79 (2013) Contents lists available at SciVerse ScienceDirect. Theriogenology. journal homepage:

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1 Theriogenology 79 (2013) Contents lists available at SciVerse ScienceDirect Theriogenology journal homepage: Pregnancy rates of lactating cows after transfer of in vitro produced embryos using X-sorted sperm S. Rasmussen a, J. Block b,c, G.E. Seidel Jr a, Z. Brink a, K. McSweeney a, P.W. Farin d, L. Bonilla c,1, P.J. Hansen c, * a Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado, USA b Ovatech L.L.C., Gainesville, Florida, USA c Department of Animal Sciences and D.H. Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, Florida, USA d Department of Population Health and Pathobiology, North Carolina State University, Raleigh, North Carolina, USA article info abstract Article history: Received 31 July 2012 Received in revised form 14 October 2012 Accepted 26 October 2012 Keywords: Embryo transfer In vitro fertilization X-sorted sperm Dairy cattle The main objective was to determine the efficacy of using X-sorted sperm to produce embryos in vitro for transfer into lactating dairy cows. Cows were bred by timed artificial insemination (TAI) using nonsorted semen or X-sorted sperm, or they received a fresh embryo produced in vitro by fertilization with X-sorted or nonsorted sperm using timed embryo transfer (TET). Pregnancy rates at approximately Day 32 averaged over all dairies were % (least-squares mean SEM) for TAI nonsorted, % for TET nonsorted fresh embryos, and % for TET X-sorted fresh embryos (TAI vs. both TET groups, P < 0.05; 206 to 233 cows per group). Pregnancy losses between approximately Day 32 and term ranged from 16% to 37%, the latter from TET with X-sorted sperm. Pregnancy losses to term were higher for cows receiving embryos produced in vitro than for cows bred by TAI. Calves produced via TET were not substantively different from AI controls in physical measurements or standard blood chemistry profiles. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Embryo transfer (ET) in cattle can be an important tool for genetic modification [1] and can improve pregnancy rates compared with AI when fertility is low, such as during heat stress [2,3] or in repeat breeder cows [4]. In addition, production of embryos in vitro is an efficient way to utilize X-sorted sperm, because one straw of semen can produce more embryos than with AI. There are few reports as to whether the use of X-sorted sperm for IVF results in production of embryos with altered competence for establishment of pregnancy after transfer to recipients. Reduced competence is possible because * Corresponding author. Tel.: þ ; fax: þ address: Hansen@animal.ufl.edu (P.J. Hansen). 1 Present address: Minitube of America, Mount Horeb, WI, USA. embryos produced with X-sorted sperm have been reported to have altered steady-state levels of specific mrna [5] and ultrastructural characteristics [6]. However, in one study, there was no difference in pregnancy rates after transfer of embryos produced with X-sorted sperm compared with embryos produced with conventional sperm [7]. Also, characteristics of calves produced by AI with X-sorted sperm did not differ from calves produced with nonsorted semen [8]. The major aims of this study were to determine the efficacy of establishing pregnancy in lactating cows on commercial dairy farms using transfer of an in vitroproduced embryo compared with AI, and to test whether pregnancy success was reduced in cows receiving an embryo produced using X-sorted sperm versus cows receiving an embryo produced with conventional sperm. So that findings could be readily applied, commercially available oocytes, semen, ET and AI services, and ovulation X/$ see front matter Ó 2013 Elsevier Inc. All rights reserved.

2 454 S. Rasmussen et al. / Theriogenology 79 (2013) synchronization procedures were used. Additional aims were to evaluate whether characteristics of calves born as the result of ET with X-sorted or conventional sperm were altered compared with calves born after AI. 2. Materials and methods 2.1. Overview Embryos were produced in vitro at Colorado State University and the University of Florida using similar, albeit not identical procedures. Embryos were transferred at two dairies in Colorado and three dairies in Florida and Georgia. There was variation among dairies in synchronization of reproductive cycles, personnel doing the commercial ET, and the specific bulls that provided semen. These differences were accounted for statistically by having dairy as a factor in all statistical analyses. Some analyses involved all five dairies, whereas others involved only dairies within a geographical region. Exact sperm sexing procedures for semen were proprietary, but likely did not deviate substantively from those described by Seidel and Garner [9] Colorado experiment Reagents Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. The oocyte maturation medium was a medium based on Tissue Culture Medium 199 (TCM-199) with Earle s salts and modified to contain hormones and other additives as described elsewhere [10]. Media for IVF and embryo culture were the series Fertilization-Chemically Defined Medium (F-CDM) (fertilization), CDM-1 (early embryo culture), and CDM-2 (later stage culture) [10].F-CDMmediumwassupplementedwith10mg/mL heparin, 25 mg/ml gentamycin sulfate, and 20 mg/ml amikacin for both X-sorted and nonsorted sperm. To maintain ph in air during brief embryo handling for vortexing and checking cleavage, low bicarbonate (5 mm) versions of CDM- 1 and -2 media were used [10]. The CDM-2 culture medium contained 0.25 mm phenazine ethosulfate to decrease lipid content of embryos [10]. Syngro medium, straws, plugs, and six-well manipulation plates were obtained from Bioniche (Pullman, WA, USA) Embryo production Unless otherwise stated, embryos were produced as described previously [10]. All culture occurred under sterile mineral oil (7.5 ml for drop system and 250 ml in a fourwell system). Dishes containing culture medium were equilibrated at least 4 hours before adding gametes or embryos in an incubator at 38.5 C containing 5% CO 2,5% O 2, and approximately 90% N 2 in a humidified atmosphere. For each weekly replicate, 250 (Dairy 1) or 150 (Dairy 2), Holstein cumulus oocyte complexes (COC) were obtained from Bomed (Madison, WI, USA). The COC were shipped in a portable incubator (Minitube, Verona, WI, USA) in oocyte maturation medium. Upon arrival, the temperature of the portable incubator was verified to be within 37 Cto39 C. The vials containing COC were then placed in the laboratory incubator (38.5 C, 5% CO 2 in humidified air) with lids loosened until 23 hours of maturation had elapsed. Matured COC were washed in F-CDM and then randomly assigned for insemination with X-sorted or nonsorted sperm from one Holstein bull. In total, three bulls were used. All three bulls were used for Dairy 1 (two from XY Inc., Fort Collins, CO, USA; one from Select Sires, Plain City, OH, USA), and two of the bulls used for Dairy 1 were used for Dairy 2 (one each from XY Inc. and Select Sires). Semen was thawed, no more than two straws at a time, in a 37 C water bath for 30 seconds. The X-sorted sperm was thawed, layered on top of a mini-percoll gradient (500 ml 45% Percoll over 450 ml 90% Percoll in a 1.5-mL conical tube), and the suspension was centrifuged at 600 g for 10 minutes. The supernatant was removed with a pipette, and 1 ml HEPES-CDM-1 (H-CDM-1) was added to resuspend the sperm pellet. The tube was then centrifuged at 300 g for 5 minutes and the supernatant removed to leave a pellet and small volume of 50 to 100 ml. The sperm concentration was then determined using a hemacytometer, and sperm was diluted to sperm per ml so that the final concentration in the fertilization drop would be sperm per ml. The fertilization drop was constituted with 30 ml of X-sorted sperm, 25 to 35 COC in 10 ml, and 110 ml F-CDM in a 60-mm dish under mineral oil. Nonsorted semen was separated using a standard Percoll gradient (2 ml 45% Percoll over 2 ml 90% Percoll) in a 15 ml conical tube. Semen was centrifuged for 20 minutes at 600 g, and supernatant removed to just above the pellet. Resuspended sperm were washed in 4 ml H-CDM-1 by centrifugation for 10 minutes at 600 g. Sperm concentration was determined with a hemacytometer, and sperm was suspended to /ml to result in a final concentration of sperm per ml for fertilization. Fertilization of COC with nonsorted sperm was performed in four-well dishes with 440 ml of F-CDM, 50 COC in 10 ml F-CDM, and 50 ml of sperm suspension. For both types of sperm, fertilization was allowed to proceed for 18 hours at 38.5 C and 5% (vol/vol) CO 2 in humidified air before vortexing the oocytes (now presumptive zygotes) to remove cumulus cells and associated sperm. For this procedure, presumptive zygotes were removed from F-CDM, placed in H-CDM-1, and vortexed in 0.5 ml snap-top tubes for 1 minute. Oocytes were immediately washed from the sides of the tube, suspended in 200 to 400 ml H-CDM-1, and washed through seven drops to separate presumptive zygotes from debris and loose cells. Thereafter, approximately 50 presumptive zygotes were cultured per four-well dish in 500 ml of CDM-1 (modified to contain fructose instead of glucose) at 38.5 C in an atmosphere of 5% CO 2,5%O 2, and approximately 90% N 2 in humidified air. Embryos were checked for cleavage and developmental status at approximately 56 hours after vortexing. Noncleaved oocytes were discarded. Embryos that had only developed to the two- to five-cell stage were considered retarded and cultured in a separate well from the more advanced, normal-appearing embryos with six or more cells. All embryos were cultured for an additional 4.5 days in CDM- 2 at 38.5 Cin5%CO 2,5%O 2, and approximately 90% N 2 in humidified air. Embryos in the retarded group only

3 S. Rasmussen et al. / Theriogenology 79 (2013) rarely developed to blastocysts and, therefore, were not used for ET. Embryos for transfer were placed in Syngro holding medium and loaded into 0.25-mL ET straws (Bioniche). Embryos used for transfer (approximately 7.5 days after onset of fertilization) were quality one or two blastocysts [11], expanded blastocysts or, rarely, early hatching, or hatched blastocysts. Embryos were placed into 0.25-mL straws for transportation (approximately 1 hour), and the straws were loaded into ET guns at the farms. The ET guns were covered with a plastic chemise and then put into gun warmers (EM Tools Inc., Rusk, TX, USA) at 38 C. Embryos were transferred into recipients assigned to treatments by a predetermined random selection method AI and ET Cows were located at two commercial dairy farms in Colorado. Cows were assigned randomly within week to one of four treatments as follows: (1) timed AI (TAI) with nonsorted semen (control); (2) TAI with X-sorted sperm; (3) timed ET (TET) with an embryo produced with nonsorted sperm; (4) and TET of an embryo produced with X-sorted sperm. Semen for cows bred by AI were from the same bulls as used to produce embryos. All three bulls (Dairy 1) or two bulls (Dairy 2) were used for AI each week. All embryos were transferred fresh. Cows in Dairy 1 (>8000 cows located near Eaton, CO, USA; 40.5N, 104.5W) were used from August to December of 2007, whereas cows at Dairy 2 (>800 cows located near Longmont, CO, USA; 40.2N, 105.1W) were used between January and May, There were 16 replicates (i.e., weeks) for Dairy 1 and 15 replicates for Dairy 2. Only first service, lactating Holstein cows in an Ovsynch program were used. Cows at Dairy 1 were housed in dry lot pens. Cows at Dairy 2 had dry lot pens and covered free stalls with clean sand bedding. Cows at both dairies were milked three times per day and fed total mixed rations. Estrous cycles of cows at least 60 days postpartum (N ¼ 354 for Dairy 1 and N ¼ 184 for Dairy 2) were synchronized using a modified Ovsynch procedure [12] as follows: ovaries and reproductive tract were scanned using an Aloka 500 ultrasound scanner with a 5 MHz linear-array transducer (Aloka America, Wallingford, CT, USA), and 100 mg gonadotropin releasing hormone (GnRH; Merial Ltd., Duluth, GA, USA) was given im in cows without ovarian or uterine pathology. Seven days later, cows were scanned and given 25 mg prostaglandin F2a (PGF; Pfizer, Kalamazoo, MI, USA) im if a CL or a follicle 13 to 19 mm in diameter was present. Cows received a second treatment of 100 mg GnRH im, 2.5 days after PGF. If structures were not observed on the day PGF was scheduled, cows were restarted with GnRH with or without a controlled internal drug releasing device (Pfizer). Some of these cows eventually re-entered the study. For TAI, cows were bred 18 hours after the second GnRH injection with X-sorted sperm or nonsorted semen. Cows selected for TET received an embryo 1 week later. Embryos were transferred 7.5 to 8.0 days after oocyte fertilization for recipients with a CL detected by ultrasonic examination. Cows were given a caudal epidural (5 ml of 2% [wt/vol] lidocaine; Mountain Veterinary Supply, Fort Collins, CO, USA) before transfer of an embryo ipsilateral to the CL. Pregnancy status was determined at Day 35 (Dairy 1) or 32 (Dairy 2) of pregnancy by ultrasound. Cows with dead or dying conceptuses (at Days 32 or 35) were considered not pregnant. Fetal deaths were determined between first check and approximately Day 60. In addition, calving data were recorded. Cows that left the herd before the first pregnancy check were excluded from the experiment. Cows with pyometritis when checked for pregnancy were included in the data (N ¼ 5 at Dairy 1) Florida experiment Reagents All materials were purchased from Sigma-Aldrich or Fisher Scientific (Fairlawn, NJ, USA), unless specified otherwise. HEPES-Tyrode s Lactate and IVF-Tyrode s Lactate were purchased from Caisson Laboratories Inc. (Logan, UT, USA). These media were used to prepare HEPES-Tyrode s albumin lactate pyruvate (TALP) and IVF-TALP [13]. Oocyte collection and maturation media for purchased oocytes was proprietary. Oocyte collection medium for oocytes collected locally was TCM-199 with Hank s salts without phenol red (Atlanta Biologicals, Norcross, GA, USA) and supplemented with 2% (vol/vol) bovine steer serum (Pel- Freez, Rogers, AR, USA), 2 U/mL heparin, 100 U/mL penicillin-g, 0.1 mg/ml streptomycin sulfate, and 1 mm glutamine. Oocyte maturation medium for oocytes collected locally was TCM-199 (Invitrogen, Carlsbad, CA, USA) with Earle s salts supplemented with 10% (vol/vol) bovine steer serum, 2 mg/ml estradiol 17-b, 20 mg/ml bovine follicle stimulating hormone (Folltropin-V; Bioniche), 22 mg/ml sodium pyruvate, 50 mg/ml gentamicin sulfate, and 1 mm glutamine. Percoll was from Amersham Pharmacia Biotech (Uppsala, Sweden). Potassium simplex optimized medium (KSOM) that contained 1 mg/ml BSA was from Caisson Laboratories Inc. On the day of use, KSOM was modified to produce KSOM-BE2 as described previously [14]. Synthetic oviductal fluid [15] was purchased as a custom formulation from Millipore (Billerica, MA, USA). Lidocaine was from Pro Labs (St. Joseph, MO, USA) Embryo production The COC were purchased from Bomed, TransOva Genetics (Sioux Center, IA, USA), or Evergen Biotechnologies (Storrs, CT, USA). After collection, COC were placed into 2 ml cryovials (approximately 50 to 100 COC per cryovial) containing maturation medium and shipped overnight, in a portable incubator set at 39 C, to the laboratory in Gainesville, FL, USA. Additional Holstein COC were collected from locally obtained ovaries (Central Packing Co., Center Hill, FL, USA) and matured as described [14]. Regardless of collection procedure, all COC were allowed to mature for 21 to 24 hours. In vitro fertilization and embryo culture were conducted as described previously [14]. After maturation, COC were washed once in HEPES-TALP and then randomly assigned to be inseminated with either nonsorted semen or X-sorted sperm from one of four Holstein bulls (XY Inc.). Non-sorted

4 456 S. Rasmussen et al. / Theriogenology 79 (2013) semen and X-sorted sperm were Percoll-purified using mini-percoll gradients (500 ml 45% [vol/vol] over 500 ml 90% [vol/vol]). Semen or X-sorted sperm was layered on top of the mini-percoll gradient and centrifuged for 20 minutes at 300 g. The sperm pellet from each group was then washed in 4 ml of HEPES-TALP and centrifuged for 5 minutes at 300 g. Sperm were resuspended in IVF-TALP to yield a final concentration of 0.5 to sperm per ml. Sperm and COC were allowed to coincubate for 6 to 8 hours at 38.5 C and 5% (vol/vol) CO 2 in humidified air. After fertilization, presumptive zygotes were vortexed to remove cumulus cells and associated sperm and then washed three times in HEPES-TALP before being placed into embryo culture medium. Presumptive zygotes were cultured in 50-mL microdrops overlaid with mineral oil in groups of 25 to 30 in a humidified atmosphere of 5% CO 2,5% O 2, and approximately 90% N 2 at 38.5 C. For Farm 1, embryos were cultured in KSOM-BE2, as described [14]. For Farms 2 and 3, embryos were cultured in synthetic oviductal fluid [15] which was modified to contain 0.5 mm fructose, 1.0 mm alanyl-glutamine, 5.3 mm sodium lactate, 0.5 mm tri-sodium citrate, 2.77 mm myo-inositol, and 1 mg/ml polyvinyl alcohol. Cleavage rate was recorded on Day 3 after insemination, and the proportion of oocytes developing to the blastocyst stage was recorded on Day 7 after insemination. For fresh ET, grade 1 [11] morula, blastocyst, and expanded blastocyst stage embryos were harvested on Day 7(N¼ 26 replicates) or Day 8 (N ¼ 2 replicates) after insemination. Harvested embryos were loaded into 0.25 ml French straws in holding medium (HEPES-TALP containing 10% [vol/vol] fetal bovine serum and 50 mm dithiothreitol [as an antioxidant]), and straws containing selected embryos were then placed horizontally into a portable incubator (Biotherm INC-12V; Cryologic, Victoria, Australia) at 39 C and transported to the respective farm. At the farm, straws containing embryos were loaded into a transfer pipette (IMV Technologies, L Aigle, France) and randomly transferred to recipients. Of the harvested embryos, nine were morula, 53 were blastocysts, and 127 were expanded blastocysts. For some replicates, some embryos produced using X-sorted sperm were vitrified. Grade 1 blastocyst and expanded blastocyst stage embryos were harvested on Day 7 after insemination and washed twice in TCM-199 containing 10% (vol/vol) fetal bovine serum. Harvested embryos were then vitrified in open-pulled straws as described [4]. On the day of transfer, vitrified embryos were thawed as described [4], loaded into 0.25-mL straws and transported to the farm as described for fresh embryos. Of the vitrified embryos that were transferred, 25 were blastocysts and 33 were expanded blastocysts AI and ET The experiment was conducted between April 2007 and January 2008 at three locations (Farm 1, approximately 2500 cows, located in Trenton, FL, USA; 29.4N, 82.5W; Farm 2, approximately 1500 cows, located in Greenville, FL, USA; 30.3N, 83.4W; Farm 3, approximately 2000 cows, located in Quitman, GA, USA; 30.5N, 83.3W). Primiparous and multiparous, first-service, lactating Holstein cows were used at each farm. Cows at all dairies were housed in freestall barns, bedded with sand, fed a total mixed ration, and milked three times per day. A total of 375 cows were synchronized for either TAI or TET using modified presynch-ovsynch protocols. At Farm 1, cows were presynchronized with two treatments of PGF (25 mg, im) given 14 days apart on Day 38 and Day 24 (Day 0 ¼ day of expected ovulation). Cows received 100 mg of GnRH (im) on Day 10 and 25 mg PGF im on Day 3. A second treatment of GnRH was administered on Day 0, 72 hours after PGF administration. Cows at Farms 2 and 3 were presynchronized with two treatments of PGF as described for Farm 1, except that the treatments occurred on Days 36 and 22, respectively. As for Farm 1, cows received 100 mg of GnRH on Day 10 followed by 25 mg PGF on Day 3. A second treatment of GnRH was administered on Day 1, 56 hours after PGF administration. For all farms, Day 0 was defined as the presumptive day of ovulation. For cows at Farm 1, Day 0 occurred between 68 and 74 days in milk. For cows at Farms 2 and 3, Day 0 occurred between 76 and 82 days in milk. A total of 28 weekly replicates were completed. For 21 replicates, cows were randomly assigned to one of three treatment groups: (1) TAI with nonsorted semen; (2) TET with a fresh embryo produced using nonsorted sperm; and (3) TET with a fresh embryo produced using X-sorted sperm. For seven replicates, a fourth treatment group, TET with a vitrified embryo produced using X-sorted sperm, was also included. At Farm 1, cows that were assigned to the TAI group were inseminated on Day 0 in conjunction with the second GnRH treatment of the Ovsynch protocol. At Farms 2 and 3, cows assigned to the TAI group were inseminated on Day 0, 16 hours after the second GnRH treatment of the Ovsynch protocol. Of 100 inseminated cows, 87 were inseminated using semen from one of the bulls used to produce embryos and 13 were inseminated with semen from one of five bulls not used for embryo production. On Day 7 after presumptive ovulation, the presence or absence of a CL was diagnosed either by transrectal palpation or by ultrasonography with an Aloka 500 ultrasound scanner with a 5 MHz linear-array transducer among cows in all treatment groups, including cows subjected to TAI. Cows in the TAI and TET groups that did not have a visible CL on Day 7 were excluded from the study. All cows in the TET groups that had a visible CL on Day 7 after presumptive ovulation received caudal epidural anesthesia (5 ml of 2% [wt/vol] lidocaine) and a single embryo was transferred to the uterine horn ipsilateral to the ovary with the CL. Pregnancy was diagnosed by ultrasonography or transrectal palpation at Days 33 to 58 of gestation. Data for calving rate and calf sex ratio were also recorded Assessment of calves Thirty-eight Holstein heifer calves were examined on the day of birth for physical development and health status. Data were collected from calves born during July to September 2008 at Farm 3 from the Florida experiment located in Quitman, GA, USA (30.5N, 83.3W). Treatment groups included calves resulting from TAI with nonsorted semen, TET with a fresh embryo produced using X-sorted

5 S. Rasmussen et al. / Theriogenology 79 (2013) sperm, TET with a vitrified embryo produced using X-sorted sperm, and calves produced at the farm as a result of AI with nonsorted semen from one of five Holstein bulls not used for other treatments (farm controls). All calves were singletons, except for one set of twins in the farm control group. Physical measurements of calves included birth weight and other parameters related to body size. Birth weight was measured using a digital scale (W.C. Redmon Co., Peru, IN, USA). Adjusted birth weight was calculated in calves who received colostrum before data collection by subtracting the estimated weight of colostrum consumed from the birth weight. Physiological measurements included rectal temperature, heart rate, respiration rate, and capillary refill time. Blood samples of calves were collected by jugular venipuncture for analysis of serum biochemical values. Blood samples were transported on ice to the laboratory where serum was harvested and stored at 20 C until analysis. Biochemical parameters in serum were determined using a chemistry analyzer (Hitachi Model 912 Analyzer, Boehringer Mannheim, Mannheim, Germany) Analysis of data Cleavage and blastocyst rates were analyzed by leastsquares ANOVA using the GLM procedure of SAS for Windows 9.0 (SAS, Cary, NC, USA). Values presented are least-squares means SEM. The proportion of oocytes that cleaved on Day 3 after insemination and that developed to the blastocyst stage on Day 7 after insemination were calculated for each replicate. The mathematical model included sperm type, bull, and interactions. Pregnancy rates, calving rates, pregnancy losses, and sex ratios for the individual Colorado and Florida data were analyzed by Chisquare with the Fisher-Yates correction. Data other than calf characteristics were analyzed as three sets. Analyses of data from Colorado, and then Florida are considered first (Tables 1 to 3). A second analysis concerns a subset of the Florida data using only one farm at which vitrified embryos were transferred (Table 4). Third, as was planned prospectively at inception of these studies, least-squares analysis of variance combining data from Florida and Colorado was performed using the GLM procedure of SAS for the treatments in common at both locations: (1) TAI with nonsorted semen (control); (2) TET with embryos produced in vitro with nonsorted sperm; and (3) TET with embryos produced in vitro with X-sorted sperm. We included individual dairies in the model, so differences in personnel and methods between Colorado and Florida were appropriately included; there were no interactions (P > 0.05) between dairies and treatments for pregnancy rates. Physical, physiologic, and biochemical measures of calves were analyzed for the effect of treatment by least-squares ANOVA using the GLM procedure of SAS. When the main effect of treatment approached significance (P < 0.10), multiple comparisons among treatments were examined using the pdiff procedure of SAS. Differences between individual treatments where P < 0.05 are reported. All values are reported as leastsquares means SEM. Table 1 Comparison of pregnancy rates for lactating cows receiving timed artificial insemination (TAI) or timed embryo transfer (TET) using nonsorted semen or X-sorted sperm (Colorado). Treatment 3. Results Pregnancy rate, Days 32 to Detailed findings from the Colorado experiment Pregnancy rate, Day 60 TAI nonsorted semen 62/133 ¼ 47% a 57/132 ¼ 43% a TAI X-sorted sperm 55/146 ¼ 38% a 45/144 ¼ 31% a TET nonsorted sperm 26/108 ¼ 24% b 22/107 ¼ 21% b TET X-sorted sperm 31/112 ¼ 28% b 20/112 ¼ 18% b Within a column, means without a common superscript letters differed (P < 0.05) Embryo production Cleavage rates were not different for oocytes fertilized with X-sorted (67 3%; N ¼ 3985) or nonsorted sperm (69 2%; N ¼ 1785), but the proportion of oocytes that became a blastocyst was lower (P < 0.05) for oocytes fertilized with X-sorted sperm (13 1%) than for oocytes fertilized with nonsorted sperm (23 2%) Pregnancy rates Results for pregnancy rates as determined at 32 to 35 and 60 days of gestation are shown in Table 1. More cows were planned to be in the AI control group than in the other treatments because costs for this treatment were lower. Pregnancy rates were lower for TET than for TAI at both Days 32 to 35 and Day 60 of gestation. There were no differences in pregnancy rate between cows receiving an embryo produced with X-sorted and cows receiving an embryo produced with nonsorted sperm. For TAI, pregnancy rates at Days 32 to 35 and 60 were not different (P > 0.1) between cows inseminated with X-sorted sperm and cows inseminated with nonsorted semen. There was, however, a nonsignificant trend for pregnancy rate at Day 60 to be lower for cows inseminated with X-sorted sperm Calving rates, pregnancy losses, and sex ratio Calving rates for the TET treatments (Table 2) were lower than for the TAI control (P < 0.05). Pregnancy loss Table 2 Calving rate, pregnancy loss, and sex ratio for lactating cows receiving timed artificial insemination (TAI) or timed embryo transfer (TET) using nonsorted semen or X-sorted sperm (Colorado). Treatment Calving rate Pregnancy loss Females born between Days 32 and 35 and term TAI nonsorted 50/130 ¼ 38% a 9/59 ¼ 15% a 24/50 ¼ 48% a semen TAI X-sorted 40/142 ¼ 28% a,b 13/53 ¼ 24% a,b 35/40 ¼ 88% b sperm TET nonsorted 18/105 ¼ 17% b 5/23 ¼ 22% a,b 8/18 ¼ 50% a sperm TET X-sorted sperm 18/112 ¼ 16% b 13/31 ¼ 32% b 16/18 ¼ 89% b Within a column, means without a common superscript letters differed (P < 0.05).

6 458 S. Rasmussen et al. / Theriogenology 79 (2013) Table 3 Pregnancy rates, pregnancy loss, calving rate, and sex ratio after timed artificial insemination (TAI) with nonsorted semen or timed embryo transfer (TET) using fresh embryos produced with nonsorted semen or X-sorted sperm (Florida). Treatment Pregnancy rate, Days 33 to 58 Calving rate Pregnancy loss between Females born Days 33 to 58 and term TAI nonsorted semen 30/100 ¼ 30% a 24/100 ¼ 24% a 6/30 ¼ 20% a 10/24 ¼ 42% a TET nonsorted sperm 27/95 ¼ 28% a 19/94 ¼ 20% a 7/26 ¼ 27% a 8/18 ¼ 44% a TET X-sorted sperm 28/94 ¼ 30% a 16/89 ¼ 18% a 7/23 ¼ 30% a 13/16 ¼ 81% b Within a column, means without a common superscript letters differed (P < 0.05). between Day 32 and term ranged from 15% to 32%, with the losses for TAI with nonsorted semen being lower (P < 0.05) than for TET using X-sorted sperm (Table 2). The percentage of calves that were female was higher (P < 0.05) for TAI and TET using X-sorted sperm than for TAI and TET using nonsorted semen (Table 2) Detailed findings from the Florida experiment Embryo production Cleavage rates were lower (P < 0.05) for oocytes fertilized with X-sorted sperm (57 4%; N ¼ 4260) than for oocytes fertilized with nonsorted sperm (71 5%; N ¼ 1906). Similarly, the proportion of oocytes that became a blastocyst was lower (P < 0.05) for oocytes fertilized with X-sorted sperm (8 2%) than for oocytes fertilized with nonsorted sperm (15 3%) Pregnancy rates, pregnancy losses, and sex ratio There was no difference in pregnancy rate or calving rate between TAI, TET using fresh embryos produced with nonsorted semen, or TET using fresh embryos produced with X-sorted sperm (Table 3). Data from one farm in Florida that included contemporaneously transferred vitrified embryos are presented in Table 4. Although not significantly different (P > 0.1), transfer of vitrified embryos resulted in numerically lower pregnancy rates than transfer of fresh embryos or TAI. There was no significant difference in pregnancy loss between initial pregnancy diagnosis and term between treatments (Table 3). The percentage of calves that were female was higher (P < 0.05) for the TET group receiving embryos produced with X-sorted sperm than for other groups in which nonsorted semen was used (Table 3) Colorado and Florida data combined Results regarding pregnancy rates, calving rates, pregnancy loss, and the percentage of female calves after combining Florida and Colorado data are presented in Table 5. There were no differences in pregnancy or calving rates between embryos produced with nonsorted semen or X-sorted sperm. However, both TET treatments had lower pregnancy and calving rates than TAI (P < 0.05). Pregnancy loss between Days 32 to 58 and term was higher (P < 0.05) for embryos produced with X-sorted sperm than for TAI. There was no difference in pregnancy loss between embryos produced with nonsorted semen versus those produced by TAI or versus embryos produced with X-sorted sperm. The percentage of female calves was higher (P < 0.05) for embryos produced using X-sorted sperm than for TAI or embryos produced using nonsorted semen Characteristics of calves There were few differences in calves because of treatments (Tables 6 and 7). Capillary refill time was longer for TAI calves than other groups (P < 0.05). Several biochemical measurements were different for FAI calves than for other groups (albumin, albumin:globulin, g-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, and magnesium), but there were no differences in biochemical measurements between TAI and TET calves (Table 7). 4. Discussion The use of X-sorted sperm, already widely employed for AI in dairy cattle [16], has the potential to alter the structure of the dairy industry by increasing the replacement heifer supply, creating opportunities for using a proportion of the dairy herd for producing beef animals, and improving the rate of genetic selection [17]. Use of X-sorted sperm in ET programs represents another use of this technology. The experiments described herein were done at five farms at varying latitudes with embryos produced in two laboratories. Procedures at the different locations were similar but not identical, personnel differed, and semen Table 4 Pregnancy rates, pregnancy loss, calving rate, and sex ratio at one Florida farm after timed artificial insemination (TAI) and timed embryo transfer (TET) using embryos produced with nonsorted semen or X-sorted sperm: results from replicates where a vitrification treatment was included. Treatment Pregnancy rate, Days 33 to 58 Calving rate Pregnancy loss between Females born Days 33 to 58 and term TAI nonsorted semen 16/38 ¼ 42% 12/38 ¼ 32% 4/16 ¼ 25% 6/12 ¼ 50% TET nonsorted sperm 11/31 ¼ 35% 8/31 ¼ 26% 3/11 ¼ 27% 4/8 ¼ 50% TET X-sorted sperm 18/50 ¼ 36% 10/47 ¼ 21% 5/15 ¼ 33% 9/10 ¼ 90% TET vitrified X-sorted sperm 14/58 ¼ 24% 10/58 ¼ 17% 3/13 ¼24% 8/12 ¼ 67% Differences between treatments were nonsignificant.

7 S. Rasmussen et al. / Theriogenology 79 (2013) Table 5 Least-squares means þ SEM rates for combined Colorado and Florida data for timed artificial insemination (TAI) and timed embryo transfer (TET; excluding vitrified embryos). Treatment Cows, N Pregnancy rate, Days 32 to 58 (%) Pregnancy loss between Days 32 and 58 and term (%) Calving rate (%) Female (%) TAI nonsorted semen a a a a TET nonsorted semen b a,b b a TET X-sorted sperm b b b b Within a column, means without a common superscript letters differed (P < 0.05). sources differed. Farms were considered as factors in statistical analyses, which also resulted in blocking for laboratory differences in procedures and personnel. There was no treatment by farm interaction for pregnancy rate (P > 0.05). Results were very similar under these varied conditions, and because of the design, were considered representative of expected results on similar dairies served by personnel providing commercial ET and AI services using commercially available semen and oocytes. Cleavage rates from the Colorado study did not differ between nonsorted and X-sorted sperm, but the sperm concentration was four times higher during IVF for the X-sorted sperm. For the Florida study, identical sperm concentrations were used for X-sorted and nonsorted sperm, and fertilization time was shorter than in Colorado. Probably as a result, cleavage rates were lower for sexed sperm. Perhaps embryos derived from X-sorted sperm would have lower competence to establish pregnancy after transfer to a recipient, because of sperm damage caused during the sorting process. Indeed, there was evidence in both Colorado and Florida studies for damage to X-sorted sperm as indicated by reduced development of fertilized oocytes to the blastocyst stage. Overall, however, there was no difference in competency to establish pregnancies for embryos produced with X-sorted sperm as compared with those produced using nonsorted semen. Similarly, Xu et al. [7] reported no decrease in calving rates with embryos from sexed sperm compared with controls. Another possible problem associated with use of X-sorted sperm for IVF is that female embryos, which make up the great majority of embryos produced using X-sorted sperm, can develop poorly in culture medium high in glucose [18,19]. However, this potential problem was avoided in the present experiments, because fructose was used as an energy source to replace glucose. Pregnancy rates at Day 60 of gestation were slightly lower for TAI using X-sorted sperm than in cows inseminated with nonsorted semen. This result mirrors results from 12 million inseminations of US Holstein cows and heifers [16]. The reduced conception rate in animals inseminated with X-sorted sperm is probably because of a reduced fertilization rate or early embryonic development, rather than to events after blastocyst formation. This conclusion was based on the observations that: (1) fewer oocytes inseminated with X-sorted sperm cleaved (in the Florida study) or became blastocysts (in both the Colorado and Florida studies) than oocytes inseminated with nonsorted sperm; and (2) there was no difference in pregnancy rate between TET cows receiving an embryo produced with X-sorted sperm or an embryo produced with nonsorted semen. In other studies too, use of X-sorted sperm in vitro was associated with reduced cleavage and competence to develop to the blastocyst stage [5,20 22]. The difference in development of embryos produced with sexed sperm occurs after the first cell cycle [20,21] although the problem might be initiated in the first cell cycle. Pregnancy success, as defined by pregnancy rates measured from Days 30 to 60 of gestation or by calving rate, was not improved by use of ET as compared with AI. This was not surprising, because in other studies, transfer of embryos produced in vivo or in vitro did not increase fertility of lactating dairy cows when pregnancy rates to AI were not compromised [23,24]. In contrast, ET improved fertility when cows were subfertile, e.g., during heat stress [2,3] or in repeat breeder cows [4]. Table 6 Physical and physiological measurements of Holstein female calves resulting from farm control artificial insemination (FAI), timed artificial insemination (TAI), or timed embryo transfer (TET). FAI TAI, nonsorted semen TET, X-sorted sperm TET, X-sorted sperm, vitrified Calves, N Body weight (kg) a a a a Body weight, adjusted (kg) a a a a Crown rump length (cm) a a a a Biparietal diameter (cm) a a a a Heart girth (cm) a a a a Fetlock diameter (cm) a a a a Right metacarpus (cm) a a a a Right metatarsus (cm) a a a a Respirations per minute a a a a Heart rate per minute a a a a Rectal temperature ( C) a a a a Capillary refill time (sec) a b a a Means without a common superscript letters differed (P < 0.05). Data are least-squares means SEM.

8 460 S. Rasmussen et al. / Theriogenology 79 (2013) Table 7 Serum biochemical values of Holstein female calves resulting from farm control artificial insemination (FAI), timed artificial insemination (TAI), or timed embryo transfer (TET). FAI TAI nonsorted semen TET X-sorted sperm TET X-sorted sperm, vitrified Calves, N Total protein (g/dl) a a a a Albumin (g/dl) a b b b Globulin (g/dl) a a a a Albumin:globulin ratio a b a,b a,b Cholesterol (mg/dl) a a a a Triglycerides (mg/dl) a a a a Glucose (mg/dl) a a a a BUN (mg/dl) a a a a Creatinine (mg/dl) a a a a BUN:creatinine ratio (mg/dl) a a a a Total bilirubin (mg/dl) a a a a g-glutamyltransferase (m/l) a b b b Alkaline phosphatase (m/l) a b b a,b Alanine aminotransferase (m/l) a a a a Aspartate aminotransferase (m/l) a b b b Amylase (m/l) a a a a Calcium (mg/dl) a a a a Phosphorus (mg/dl) a a a a Magnesium (meq/l) a b b,c b Sodium (meq/l) a a a a Potassium (meq/l) a a a a Chloride (meq/l) a a a a Means without a common superscript letters differed (P < 0.05 unless otherwise indicated). Data are least-squares means SEM. Abbreviation: BUN, blood urea nitrogen. c Different from FAI, P ¼ An important question regarding the use of transfer of in vitro produced embryos is whether the alterations in molecular, biochemical, and ultrastructural properties of the embryo associated with culture [25] results in a conceptus with reduced ability to complete normal development in utero. There are reports that pregnancy losses after initial pregnancy diagnosis are greater for recipients of in vivo-produced embryos [26,27] or in vitroproduced embryos [3] than for cows bred by AI. In other reports, however, there were no differences in pregnancy loss between cows pregnant as a result of ET or AI when embryos produced in vivo [24] or in vitro [4] were transferred. In the combined data set for the present study, pregnancy loss for cows receiving embryos produced with X-sorted sperm was higher than for TAI cows, but there was no difference in pregnancy loss between cows that received embryos produced with nonsorted semen and TAI. There are also a variety of abnormalities reported for calves born as the result of transfer of an in vitro-produced embryo [28]. In the current study, there were no differences between TAI and TET calves Conclusions Embryos were produced in vitro using X-sorted sperm without loss in competence to establish and maintain pregnancy after transfer into recipients, compared with embryos produced with nonsorted sperm. However, pregnancy losses tended to be higher for cows receiving embryos produced in vitro than for cows bred by TAI. Therefore, this emphasized the importance of optimizing culture systems to allow embryonic development in vitro to more closely match events occurring in vivo. Acknowledgments Research was supported by National Research Initiative Competitive Grant no from the USDA National Institute of Food and Agriculture. We very much appreciate the collaboration of the dairies involved. The authors thank Scott Purcell and especially James Zumbrunnen for help with statistical analyses, Jessica Hicks for assistance with collection of calf data, William Rembert and Central Packing for assistance in obtaining ovaries, XY Inc. and Select Sires, Inc. for donation of semen, Bioniche Life Sciences for donation of plasticware, and Char Farin for help with data retrieval. References [1] Hansen PJ, Block J. Towards an embryocentric world: the current and potential uses of embryo technologies in dairy production. Reprod Fertil Dev 2004;162:1 14. [2] Hansen PJ. Exploitation of genetic and physiological determinants of embryonic resistance to elevated temperature to improve embryonic survival in dairy cattle during heat stress. Theriogenology 2007;68(Suppl. 1):S [3] Stewart BM, Block J, Morelli P, Navarette AE, Amstalden M, Bonilla L, et al. Efficacy of embryo transfer in lactating dairy cows during summer using fresh or vitrified embryos produced in vitro with sexsorted semen. J. Dairy Sci 2011;94: [4] Block J, Bonilla L, Hansen PJ. Efficacy of in vitro embryo transfer in lactating dairy cows using fresh or vitrified embryos produced in a novel embryo culture medium. J Dairy Sci 2010;93: [5] Morton KM, Herrmann D, Sieg B, Struckmann C, Maxwell WM, Rath D, et al. Altered mrna expression patterns in bovine blastocysts after fertilisation in vitro using flow-cytometrically sex-sorted sperm. Mol Reprod Dev 2007;74: [6] Palma GA, Olivier NS, Neumüller Ch, Sinowatz F. Effects of sexsorted spermatozoa on the efficiency of in vitro fertilization and ultrastructure of in vitro produced bovine blastocysts. Anat Histol Embryol 2008;37:67 73.

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