Sex-Sorted Semen for Timed Artificial Insemination of Beef Heifers

Theriogenology 98 (2017) 88e93

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Theriogenology

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Effective use of SexedULTRA™ sex-sorted semen for timed artiﬁcial insemination of beef heifers

J.M. Thomas a, J.W.C. Locke a, b, R. Vishwanath c, J.B. Hall d, M.R. Ellersieck e, M.F. Smith a, * D.J. Patterson a, a Division of Animal Sciences, University of Missouri, Columbia, MO 65211, United States b College of Veterinary Medicine, University of Missouri, Columbia, MO 65211, United States c Sexing Technologies, Navasota, TX 77868, United States d Department of Animal and Veterinary Science, Nancy M. Cummings Research Extension and Education Center, University of Idaho, Carmen, ID 83462, United States e Agriculture Experiment Station Statistician, University of Missouri, Columbia, MO 65211, United States article info abstract

Article history: An experiment was designed to evaluate the relative fertility of SexedULTRATM sex-sorted semen Received 30 November 2016 compared to conventional, non-sex-sorted semen when used among beef heifers in conjunction with Received in revised form split-time AI following the 14-d CIDR-PG protocol. Units of conventional semen were generated with 17 March 2017 25.0 106 live cells per 0.5 ml straw prior to freezing, and units of sex-sorted semen were generated Accepted 20 March 2017 using the SexedULTRATM Genesis III sorting technology with 4.0 106 live cells per 0.25 ml straw prior to Available online 23 March 2017 freezing. Sex-sorted units were sorted to contain X chromosome-bearing sperm cells at an accuracy level of >90%. Estrus was synchronized in 851 heifers at four locations using the 14-d CIDR-PG protocol: Keywords: Sexed semen controlled internal drug release (CIDR) insert (1.38 g progesterone) on Day 0, CIDR removal on Day 14, Timed artiﬁcial insemination and administration of prostaglandin F2a (PG; 25 mg im) on Day 30. Estrus detection aids were applied at Beef heifer PG on Day 30 to evaluate estrous response rate, and split-time AI was performed based on estrous Estrus synchronization response. At 66 h after PG (Day 33), heifers having expressed estrus received timed AI. Heifers failing to express estrus by 66 h received timed AI 24 h later (90 h after PGF2a on Day 34). Heifers failing to express estrus by 90 h were administered gonadotropin-releasing hormone (GnRH; 100 mg im) concurrent with AI. Heifers were preassigned to treatment (insemination with either conventional or SexedULTRATM sex- sorted semen), and treatments were balanced within each location based on source, reproductive tract score, and weight. Heifers were exposed for natural service beginning 14 d after AI for the remainder of a 60 d breeding season. Pregnancy rates to AI across locations tended to be higher (P ¼ 0.09) for heifers inseminated with conventional semen (60%; 257/429) compared to sex-sorted semen (52%; 218/422). Higher pregnancy rates to AI (P < 0.0001) were obtained among heifers that expressed estrus prior to AI than among heifers that failed to express estrus prior to AI at 90 h. Total pregnancy rates at the end of the 60 d breeding season did not differ between heifers that received sex-sorted semen at AI (89%; 376/422) and heifers that received conventional semen at AI (89%; 382/429). In summary, the pregnancy rates observed suggest that SexedULTRATM sex-sorted semen can be used effectively for timed AI of beef heifers when split-time AI is performed following the 14-d CIDR-PG protocol. © 2017 Elsevier Inc. All rights reserved.

1. Introduction based on differences in DNA content between X- and Y- chromosome-bearing sperm cells [1,2]. This technology has been Using a flow cytometer, sperm cells can be effectively sorted used successfully to produce sex-sorted units of bovine sperm cells for commercial use [3,4]. However, sex-sorted sperm produced using this method have reduced viability and overall quality after cryopreservation and thawing [5]. Recent research efforts have * Corresponding author. Animal Science Research Center S132, 920 E Campus Dr, resulted in the development of optimized equipment and proced- Columbia, MO 65211, United States. E-mail address: [email protected] (D.J. Patterson). ures that decrease the degree of cellular damage occurring to sperm http://dx.doi.org/10.1016/j.theriogenology.2017.03.018 0093-691X/© 2017 Elsevier Inc. All rights reserved. J.M. Thomas et al. / Theriogenology 98 (2017) 88e93 89 cells during the sex-sorting process [6e8]. Sex-sorted semen pro- been recommended that sex-sorted semen not be used in duced using this next generation technology is presently available conjunction with FTAI or that use of sex-sorted semen should be and marketed commercially for both dairy and beef breeds under restricted to only females expressing estrus prior to FTAI [24]. the trade name SexedULTRATM (Sexing Technologies, Navasota, TX). In an effort to improve pregnancy rates to timed AI, Thomas In a large trial using semen from eight Holstein bulls for et al. [20] developed a modified timed AI approach in which timing insemination of over 6000 heifers in 41 commercial locations, of insemination is delayed for non-estrous females until 20e24 h pregnancy rates to AI following detected estrus were improved by after fixed-time AI of cows in estrus. This approach, termed “split- 4.5% among dairy heifers inseminated with SexedULTRATM sex- time AI,” was found to improve pregnancy rates when using sex- sorted semen (46.1%) compared to sex-sorted semen produced sorted semen among mature beef cows following the 7-d CO- using the previous generation XY technology (41.6%) [9]. Synch þ CIDR protocol [20]. When evaluated using conventional Furthermore, the improved speed and reduced sperm cell loss semen, split-time AI was found to improve pregnancy rates among associated with the new technology facilitates use of higher beef heifers following the 14-d CIDR-PG protocol [25] as well as sperm cell numbers per unit [9], above the typical 2.0 106 among mature cows following the 7-d CO-Synch þ CIDR protocol concentration that became standard for units of sex-sorted semen [26], provided that GnRH was administered to non-estrous cows produced using the XY method [3,10,11]. A separate trial using concurrent with the delayed insemination [27]. To date, however, semen from five bulls for insemination of over 6000 dairy animals split-time AI has not been evaluated using sex-sorted semen among based on detected estrus indicated no difference between preg- beef heifers following the 14-d CIDR-PG protocol. Moreover, the nancy rates to SexedULTRATM semen at a concentration of original split-time AI work with sex-sorted semen [20] predated 4.0 106 cells per unit and pregnancy rates to conventional the development of the SexedULTRATM sorting process. This semen at a concentration of 15 106 cells per unit [9]. However, experiment was designed to evaluate the relative fertility of Sex- SexedULTRATM semen has not been evaluated among beef animals edULTRATM sex-sorted semen compared to conventional, non-sex- or, more critically, in the context of timed AI. sorted semen when used among beef heifers in conjunction with When using protocols designed to facilitate fixed-time artificial split-time AI following the 14-d CIDR-PG protocol. insemination (FTAI), insemination is performed for all females at a predetermined time rather than based on detected estrus. Since 2. Materials and methods FTAI does not require estrus detection, protocols that facilitate FTAI significantly reduce the labor and time commitment associated All experimental procedures were approved by the University of with carrying out an AI program. This likely enhances adoption of Missouri Animal Care and Use Committee. AI in the industry, as time and labor considerations are a barrier to adoption of AI [12]. However, there is potential for reduced preg- 2.1. Animals nancy rates when using FTAI compared to AI based on detected estrus, as insemination occurs at a less-than-optimal time point for Individual animal weights and reproductive tract scores (RTS) some females. Pregnancy rate to AI is significantly affected by [28e30] were obtained for beef replacement heifers (14e15 timing of insemination relative to estrus onset and ovulation months of age) at four locations. In each location, heifers with [13e16]. Females failing to express estrus prior to FTAI achieve abnormal or infantile (RTS 1) reproductive tracts were culled and much lower pregnancy rates than those having expressed estrus, not exposed for estrus synchronization and AI (Location 1: n ¼ 3; with a recent meta-analysis [17] finding pregnancy rates were 27% Location 2: n ¼ 5; Location 3: n ¼ 0; Location 4: n ¼ 2). lower for non-estrous beef cows and heifers inseminated with conventional, non-sex-sorted semen. 2.2. Estrus synchronization When using sex-sorted semen produced using the XY technology, pregnancy rates to FTAI were particularly low among beef cows Estrus was synchronized using the 14-d CIDR-PG protocol and heifers that had not expressed estrus prior to FTAI [18e20]. (Fig. 1). Heifers received an Eazi-Breed CIDR insert (1.38 g proges- Overall, lower pregnancy rates were obtained when sex-sorted terone; Zoetis, Madison, NJ) on Day 0, and CIDRs were removed on semen was used in FTAI [18,20e22]. Therefore, it had generally Day 14. Prostaglandin F2a (PG; 25 mg im; Lutalyse, Zoetis, Madison,

GnRH Non-Estrous Heifers

AI AI Estrous Remaining PGF2α Heifers Heifers CIDR … 16 d … … 66 h … … 24 h … 0 14 30 33 34 Treatment days

Fig. 1. Treatment schedule for the 14-d CIDR-PG protocol. Heifers received an Eazi-Breed CIDR insert (1.38 g progesterone; Zoetis, Madison, NJ) on Day 0, and CIDRs were removal on

Day 14. Prostaglandin F2a (PG; 25 mg im; Lutalyse, Zoetis, Madison, NJ) was administered on Day 30, and split-time artiﬁcial insemination (STAI) was performed at 66 h (Day 33) or 90 h (Day 34) after PG administration based on estrous response. Heifers failing to express estrus by 90 h after PG were administered gonadotropin-releasing hormone (GnRH; 100 mg im; Cystorelin, Merial, Athens, GA) concurrent with AI at 90 h. 90 J.M. Thomas et al. / Theriogenology 98 (2017) 88e93

NJ) was administered on Day 30, and split-time artificial insemi- fetal size [31], as heifers were not exposed to natural service bulls nation (STAI) was performed at 66 h (Day 33) or 90 h (Day 34) after until 14 d after AI. In Locations 2e4, fetal sex was determined for PG administration based on estrous response. Heifers failing to pregnancies resulting from AI. express estrus by 90 h after PG were administered gonadotropin- releasing hormone (GnRH; 100 mg im; Cystorelin, Merial, Athens, 2.7. Statistical analysis GA) concurrent with AI. Time of PG administration and time of AI were recorded for each heifer. Treatment differences for RTS, weight, and timing of insemination were analyzed using the TTEST procedure of SAS (SAS Inst. 2.3. Estrus detection Inc., Cary, NC). Chi-square contingency tables (PROC FREQ; SAS Inst. Inc., Cary, NC) were used to analyze treatment differences in estrous Estrus detection aids (Estrotect, Rockway Inc, Spring Valley, WI) response and final pregnancy rates at the end of the breeding were applied at PG administration on Day 30 and evaluated at 66 h season, as well as differences between AI technicians. Pregnancy after PG on Day 33 and at 90 h after AI on Day 34 to determine rate rates to STAI were analyzed using a generalized linear model with of estrous response. Estrus was defined as removal of >50% of the the GLIMMIX procedure of SAS (SAS Inst. Inc., Cary, NC) using the scratch-off coating on the Estrotect. binomial distribution, link logit function. The error term was specified as the treatment location interaction. The following 2.4. Semen collection variables were tested for inclusion in the model: treatment, location, bull, technician, heifer source, heifer RTS, heifer weight, Semen was collected from two commercially available two- estrous response (estrous by 66 h, estrous by 90 h, or non-estrous year-old AI bulls, and units of sex-sorted and conventional semen by 90 h), treatment bull interaction, treatment x estrous were produced from contemporaneous ejaculates. Semen was response interaction, bull x estrous response interaction, and collected over an eight day period (March 18th through March treatment x bull x estrous response interaction. Variables that did 25th, 2016) for Bull A and over a 22 day period (February 15th not significantly (P > 0.10) affect pregnancy rate to STAI were through March 7th, 2016) for Bull B. Semen from three separate removed from the model, and the final model contained treatment ejaculates per bull were used, with two ejaculates used for pro- and estrous response. Least squares means were generated using duction of sex-sorted semen and one for conventional. All the this model and compared using Fisher's least significant difference. semen passed the standard quality control criteria use for sex sorted and conventional semen respectively. Sex sorted semen and 3. Results conventional semen from all ejaculates within bull were used in equal proportions across locations. Units of conventional semen For heifers expressing estrus prior to 66 h after PG, the interval were generated with 25.0 106 live cells per 0.5 ml straw prior to from PG to AI was 66.0 ± 0.1 h for heifers inseminated with con- freezing, based on sperm cell concentrations commonly used in ventional semen and 65.9 ± 0.08 h for heifers inseminated with industry. Units of sex-sorted semen were generated using the sex-sorted semen. For the remaining heifers receiving delayed SexedULTRATM Genesis III sorting technology (Sexing Technologies, insemination at 90 h after PG, the interval from PG to AI was Navasota, TX) with 4.0 106 live cells per 0.25 ml straw prior to 90.0 ± 0.09 h for heifers inseminated with conventional semen and freezing, based on sperm cell concentrations typically used in 90.0 ± 0.09 h for heifers inseminated with sex-sorted semen. Heifer SexedULTRATM 4M sex-sorted semen. Sex-sorted units were sorted weight, RTS, and estrous response rates for each treatment and to contain X chromosome-bearing sperm cells at an accuracy level location are reported in Table 1. The proportion of heifers of >90%, the typical accuracy level at which SexedULTRATM sex- expressing estrus prior to 66 h did not differ between treatments, sorted semen is produced and marketed. nor did the proportion of heifers expressing estrus during the delay period. Heifer weight and RTS did not differ based on treatment. 2.5. Artificial insemination In locations with more than one AI technician (Location 2 and 3), pregnancy rates to STAI did not differ based on technician. Preg- Within each location, heifers were preassigned to balanced nancy rates by location, treatment, and bull are presented in treatments (conventional versus sex-sorted semen) based on Table 2. Across locations, pregnancy rates to AI tended to be higher source, RTS, and weight. Location 1 used Bull A exclusively. Loca- (P ¼ 0.09) for heifers inseminated with conventional semen (60%; tions 2 and 3 used Bulls A and B equally, with heifers preassigned to 257/429) compared to sex-sorted semen (52%; 218/422). Pregnancy bull within treatment based on source, RTS, and weight. In Loca- rates to AI did not differ between Bull A (58%; 282/487) and Bull B tions 1 and 4, AI was performed by a single technician. Three (53%; 193/364), and there was no significant interaction of bull x technicians performed AI in Locations 2, and two technicians per- treatment, bull x estrous response, or bull x treatment x estrous formed AI in Locations 4. To minimize potential for confounding response. Pregnancy rate to AI based on treatment and estrous effects of technician in locations with more than one technician, response are presented in Table 3. Pregnancy rates were greater technicians were preassigned and balanced within treatment and (P < 0.0001) for heifers that expressed estrus prior to AI than for bull on the basis of heifer source, RTS, and weight. Fourteen d after heifers that failed to express estrus prior to AI at 90 h. AI, heifers were exposed to fertile bulls for the remainder of the In Location 1, in which pregnancy diagnosis was performed both breeding season. at Day 62 and Day 120 after AI, one heifer in each treatment had a pregnancy resulting from AI that was not present at Day 120. Due to 2.6. Pregnancy diagnosis this pregnancy loss, these heifers are reported as not having become pregnant to AI in Tables 2 and 3. Total pregnancy rates at Pregnancy rate to AI was determined by transrectal ultraso- the end of the 60 d breeding season did not differ between heifers nography (Aloka 500V equipped with a 5.0-MHz linear-array that received sex-sorted semen at AI (89%; 376/422) and heifers transducer; Aloka, Wallingford, CT) 62 d and 120 d after AI in that received conventional semen at AI (89%; 382/429). For loca- Location 1, 95 d after AI in Location 2, 89 d after AI in Location 3, and tions in which fetal sex was determined at pregnancy diagnosis 88 d after AI in Location 4. Pregnancies resulting from AI were (Locations 2e4), fetal sex was determinable for 389 of the 401 distinguished from those resulting from natural service based on heifers that became pregnant to AI. Of these, heifers inseminated J.M. Thomas et al. / Theriogenology 98 (2017) 88e93 91

Table 1 to mating. Through use of sex-sorted semen in artificial insemi- Heifer weight (Wt), reproductive tract score (RTS), and estrous response by treat- nation programs, beef and dairy producers can produce calves of ment and location. the desired sex at accuracies over 90% [9,32,33]. However, although Treatmenta N Wt, kg RTSb Estrous by 66 hc Total Estrousd the direct cost of sex-sorted semen is fairly moderate (generally $15 Location Proportion % Proportion % - $30 US more per unit than the cost of conventional semen), indirect costs have limited adoption of this technology, particularly in Conventional 429 390 ± 2 4.2 ± 0.1 286/429 67 384/429 90 the beef industry [34]. Indirect costs stem from reduced pregnancy ± ± Location 1 60 358 5 3.6 0.1 41/60 70 52/60 87 rates obtained when using sex-sorted compared to conventional Location 2 212 395 ± 2 4.4 ± 0.1 141/212 67 190/212 90 Location 3 60 380 ± 4 4.1 ± 0.1 50/60 83 58/60 97 semen [23,35]. Using sex-sorted semen produced with the previous Location 4 97 404 ± 3 4.4 ± 0.2 54/97 56 84/97 87 generation technology, prior research has reported pregnancy rates of a factor of 0.75 relative to the pregnancy rate obtained with Sex-sorted 422 391 ± 2 4.3 ± 0.1 259/422 61 373/422 88 conventional semen [3,24]. For example, a producer would have Location 1 61 361 ± 5 3.6 ± 0.1 43/61 70 53/61 87 fi ± ± faced a choice between a 60% rst service pregnancy rate to con- Location 2 209 396 2 4.5 0.1 128/209 61 182/209 87 fi Location 3 57 381 ± 4 4.1 ± 0.1 40/57 70 53/57 93 ventional semen and a 45% rst service pregnancy rate to sex- Location 4 95 405 ± 3 4.4 ± 0.1 48/95 51 85/95 89 sorted semen. Low first service pregnancy rates following the 14-

a Heifers received an Eazi-Breed CIDR insert (1.38 g progesterone; Zoetis, Madi- d CIDR-PG protocol would increase days-on-feed in dairy heifer son, NJ) on Day 0, and CIDRs were removal on Day 14. Prostaglandin F2a (PG; 25 mg development operations [36], but cost considerations are more im; Lutalyse, Zoetis, Madison, NJ) was administered on Day 30, and split-time complex for beef producers given the seasonal constraints of beef artificial insemination (STAI) was performed at 66 h (Day 33) or 90 h (Day 34) af- production systems. Early conception significantly increases ter PG administration based on estrous response. Heifers failing to express estrus by average age of calves at weaning and therefore pounds of calf 90 h after PG were administered gonadotropin-releasing hormone (GnRH; 100 mg im; Cystorelin, Merial, Athens, GA) concurrent with AI at 90 h. Heifers in the con- weaned [37], and age at conception also has implications for life- ventional treatment were inseminated with conventional, non-sex-sorted semen, time productivity of beef heifers [38,39]. Therefore, the indirect and heifers in the sex-sorted treatment were inseminated with sex-sorted semen. cost of low first service AI pregnancy rates is generally quite high in b e ¼ ¼ RTS (1 5 scale, where 1 immature and 5 luteal phase) of the heifers, the beef cow-calf operations. evaluated at CIDR insertion on Day 0. Likewise, lower pregnancy rates to sex-sorted semen limit the c Estrous response by 66 h after PG administration, as determined by activation of an estrus detection aid (Estrotect). degree to which progeny sex ratio can be effectively skewed. For d Total estrous response by 90 h after PG administration, as determined by acti- example, inseminating 100 heifers with conventional semen at a vation of an estrus detection aid (Estrotect). 60% pregnancy rate would result in 30 pregnancies of the favored sex, while a 45% pregnancy rate to sex-sorted semen would result in 41 pregnancies of the desired sex (assuming a 50:50 and 92:8 sex with sex-sorted semen carried 96% (171/178) female calves, ratio for conventional and sex-sorted semen respectively). While a whereas heifers inseminated with conventional semen carried 49% modest increase in the number of desired sex progeny, the relative (104/211) female calves. value of desired sex progeny has not offset the costs in many previous efforts [19,23,34,35]. Moreover, previous attempts to use sex- 4. Discussion sorted semen in FTAI treatments have generally resulted in low pregnancy rates relative to conventional semen [18,20e23]. Generally, one sex of progeny resulting from a planned mating Increased adoption of advanced reproductive technologies in the has higher value than the alternative sex, though the sex desired beef industry is likely contingent upon the development of timed and its relative value differ from operation to operation and mating protocols that do not require the labor and time commitment of estrus detection [12]. To be economically viable for widespread adoption in the beef industry, therefore, sex-sorted semen must Table 2 achieve pregnancy rates comparable to conventional semen and a b Pregnancy rates to split-time AI (STAI) by location, treatment , and bull. must be suitable for use in timed AI protocols. TM Location Treatment Bull A Bull B Overall The results presented here suggest that SexedULTRA sex-

Proportion % Proportion % Proportion % sorted semen can be used effectively in timed AI of beef heifers following the 14-d CIDR-PG protocol with STAI. It is noteworthy Location 1 Conventional 37/60 62 37/60 62 that the mean pregnancy rates to AI with SexedULTRATM semen are Sex-sorted 37/61 61 37/61 61 Location 2 Conventional 65/107 61 63/105 60 128/212 60 effectively a factor of 0.86 relative to conventional semen in this Sex-sorted 54/104 52 54/105 51 108/209 52 dataset (52% versus 60%). While a much larger dataset would be Location 3 Conventional 17/29 59 19/31 61 36/60 60 required to truly evaluate relative equivalence, these results sup- Sex-sorted 19/29 66 14/28 50 33/57 58 port previous suggestions of improved fertility using the Sex- Location 4 Conventional 32/49 65 24/48 50 56/97 58 TM Sex-sorted 21/48 44 19/47 40 40/95 42 edULTRA process compared to the previous generation XY Total Conventional 151/245 62 106/184 58 257/429 60a technology for sex-sorting of sperm cells (reviewed by Vishwanath Sex-sorted 131/242 54 87/180 48 218/422 52b [9]). Moreover, the present results detail effective use of SexedUL- TM abOverall pregnancy rates to AI tended to be higher (P ¼ 0.09) among heifers TRA sex-sorted semen in a timed AI approach, which is of sig- inseminated with conventional compared to sex-sorted semen. niﬁcant interest to the beef industry. a Pregnancy rate to AI determined by transrectal ultrasonography 62 de95 d after Compared to FTAI, STAI enhances use of sex-sorted and con- STAI. ventional semen for several reasons. When using STAI, timing of b Heifers received an Eazi-Breed CIDR insert (1.38 g progesterone; Zoetis, Madi- insemination is more optimally aligned with ovulation among son, NJ) on Day 0, and CIDRs were removal on Day 14. Prostaglandin F2a (PG; 25 mg im; Lutalyse, Zoetis, Madison, NJ) was administered on Day 30, and split-time heifers that have not expressed estrus prior to the standard time of artiﬁcial insemination (STAI) was performed at 66 h (Day 33) or 90 h (Day 34) af- FTAI, 66 h after PG [20,25]. Timing of insemination relative to ter PG administration based on estrous response. Heifers failing to express estrus by ovulation is critical when using frozen-thawed semen that may not 90 h after PG were administered gonadotropin-releasing hormone (GnRH; 100 mg retain fertility for long timespans in the female reproductive tract, im; Cystorelin, Merial, Athens, GA) concurrent with AI at 90 h. Heifers in the conventional treatment were inseminated with conventional, non-sex-sorted semen, such as semen from bulls with lower fertility [40]. Efforts evalu- and heifers in the sex-sorted treatment were inseminated with sex-sorted semen. ating timing of AI with sex-sorted semen have suggested that AI 92 J.M. Thomas et al. / Theriogenology 98 (2017) 88e93

Table 3 Pregnancy ratesa to split-time artiﬁcial insemination (STAI) based on treatmentb and estrous response during STAI.

Treatmentb Overall Estrous by 66 hc Estrous 66 h - 90 hd Non-Estrouse

Proportion % Proportion % Proportion % Proportion %

Conventional 257/429 60a 177/286 62 67/98 68 13/45 29 Bull A 151/245 62 99/161 61 43/57 75 9/27 33 Bull B 106/184 58 78/125 62 24/41 59 4/18 22 Sex-sorted 218/422 52b 136/259 53 68/114 60 14/49 29 Bull A 131/242 55 81/150 54 38/61 62 12/31 39 Bull B 87/180 48 55/109 50 30/53 57 2/18 11 abOverall pregnancy rates to AI tended to be higher (P ¼ 0.09) among heifers inseminated with conventional compared to sex-sorted semen. a Pregnancy rate to AI determined by transrectal ultrasonography 41 de95 d after STAI. b Heifers received an Eazi-Breed CIDR insert (1.38 g progesterone; Zoetis, Madison, NJ) on Day 0, and CIDRs were removal on Day 14. Prostaglandin F2a (PG; 25 mg im; Lutalyse, Zoetis, Madison, NJ) was administered on Day 30, and split-time artificial insemination (STAI) was performed at 66 h (Day 33) or 90 h (Day 34) after PG administration based on estrous response. Heifers failing to express estrus by 90 h after PG were administered gonadotropin-releasing hormone (GnRH; 100 mg im; Cystorelin, Merial, Athens, GA) concurrent with AI at 90 h. Heifers in the conventional treatment were inseminated with conventional, non-sex-sorted semen, and heifers in the sex-sorted treatment were inseminated with sex-sorted semen. c Expression of estrus by 66 h after PG administration, as determined by activation of an estrus detection aid (Estrotect). Insemination was performed at 66 h after PG administration. d Expression of estrus between 66 h and 90 h after PG administration, as determined by lack of activation of an estrus detection aid at 66 h but activation at90h. Insemination was performed at 90 h after PG administration. e Failure to express estrus by 90 h after PG, as determined by lack of activation of an estrus detection aid both at 90 h after PG administration. should take place at a time point close to ovulation for optimal when performing split-time AI, numerical differences among non- pregnancy rates [41,42]. estrous heifers with respect to bull should be interpreted The STAI approach used in this study (described by Bishop et al. cautiously. [27]) also decreases the reliance on GnRH administration for in- Lastly, these results do not provide insight into the relative duction of ovulation, instead allowing for maximum estrus performance of SexedULTRATM sex-sorted semen versus conven- expression. Across treatments, 64% (545/851) of heifers expressed tional semen in the context of FTAI. Due to higher estrous response estrus prior to 66 h after PG, the recommended time for FTAI rates prior to AI, higher pregnancy rates result from STAI compared following the 14-d CIDR-PG protocol. Due to the delay of insemi- to FTAI, even when AI is performed with conventional semen that nation when using STAI, non-estrous heifers at this time are has not undergone sorting [20,25e27]. Maximum estrous response afforded an additional 24 h to express estrus prior to AI. This prior to AI is likely even more critical when using sex-sorted semen dataset illustrates the resulting improvement in total estrous due to the reduced number of sperm cells per unit and the potential response, as 69% (212/306) of heifers that had failed to express for reduced post-thaw longevity of sorted sperm. Therefore, STAI estrus by 66 h expressed estrus prior to insemination at 90 h, for a provides an effective platform for use of sex-sorted semen in timed total estrous response of 89% (757/851). The estradiol production AI, as considerations related to non-estrous females are mitigated associated with estrus coordinates several physiological processes by high estrous response rates. critical for establishment and maintenance of pregnancy, including effects on follicular cells, the oocyte, gamete transport, and prep- Acknowledgements aration of the uterine environment [43]. Consequently, estrus expression is associated with higher pregnancy rates to timed AI The Missouri Agricultural Experiment Station (Columbia, MO) [17]. Whereas administration of GnRH to non-estrous females at contributed. The authors gratefully acknowledge Sexing Technol- FTAI likely limits subsequent estradiol production via down- ogies (Navasota, TX) for providing semen and funding for this regulation of aromatase [44], both AI and GnRH administration may project; Zoetis (Madison, NJ) for providing the Lutalyse sterile so- be delayed 20e24 h for non-estrous heifers when using STAI. As a lution and EAZI-Breed CIDR cattle inserts; Merial (Athens, GA) for result, the total proportion of animals having expressed estrus prior providing the Cystorelin; Estrotect Inc. (Spring Valley, WI) for to AI is increased in STAI [27], and this results in corresponding providing estrus detection aids; and the Mason-Knox Ranch increases in pregnancy rate compared to FTAI [26]. (Frankfort, SD), D&R Ogren Farm (Langford, SD), and University of Based on bull-to-bull variation in pregnancy rates to sex-sorted Idaho Nancy M. Cummings Research Extension and Education semen, it had been suggested that the previous generation XY Center (Carmen, ID) for use of the animals in this project. sorting procedure detrimentally affected fertility of some bulls more than others [45]. It is unclear at this time whether the newer References SexedULTRATM sorting procedure detrimentally affects fertility in a bull dependent manner or to the same degree for a respective bull. 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