C.J. Mortensen

The WNT signaling antagonist Dickkopf-1 directs lineage commitment and promotes survival of the preimplantation embryo

Successful embryonic development is dependent on factors secreted by the reproductive tract. Dickkopf‐1 (DKK1), an antagonist of the wingless‐related mouse mammary tumor virus (WNT) signaling pathway, is one endometrial secretory protein potentially involved in maternal‐embryo communication. The purpose of this study was to investigate the roles of DKK1 in embryo cell fate decisions and competence to establish pregnancy. Using in vitro‐produced bovine embryos, we demonstrate that exposure of embryos to DKK1 during the period of morula to blastocyst transition (between d 5 and 8 of development) promotes the first 2 cell fate decisions leading to increased differentiation of cells toward the trophectoderm and hypoblast lineages compared with that for control embryos treated with vehicle. Moreover, treatment of embryos with DKK1 or colony‐stimulating factor 2 (CSF2; an endometrial cytokine known to improve embryo development and pregnancy establishment) between d 5 and 7 of development improves embryo survival after transfer to recipients. Pregnancy success at d 32 of gestation was 27% for cows receiving control embryos treated with vehicle, 41% for cows receiving embryos treated with DKK1, and 39% for cows receiving embryos treated with CSF2. These novel findings represent the first evidence of a role for maternally derived WNT regulators during this period and could lead to improvements in assisted reproductive technologies.—Denicol, A. C., Block, J., Kelley, D. E., Pohler, K. G., Dobbs, K. B., Mortensen, C. J., Ortega, M. S., Hansen, P. J. The WNT signaling antagonist Dickkopf‐1 directs lineage commitment and promotes survival of the preim‐plantation embryo. FASEB J. 28, 3975‐3986 (2014). www.fasebj.org

The flipped classroom stimulates greater learning and is a modern 21st century approach to teaching today's undergraduates

Many classrooms in higher education still rely on a transformative approach to teaching where students attend lectures and earn course grades through examination. In the modern age, traditional lectures are argued by some as obsolete and do not address the learning needs of today’s students. An emerging pedagogical approach is the concept of the flipped classroom. The flipped classroom can simply be described as students viewing asynchronous video lectures on their own and then engaging in active learning during scheduled class times. In this study, we examined the flipped classroom teaching environment on student learning gains in an Introduction to Equine Science course. Students (n = 130) were asked to view 7.5 h of recorded lectures divided into 8 learning modules, take online quizzes to enforce lecture viewing, take 3 in-class exams, and prepare to participate in active learning during scheduled class times. Active learning approaches included individual activities, paired activities, informal small groups, and large group activities. When compared to students in the traditional lecture format in earlier years, students in the flipped format scored higher on all 3 exams (P < 0.05), with both formats taught by the same instructor. Analysis of ACT scores demonstrated no intellectual capacity differences between the student populations. To evaluate any gains in critical thinking, flipped format students were asked to take the Cornell Critical Thinking Exam (version X). Scores improved from the pretest (50.8 ± 0.57) to the posttest (54.4 ± 0.58; P < 0.01). In the flipped course, no correlations were found with student performance and interactions with online content. Students were asked in class to evaluate their experiences based on a 5-point Likert scale: 1 (strongly disagree) to 5 (strongly agree). The flipped classroom was ranked as an enjoyable learning experience with a mean of 4.4 ± 0.10, while students responded positively to other pointed questions. In formal course evaluations, flipped format students ranked the following higher (P < 0.05): instructor availability to assist students; encouragement of independent, creative, and critical thinking; and amount learned. Overall, the flipped classroom proved to be a positive learning experience for students. As the classroom continues to modernize, pedagogical approaches such as the flipped classroom should be considered for many lecture-style courses taught in the animal sciences.

Exercise affects both ovarian follicular dynamics and hormone concentrations in mares

The objectives were to evaluate the effects of exercise on ovarian folliculogenesis and related hormones in mares. Mares (n = 11) were randomly assigned into a control (non-exercised) or treatment (exercised) group. Treatment mares (n = 5) were moderately exercised for 30 min, 6 d/wk. All mares underwent daily transrectal ultrasonographic examinations and ovarian follicles > 6 mm were measured. Blood samples were collected during the first (Cycle 1) and last (Cycle 4) cycle, and serum concentrations of cortisol, LH, and FSH were determined. Mean cortisol concentrations were elevated (P < 0.05) in exercised mares, 6.29 ± 0.22 compared with 5.62 ± 0.16 ng/dL (mean ± SEM), 30 min post exercise. There were no significant differences between groups in mean FSH concentrations; however, exercised mares had lower (17.3 ± 6.4 vs 41.1 ± 5.5 ng/mL; P < 0.05) peak LH concentrations. Furthermore, exercised mares experienced a longer (24.7 ± 0.8 vs 22.2 ± 0.8 d; P < 0.05) mean interovulatory interval for all cycles combined, fewer (P < 0.05) follicles 6 to 20 mm in diameter, and an increased (P < 0.05) number of follicles >20 mm following deviation. The dominant and largest subordinate follicle in exercised mares had a greater (P < 0.05) mean diameter on the day of deviation, suggesting delayed deviation. Exercised mares also tended (P = 0.06) to have an increased number of cycles with at least two dominant follicles compared to control (62 vs 36%, respectively), indicating a decreased ability of the largest follicle to assert dominance. Under the conditions of this study, moderately exercising mares induced higher cortisol concentrations, lowered peak LH concentrations, and altered ovarian follicular dynamics.

Impact of moderate exercise on ovarian blood flow and early embryonic outcomes in mares

The advent of embryo transfer has allowed horses to continue to train and compete during the breeding season. However, the associated stress of exercise may be detrimental to reproduction. The objectives of this study were to evaluate differing exercise protocols on reproductive blood flow and embryonic outcomes in mares. Light-horse mares were randomized into control (n = 4), partial-exercised (n = 6), and full-exercised (n = 6) groups. Partial-exercised mares were moderately exercised 30 min daily during the periovulatory period and rested after ovulation for 7 d. Full-exercised mares were exercised for 30 min daily throughout the reproductive cycle. Mares were artificially inseminated during estrus and subjected to uterine flush for embryo recovery on d 7 post ovulation. Blood flow through both ovarian arteries and vascular perfusion of the wall of the preovulatory follicle were examined by color Doppler ultrasonography. Results indicated exercise induced greater serum cortisol concentrations (P < 0.05). Embryo recovery rates were reduced in exercised (20/46, 43%) compared with control (14/21, 67%) mares (P < 0.10). When examined separately, embryo recovery rates for partial-exercised (11/25, 44%) and full-exercised (9/21, 43%) mares were not significantly different. Additionally, fewer quality Grade 1 embryos were recovered from partial-exercised mares compared with both control and full-exercised mares (P < 0.05). Blood flow through both ovarian arteries was greater in both exercised groups in the days leading up to ovulation (P < 0.05). However, vascular perfusion of the wall of the preovulatory follicle on the day before ovulation was less in both partial-exercised (45.9 ± 3.0%) and full-exercised (44.8 ± 3.4%) mares vs. control (54.9 ± 3.6%; P < 0.05). In exercised mares, vascular perfusion of the follicle wall was greater when an embryo was recovered (P < 0.01). No differences were found in follicle ovulatory diameter among exercised and non-exercised mares. When groups were combined, follicle diameter was greater when an embryo was recovered (44.9 ± 1.0 mm) compared with an unsuccessful embryo recovery attempt (42.8 ± 0.7 mm; P < 0.05). In conclusion, these data demonstrated that exercise increased ovarian arterial blood flow leading up to ovulation and decreased vascular perfusion of the wall of the preovulatory follicle. Mares given rest the day after ovulation up until an embryo collection attempt did not improve embryo recovery rates.

Oral L-arginine supplementation impacts several reproductive parameters during the postpartum period in mares.

L-arginine is an amino acid which can alter pituitary function and increase blood flow to the reproductive tract. The objective was to determine the effect of supplementing 100g of L-arginine on plasma arginine concentrations, follicular dynamics and ovarian and uterine artery blood flow during the estrus that occurs subsequent to foaling. In Experiment 1, mares were fed 100g L-arginine for 1 day during the last 3 weeks of pregnancy and plasma samples taken for every hour for the first 4h and every other hour until 12h.L-arginine supplementation elevated plasma arginine concentrations from 1 to 8h post feeding; arginine peaked at 6h (arginine: 515±33μmol/L; control: 80±33μmol/L). In Experiment 2, mares received either 100g L-arginine or control diets beginning 21 d before the expected foaling date and continued for 30 d postpartum. The reproductive tract was evaluated by transrectal Doppler ultrasonography from Day 1 postpartum through Day 30. There were no differences in ovarian follicular dynamics, ovarian or uterine resistance indices between groups. Vascular perfusion of the F1 follicular wall was greater in L-arginine supplemented mares (37.3±2.6%) than controls (25.4±2.7%; P<0.05). L-arginine supplemented mares had a smaller uterine body and horns and accumulated less uterine fluid than controls (P<0.05). The combination of reducing uterine fluid accumulation, while not altering follicular development, raises the possible use of L-arginine supplementation as a breeding management tool during the postpartum period to increase reproductive success.

Influence of L-arginine supplementation on reproductive blood flow and embryo recovery rates in mares

Supplementation with L-arginine can increase uterine arterial blood flow and vascular perfusion of the preovulatory follicle in mares. Increased vascular perfusion of the preovulatory follicle has been correlated with successful pregnancy in mares. The objective of this study was to determine if supplemental L-arginine would increase ovarian arterial blood flow, vascular perfusion of the preovulatory follicle, and embryo recovery rates in mares. Mares were blocked by age and breed and assigned at random within block to L-arginine supplementation or control groups. Mares were fed L-arginine beginning 17 days before and through the duration of the study. Transrectal Doppler ultrasonography was used to measure ovarian arterial blood flow and vascular perfusion of the preovulatory follicle daily when it reached 35 mm and subsequent CL on Days 2, 4, and 6. Mares, on achieving a follicle of 35 mm or more were bred via artificial insemination and an embryo collection was attempted 7 days after ovulation. Treatment did not affect interovulatory interval (arginine-treated, 18.1 ± 2.6 days; control, 20.7 ± 2.3 days) or embryo recovery rate (arginine-treated, 54%; control, 48%). Mares treated with l-arginine had a larger follicle for the 10 days preceding ovulation than control mares (30.4 ± 1.2 and 26.3 ± 1.3 mm, respectively; P < 0.05) and vascular perfusion of the dominant follicle tended (P = 0.10) to be greater for the 4 days before ovulation. No differences were observed between groups in diameter or vascular perfusion of the CL. Resistance indices, normalized to ovulation, were not significantly different between groups during the follicular or luteal phase. Oral l-arginine supplementation increased the size and tended to increase perfusion of the follicle 1, but had no effect on luteal perfusion or embryo recovery rates in mares.

Orally supplemented l-arginine impairs amino acid absorption depending on dose in horses1

The beneficial effect of L-arginine (L-Arg) supplementation, on the physiology of several species, has generated an interest in the use of L-Arg as a nutraceutical in horses, but dosage and absorption of orally supplemented L-Arg must be inferred from other species. The study objective was to determine the effect of 2 oral L-Arg doses on plasma arginine concentrations and the effect on absorption of other amino acids in mares. In Experiment 1, mares were blocked by age and breed and were fed L-Arg supplemented (supplemented with 0.025% BW L-Arg; n=6) or control (no supplement; n=6) concentrate on a single day with blood samples taken at 0, 0.5, 1, 2, 3, 4, and 5 h relative to feeding. In Experiment 2, mares (n=6) were used in a 3×3 Latin square design with L-Arg (0.0125% of BW), urea (0.0087% of BW), and control (no supplement) fed mixed into a grain concentrate as single meal with blood samples taken at 0, 1,2, 4, 6, 8,10, and 12 h relative to feeding. In Experiment 1, L-Arg supplementation increased (P<0.05) plasma L-Arg and ornthine concentrations and decreased (P<0.05) lysine and methionine concentrations compared with the control group. At 1 h post feeding, L-Arg mares had lower (P<0.05) plasma concentrations of histidine, glutamic acid, proline, isoleucine, threonine, phenylalanine, leucine, valine, alanine, and taurine. In Experiment 2, L-Arg supplementation increased (P<0.05) arginine and ornithine concentrations compared with urea and control; there was no difference among other amino acids. These experiments indicate that L-Argis absorbed and, dependent on the dose, alters the absorption of other amino acids in mares.

l-Arginine supplementation 0.5% of diet during the last 90 days of gestation and 14 days postpartum reduced uterine fluid accumulation in the broodmare

L-Arginine is an essential amino acid in many species that has been shown to influence reproduction. However, in horses a dose of 1% L-arginine of total dietary intake impaired absorption of other amino acids, whereas a dose of 0.5% did not. The objectives of this experiment were to evaluate postpartum parameters on mares supplemented with 0.5% L-arginine through the last 90d of gestation and 14d postpartum. Sixteen light-horse mares were randomly divided in two groups: 8 mares supplemented with 0.5% L-arginine and 8 mares fed an isonitrogenous equivalent. Gestation length, days to uterine clearance and days to first ovulation were compared. Uterine body depth, diameter of uterine horns, and length of largest pocket of uterine fluid were recorded daily via transrectal ultrasound. Measurements of foal weight, height, and cannon bone circumference were recorded for 9 weeks. Arginine treatment had no effect on gestation length (P=0.58). Supplemented mares cleared fluid quicker postpartum (6.8±0.53d; P=0.026) compared to control (9.0±0.38d). Mares supplemented with L-arginine had smaller diameter of fluid present in the postpartum uterus (P≤0.05). Days to first postpartum ovulation were not affected by treatment nor any influence on uterine involution. Finally, treatment had no effect on any foals measured parameters. L-Arginine supplementation fed at 0.5% of daily intake during the last 90d of gestation and early postpartum in mares decreased uterine fluid accumulation, yet did not appear to have any effect on any other parameters measured.

Using Doppler ultrasonography on day 34 of pregnancy to predict pregnancy loss in lactating dairy cattle

The objective of this experiment was to determine whether uterine or ovarian vascular dynamics could be used to identify cows at risk for pregnancy loss. Our hypothesis was that cows that subsequently lose their pregnancy will have decreased corpus luteal (CL) perfusion, or an increased resistance index (RI; reduced blood flow), or both, at d 34 of pregnancy. Day 34 was chosen because it is a common time for dairy cattle to be checked for pregnancy. This experiment was performed in 2 replicates from November 2011 to April 2012 (n = 69) and from November 2012 to April 2013 (n = 53). Cows were bred via timed artificial insemination using Ovsynch-56 and checked for pregnancy on d 32 after artificial insemination. At d 34, cows confirmed pregnant were examined via transrectal Doppler ultrasonography. Blood samples collected via coccygeal vein were used to measure circulating plasma progesterone concentrations. Diameter of the corpus luteum and crown-rump length were measured. Color power Doppler ultrasonography was used to determine vascular perfusion to the CL, and RI was measured for the uterine arteries just after branching from the umbilical artery. Records were later examined to identify pregnancy status of cows after reconfirmation. Abortion rate did not differ between replicates (11.6% in replicate 1, 9.4% in replicate 2). Mean crown-rump length of embryos that were carried to term was greater on d 34 than that in cows that aborted (14.23 ± 0.27 vs. 13.21 ± 0.53 mm). Circulating progesterone concentration at d 34 was greater for cows that carried pregnancies to term than for those that aborted (9.1 ± 0.7 vs. 7.5 ± 1.0 ng/mL). The final logistic regression model consisted of crown-rump length, progesterone concentration, and RI of the uterine artery contralateral to pregnancy. Decreased crown-rump length and progesterone concentration tended to be associated with increased odds ratio for pregnancy loss, whereas CL perfusion and uterine blood flow were not associated with increased odds ratio of pregnancy loss. In conclusion, examining CL perfusion and RI of the uterine arteries on d 34 of pregnancy does not offer a method to identify lactating Dairy cattle at risk for pregnancy loss after d 34.

Identifying the optimal course delivery platform in an undergraduate animal behavior research course1

Abstract There is a critical need to evaluate pedagogical delivery platforms best suited for undergraduates in the information age. Our goal was to identify the optimum course delivery platform for a basic research course based on student performance and critical thinking scores. Students were expected to plan, conduct, and report on an animal behavior research project of their own design. The course was taught in three different formats: traditional, online, and flipped, over 2 yr by the same instructor at both the University of Florida and Santa Fe College. Student assessments included weekly quizzes, assignments, a written report, a poster presentation, and attendance. We conducted pre- and postassessments using the Cornell Critical Thinking Test (CCTT). Students’ grades differed depending on format delivery and were also different between University of Florida and Santa Fe College students. For students at the University of Florida, quiz grades and poster grades did not differ (P < 0.50) between formats. However, assignment grades (P = 0.04) and report grades (P < 0.001) differed by format and were higher in the flipped and online-only version of the course, compared with traditional. For students at the Santa Fe College, quiz grades (P = 0.71) did not differ, but assignment (P < 0.001), report (P = 0.003), and poster (P < 0.001) grades were higher in the flipped and traditional format of the course. Within the flipped format at the University of Florida, student CCTT scores increased (P < 0.001) between pre- and posttest, whereas the scores within the other formats did not differ. When we compared the magnitude of change between pre- and posttest scores across formats, students at the University of Florida in the flipped format tended (P = 0.060) to have a greater gain than students in the online format. For students at Santa Fe College, there was no difference between pre- and posttest CCTT scores for any format, and the magnitude of change in scores did not differ between formats. Overall, our results suggest that teaching format influences student grades and critical thinking scores. Different effects were seen in different student populations; however, positive effects of the flipped format on student grades were seen at both institutions. In conclusion, flipped format courses may improve learning and critical thinking in an early research-based course.