Procedures to maintain viability of mammalian gametes and embryos in vitro, including cryopreservation, have been exceedingly valuable for my research over the past 55 years. Keeping sperm viable in vitro enables artificial insemination, which, when combined with selective breeding, often is the most effective approach to making rapid genetic change in a population. Superovulation and embryo transfer constitute a parallel approach for amplifying reproduction of female mammals. More recent developments include sexing of semen, in vitro fertilization, cloning by nuclear transfer, and genetic modification of germline cells, tools that are enabled by artificial insemination and/or embryo transfer for implementation. I have been fortunate in being able to contribute to the development of many of the above techniques, and to use them for research and applications for improving animal agriculture. Others have built on this work to circumvent human infertility, assist reproduction of companion animals, and rescue endangered species. It also has been a privilege to teach, mentor, and be mentored in this area. Resulting worldwide friendships have enriched me personally and professionally.

This study was conducted to determine effects of pre-synchronization of ovulation timing among heifers and delayed fixed-time artificial insemination (TAI) with sex-sorted semen on proportion of heifers pregnant after TAI (PR/AI). Heifers were assigned to one of eight treatments: 1 and 2), 7-d CO-Synch + CIDR treatment regimen with administration of gonadotropin-releasing hormone and a CIDR insert on Day 0, prostaglandin F2a (PGF) at CIDR removal on Day 7, and TAI occurring 54 h later with conventionally processed (CTRL54-CNV) or sex-sorted semen (CTRL54-SEX); 3 and 4), same as CTRL54 but TAI delayed to 72 h with conventionally processed (CTRL72-CNV) or sex-sorted semen (CTRL72-SEX); 5 and 6), same as CTRL54 but additional administration of PGF on Day -7 and TAI with conventionally processed (PRE54-CNV) or sex-sorted semen (PRE54-SEX); 7 and 8), same as PRE54 treatments but TAI delayed to 72 h with conventionally processed (PRE72-CNV) or sex-sorted semen (PRE72-SEX). Proportion of heifers pregnant after TAI was greater (P = 0.02) with conventionally processed semen compared with sex-sorted semen, yet PR/AI did not differ (P = 0.14) between heifers in PRE72-CNV and PRE72-SEX groups. There were greater PR/AI in the PRE72-SEX (P = 0.03) than CTRL54-SEX group (46.1 % and 36.9 %) and there was no difference (P = 0.31) in PR/AI between CTRL54-CNV and PRE72-SEX groups (50.4 % and 46.1 %). In conclusion, pre-synchronization of ovulation timing among heifers combined with delayed TAI resulted in increased PR/AI with sex-sorted semen compared with the 7-d CO-Synch+CIDR treatment regimen.

To determine effects of delaying the injection of prostaglandin F2α (PGF) and fixed-time artificial insemination (TAI) in the 14-d CIDR-PG protocol, 1,049 Angus heifers at six locations were enrolled in a completely randomized design. Within location heifers were randomly assigned to one of two treatment groups: 1) PG16 (n = 518), heifers received a controlled internal drug release (CIDR) insert on d 0 for 14 d, a 25-mg injection of PGF 16 d after CIDR removal (d 30), and a 100-µg injection of gonadotropin-releasing hormone concurrent with TAI 66 ± 2 h later; or 2) PG17 (n = 531), heifers were treated the same as PG16, however, PGF was administered 17 d after CIDR removal (d 31), and heifers were TAI 66 ± 2 h later. Estrus detection patches were applied to a subset (n = 482) of heifers at the time of PGF administration and were examined for activation at TAI. Dominant follicle diameter was determined via transrectal ultrasonography at PGF administration and TAI in a subset of heifers (n = 116). Transrectal ultrasonography was performed to determine pregnancy rates to TAI (PR/AI) between 30 and 45 d after TAI. Estrus expression prior to TAI differed by treatment where PG17 heifers had greater (P < 0.01) expression of estrus than PG16 heifers (57.8 ± 6.1% vs. 43.4 ± 6.1%, respectively). Nevertheless, dominant follicle diameters at PGF and at TAI were similar (P ≥ 0.59) between PG16 and PG17 heifers. In addition, PR/AI did not differ (P = 0.29) between PG16 and PG17 treatments (50.5 ± 3.2% vs. 45.7 ± 3.1%, respectively). Results of this experiment indicate that delaying the injection of PGF and TAI in the 14-d CIDR-PG protocol increased estrus expression prior to TAI yet did not improve fertility in beef heifers.

For most traits in farm animals, the environment and chance influence phenotype much more than genetics. However, genetic tools including genetic modification (GM) are extremely powerful approaches to changing average phenotypes of populations. In nearly all cases, adding a new genotype to a population via conventional or GM approaches will increase biodiversity initially. The ultimate effect of the new genotype on the biodiversity of the general population will depend on how the new genotype is used in breeding programs. A special case, often termed gene editing, is using GM approaches to move genotypes within a species, e.g., from beef to dairy cattle. The end product will be identical whether produced conventionally or via GM, so the only real difference is that the GM approach will be more rapid, in some cases producing the change a decade sooner. To date, there are no GM farm animals outside of laboratory settings, university farms, etc., so at present, there has been no practical effect on biodiversity or ecosystems. How GM farm animals are used in the future will determine whether resulting biodiversity will increase, decrease, or stay about the same.

The overall aim of this research was to improve fertility of cattle inseminated with sexed spermatozoa by improving sperm sorting procedures. Six field trials were conducted in which 4,264 heifers were inseminated into the uterine body with cryopreserved sexed or unsexed control spermatozoa. Pregnancy or calving rates with doses of 2 x 10(6) sexed spermatozoa ranged from 32 to 51%; these averaged 69% of the pregnancy rates with 20 x 10(6) unsexed, control spermatozoa (range 53 to 79% of controls). Fertility of sexed spermatozoa was especially low on farms where control fertility was low. Accuracy of sexing ranged from 86 to 91%. Laser power of 150 mW for interrogating spermatozoa did not result in lower pregnancy rates (43%) than when power was decreased as much as possible for a particular sorting batch (50 to 130 mW) to still achieve sexing accuracy (38% pregnant). Addition of catalase to fluids containing spermatozoa was beneficial when thawed spermatozoa were incubated in vitro for 2 h but had no effect on pregnancy rates. There also was no effect on pregnancy rates between two concentrations of Hoechst 33342 for staining spermatozoa. Freezing 2 x 10(6) sexed spermatozoa at 20 x 10(6)/ml resulted in a slightly higher rate of pregnancy (P < 0.05) than at 10 x 10(6)/ml. The information obtained in these trials, along with other improvements, notably lowering pressure in the sorting system from 50 to 40 psi, has been used to improve procedures for sexing spermatozoa commercially.

Successful programs to manipulate estrus and ovulation to maximize pregnancy out­comes in suckled beef cattle have been developed to limit animal handling and to elimi­nate the need to detect estrus, thereby providing more opportunity to incorporate AI to start the breeding season. The most successful and consistent synchronization scheme employs an intravaginal progesterone insert (controlled internal drug release, CIDR) in place for 7 d concurrent with GnRH treatment, and, upon removal of the insert, injection of PGF2α, followed by timed AI at 60 to 66 h concurrent with a second dose of GnRH (CO-synch + CIDR).

To determine the effects of administration of 25 mg of PGF2α 7 d prior to the initiation of the 7-d CO-Synch + controlled internal drug release (CIDR) fixed-time AI (TAI) protocol, 985 Bos taurus beef heifers were enrolled in a completely randomized design at 9 locations from April to July of 2016. Within location, all heifers were randomly assigned to 1 of 2 treatments: 1) CONTROL (n = 496); 100 µg injection of GnRH and a CIDR insert for 7 d [day 7], administration of 25 mg of PGF2α at CIDR removal [day 0], followed by a second injection of GnRH and TAI 54 ± 2 h later; or 2) PRESYNCH (n = 489); same as CONTROL but heifers received an additional injection of 25 mg of PGF2α 7 d prior [day 14] to CIDR insertion. Estrous detection patches were applied to all heifers on day 14 and were evaluated for estrual activity on day 7. Similarly, estrus alert patches were placed on all heifers on day 0 and evaluated for estrual activity at the time of TAI. Pregnancy was diagnosed via transrectal ultrasonography between 35 and 55 d after TAI. The percentage of heifers exhibiting estrus between days 14 and 7 was greater (P < 0.001) for the PRESYNCH (70.1 ± 2.4%) than the CONTROL (41.1 ± 2.3%) treatment, whereas the percentage of heifers exhibiting estrus between day 0 and TAI was greater (P < 0.001) for the CONTROL (55.6 ± 2.4%) than the PRESYNCH (39.7 ± 2.5%) treatment. Estrus response rates differed (P < 0.001) among locations. Pregnancy rates to TAI differed (P = 0.023) among locations; however, they did not differ (P = 0.739) between CONTROL and PRESYNCH treatments (45.4 ± 2.5 vs. 43.2 ± 2.5%, respectively). Final breeding season pregnancy rates did not differ (P = 0.811) between treatments. Therefore, an injection of PGF2α 7 d prior to initiation of the 7-d CO-Synch + CIDR protocol failed to improve pregnancy rates to TAI in replacement beef heifers.

Increasing production costs prompted efforts to improve beef production efficiency. The All Heifer, No Cow (AHNC) production system involves insemination of nulliparous heifers with sexed semen to produce primarily female calves that are early weaned 3 months after parturition. Dams are finished on a high concentrate diet (105 ± 12d) and harvested before 30 months of age. Every animal in the enterprise is growing, which eliminates mature cow maintenance costs and improves efficiency. The objective was to assess AHNC production parameters. In years 1 (n = 53) and 2 (n = 57) Angus-based heifers were purchased and managed under the AHNC system. Female calves started in years 1 and 2, plus purchased replacements, became dams in years 3 (n = 53) and 4 (n = 56), respectively. Pregnancy rate at 30 d post fixed time AI with sexed semen was 46.4 ± 4.8 %, and 92.3 ± 1.1 % were pregnant after a second round of AI and bull exposure. Using AHNC, 67.0 ± 15.8 % of females replaced themselves with a heifer. The remaining 33.0 % were purchased from outside the system. During finishing, ADG was 1.8 ± 0.8 kg•d-1 and DMI was 15 ± 3.5 kg•d-1. Dressing percent was 60.0 ± 1.9 %. The USDA quality grading system classified 28.4 % of the carcasses (n = 176) as C-maturity. However, lean maturity (A00 = 100, B00 = 200, etc.) and marbling score (slight00 = 300, small00 = 400, etc.) for A and B versus C maturity were 165 ± 24 and 165 ± 26 (P = 0.83) and 456 ± 93 and 456 ± 76 (P = 0.78), respectively. In conclusion, AHNC produced quality carcasses. Calving at a younger age, starting the finishing phase earlier, and innovative marketing strategies may help reduce incidence of C-maturity carcasses.

Confounding interests between biological and economic efficiencies of the All Heifer, No Cow (AHNC) beef production system makes system evaluation difficult. In a conventional cow/calf system, emphasis is placed on reproduction, longevity, and maternal behavior with the goal of achieving a cow that successfully breeds back and consistently weans a healthy calf each year. With AHNC, calving ease and carcass quality are emphasized. In the AHNC system, yearling heifers are inseminated with sexed semen to produce primarily females; calves are weaned 3 months after parturition; heifers are finished on a high concentrate diet; and are harvested before 30 months of age. By eliminating the mature cowherd, every animal in the enterprise is growing, offering improved biological efficiency. However, the reduction in feed use does not necessarily equate with economic efficiency. The carcasses produced are high marbling, but payment does not always reflect quality due to premature bone ossification caused by increased hormone concentrations during pregnancy. A model was developed using system dynamics methodology to simulate an AHNC production system. The objective was to increase understanding of interactions and trade-offs between biological and economic efficiencies in the AHNC system. Biological efficiency was considered as lifetime kg of TDN (input) per kg of hot carcass weight produced (output). Profitability was evaluated using net income. The model was calibrated using data from the period 2013 to 2017 collected from the management of an AHNC herd. Efficiency was evaluated by changing the age at harvest and adjusting diet type. Profit leverage points were identified by changing selected variables ±10 % from the observed values while all other variables were held constant. Net income was sensitive to the percent female calves born; a greater number of females coincide with greater profitability. Results suggested AHNC could be managed to balance biological and economic efficiency.

To determine the effects of administration of PGF2α (25 mg of dinoprost tromethamine/mL, Lutalyse; Zoetis Animal Health) 7 days prior to the initiation of the 7-day CO-Synch + CIDR fixed-time AI (TAI) protocol, 985 Bos taurus beef heifers were enrolled at 9 locations from April 1 to July 15. Within location, all heifers were randomly assigned to one of two treatments: 1) CONTROL (n = 496); 100 µg injection of GnRH (Factrel; gonadorelin hydrochloride; Zoetis Animal Health) and a controlled internal drug releasing (EAZI-BREED CIDR; 1.38 g of progesterone; Zoetis Animal Health) insert for 7 d [d -7], administration of 25 mg of PGF2α (Lutalyse, dinoprost tromethamine; Zoetis Animal Health) at CIDR removal [d 0], followed by a second injection of GnRH and fixed-time AI (TAI) 54 ± 2 h later; or 2) PRESYNCH (n = 489); same as CONTROL but heifers received an additional injection of 25 mg of PGF2α 7 d prior [d -14] to CIDR insertion. Estrotect (Rockway Inc., Spring Valley, WI) estrus detection patches were applied to all heifers on d -14 and were evaluated for estrual activity on d -7. Similarly, estrus alert patches were placed on all heifers on d 0 and evaluated for estrual activity at the time of TAI. Pregnancy was diagnosed via transrectal ultrasonography between 35 and 55 d after TAI to determine the presence of a viable embryo, thereby assessing AI pregnancy rates The percentage of heifers exhibiting estrus between d -14 and d -7 was greater (P < 0.001) for the PRESYNCH (70.3%) than the CONTROL (40.0%) treatment, whereas the percentage of heifers exhibiting estrus between d 0 and TAI was significantly (P < 0.001) greater for the CONTROL (57.9%) than the PRESYNCH (41.4%) treatment. Estrus response rates differed (P < 0.001) among locations. Pregnancy rates to TAI differed (P = 0.023) among locations, however, did not differ (P =0.739) between CONTROL and PRESYNCH treatments (46.7 vs 45.5%, respectively). Final breeding season pregnancy rates did not differ (P = 0.811) between treatments. Therefore, an injection of PGF2α 7 days prior to initiation of the 7-day CO-Synch + CIDR protocol failed to improve pregnancy rates to TAI in replacement beef heifers.

Our objective was to determine which of 2 split-time AI programs applied to suckled beef cows would result in greater pregnancy risk. Suckled beef cows (n = 1,062) at 12 locations in 4 states (CO, KS, MY, and WA) were enrolled. Cows were treated on d -7 with a progesterone insert concurrent with 100 µg GnRH and on d 0 with 25 mg PGF plus removal of the insert. Estrus-detection patches were affixed to cows at insert removal. The study was designed as a completely randomized experiment of 2 treatment combinations. Within location and balanced for parity (primiparous vs. multiparous), cows were assigned randomly to 2 treatment times (55 vs. 65 h after CIDR insert removal) at which time estrus-detection patches were assessed. Estrus was defined to have occurred when an estrus-detection patch was > 50% colored (activated). Cows determined to be in estrus were inseminated at either 55 or 65 h, whereas the residual nonestrous cows in both treatment times received GnRH at 55 or 65 h but were inseminated 20 h later at 75 or 85 h, respectively. Pregnancy outcomes were determined at 36 d after AI and at the end of the breeding season. Thus, pregnancy outcomes of interest were compared between the 55 + 75-h treatment combination and that of the 65+85-h combination. Expression of estrus was greater ( = 0.001) by 65 h after PGF than by 55 h (62.0% vs. 41.9%), respectively, and this proportion was influenced by parity (time x parity interaction; = 0.006). As a result, proportionally more ( < 0.001) cows received the timed AI at 75 than 85 h (59.4% vs. 40.6%). Similar proportions of cows not in estrus by 55 or 65 h were detected in estrus by 75 or 85 h (40.1% vs. 39.3%), respectively. The cumulative proportion of cows in estrus by 75 h was less ( < 0.001) than that by 85 h (66.7% vs. 76.7%), respectively. Pregnancy risks at 36 d differed among treatments, with cows detected in estrus and inseminated at 55 or 65 h having greater pregnancy risks than their time-inseminated herd mates at 75 or 85 h (62.3% vs.49.7%), respectively. Overall pregnancy risk for cows in the 65+85-h treatment combination was greater at 36 d than for cows in the 55 + 75-h treatment combination (61.0% vs. 51.4%), respectively. We conclude that the 65 + 85-h treatment combination produced more pregnancies than the 55 + 75-h combination, but its implementation may be somewhat less convenient in terms of cow handling times.

The single-calf heifer model (SCHM) harvests females after early-weaning their first calf, reducing average age and maintenance requirements of the herd, hence increasing biological efficiency of beef production. However, pregnancy estrogens accelerate bone ossification, which might affect carcass value of SCHM females. This study evaluated: overall maturity (OM), bone maturity (BM), lean maturity (LE), marbling (MA), Warner-Bratzler (WBSF) and slice shear force (SSF), and cooking loss (CL) of carcasses of SCHM females. Fifty-three Angus-based yearling heifers (BW = 353 ± 38.8 kg) and a second set of 58 (BW = 307 ± 29.9 kg), were synchronized and inseminated with sexed semen during first and second year of the project, respectively, to calve at approximately 24 mo of age. At weaning, average age of calves was 106 ± 22 and 120 ± 21 d, and first-calf heifers (43 each year) were fed for 88 and 90 d at a feedlot, for years 1 and 2, respectively. At harvest, carcasses were scored for LE, BM, and MA (slight = 300, small = 400, and modest = 500); OM was estimated from BM and LE (A00, B00and C00 maturities corresponded to scores of 100, 200, and 300, respectively). One LM sample was removed for SSF, WBSF and CL measurements. Carcasses were sorted by OM as < 300 or ≥ 300, and the resulting means for carcass traits were compared with a t test. Data were combined across years, since same significant differences (P < 0.05) between OM groups were obtained for both years. Means ± SD for the 66% of the carcasses classified as < 300 OM were: 192 ± 39.3 OM, 211 ± 53 BM, 165 ± 29 LE, 446 ± 84 MA, 25.4 ± 8.6 kg SSF, 4.94 ± 1.19 kg WBSF, and 25.4 ± 4.1% CL. Remaining carcasses ( ≥ 300 OM) were 305 ± 18 OM, 346 ± 46 BM, 167 ± 27 LE, 462 ± 78 MA, 27.6 ± 9.1 kg SSF, 4.96 ± 0.84 kg WBSF, and 26.1 ± 4.2% CL. Significant differences between the 2 OM groups were found for BM (P < 0.001). However, no differences were detected for LE (P = 0.81), MA (P = 0.39), WBSF (P = 0.96), SSF (P = 0.29) or CL (P = 0.47). Therefore, differences in OM and BM did not affect palatability characteristics of carcasses of primiparous SCHM females approximately 30 mo of age.

We hypothesized that GnRH would increase pregnancy risk (PR) in a split-time AI program for cows in which estrus was not detected. A total of 1,236 suckled beef cows at 12 locations in 3 states (Colorado, Kansas, and North Dakota) were enrolled. Before applying the fixed-time AI program, BCS was assessed. Cows were treated on d -7 with a progesterone insert concurrent with 100 μg GnRH and on d 0 with 25 mg PGF plus removal of the insert. Estrus-detection patches were affixed to cows at insert removal. Estrus was defined to have occurred when an estrus-detection patch was >50% colored (activated). Cows in estrus by 65 h ( = 758; 61.3% of all cows) were randomly allocated to 2 treatments: 1) 100 μg GnRH and early + GnRH (E+G; = 373) or 2) AI only at 65 h (early - no GnRH [E-G]; = 385). The remaining cows were randomly allocated to 2 treatments: 1) 5(L+G; = 252) or 2) AI only at 84 h (late no GnRH [L-G]; = 226). Pregnancy was determined 35 d after AI via transrectal ultrasound. Pregnancy risk did not differ ( = 0.68) between E+G and E-G cows (61.9 vs. 60.4%, respectively). Conversely, for cows inseminated at 84 h, PR was greater ( = 0.01) in cows that received GnRH (L+G) compared with their herd mates not receiving GnRH (L- G; 41.7 vs. 30.8%, respectively). Of those cows not detected in estrus by 65 h, 42.1% were detected by 84 h, for a total expression of estrus by all cows of 77.6%. Administration of GnRH increased ( < 0.01) PR in cows not detected in estrus by 84 h (+GnRH = 33.4% [ = 146] vs. no GnRH = 15.0% [ = 128]) but had no effect in cows expressing estrus by 84 h (+GnRH = 65.3% [ = 103] vs. no GnRH = 61.7% [ = 97]). Neither estrus expression by 65 or 84 h nor PR was influenced by BCS, parity, or days postpartum at AI. Cows had greater PR when they had been detected in estrus before AI, and PR was improved by administration of GnRH at 65 h after insert removal in cows that were not detected in estrus and inseminated at 84 h.

Principles for selecting future research projects include interests of investigators, fundability, potential applications, ethical considerations, being able to formulate testable hypotheses and choosing the best models, including selection of the most appropriate species. The following 10 areas of assisted reproduction seem especially appropriate for further research: efficacious capacitation of bovine spermatozoa in vitro; improved in vitro bovine oocyte maturation; decreasing variability and increasing efficacy of bovine superovulation; improved fertility of sexed semen; improving equine IVF; improving cryopreservation of rooster spermatozoa; understanding differences between males in success of sperm cryopreservation and reasons for success in competitive fertilisation; mechanisms of reprogramming somatic cell nuclei after nuclear transfer; regulation of differentiation of ovarian primordial follicles; and means by which spermatozoa maintain fertility during storage in the epididymis. Issues are species specific for several of these topics, in most cases because the biology is species specific.

We hypothesized GnRH would induce ovulation in a split-time AI program by increasing pregnancy risk (PR) when estrus was not detected. A total of 1236 suckled beef cows at 12 locations in 3 states (CO, KS, and ND) were enrolled. Before applying the fixed-time AI program, BCS was assessed. Cows were treated on d −7 with a CIDR insert concurrent with 100 µg GnRH and on d 0 with 25 mg PGF2α plus removal of the insert. Estrotect patches were affixed to cows at CIDR insert removal. Estrus was defined to have occurred when an estrus-detection patch was > 50% colored (activated). Cows in estrus by 65 h (n = 758; 61.3% of all cows) were allocated randomly to 2 treatments: 1) GnRH and early AI at 65 h (E+G; n = 373), or 2) AI only at 65 h (E–G; n = 385). Remaining cows were allocated randomly to 2 treatments: 1) GnRH injection at 65 h and late AI at 84 h (L+G; n = 252), or 2) AI only at 84 h (L–G; n = 226). Pregnancy was determined 35 d after AI via transrectal ultrasound. Pregnancy risk did not differ (P = 0.68) between E+G and E–G cows (61.9 vs. 60.4%), respectively. Conversely, for cows inseminated at 84 h, PR was greater (P = 0.01) in cows that received GnRH at 65 h compared with their herd mates not receiving GnRH (41.7 vs. 30.8%), respectively. Of those cows not in estrus by 65 h, 57.7% displayed estrus by 84 h for a total expression of estrus by all cows of 77.6%. Pregnancy risk was greater (P < 0.01) in cows not detected in estrus by 84 h when treated with GnRH at 65 h compared with no GnRH (+G = 33.4% [n = 146] vs.–G = 15.0% [n = 128]), whereas no difference in PR was detected for cows detected in estrus (+G = 65.3% [n = 103] vs.–G = 61.7% [n = 97]). Neither estrus expression by 65 or 84 h nor pregnancy risk was influenced by BCS, parity, or days postpartum at AI. Cows had greater PR when they displayed estrus before AI and cows that did not display estrus by 65 h benefited from an injection of GnRH at 65 h before insemination occurred at 84 h.