B.L. Bradbury, R.C. Vann, L.C. Mapel, A.W. Lewis, T.H. Welsh Jr., R.D. Randel

Temperamental cattle have greater serum concentrations of cortisol (CS) which mediates glucose metabolism. The objective was to determine the effects of temperament on blood glucose (G) and insulin (I) following a stressor and a subsequent glucose challenge. Angus crossbred heifers (200-300 kg) were evaluated for temperament and 6 calm (C) and 6 temperamental (T) heifers were fitted with jugular catheters and placed in individual stalls. Blood was collected at cannulation and then via a cannula at 0, 30, 60, and 90 min. Following 90 min dextrose was infused via the cannula at 0.5 mg/kg BW. Blood samples were collected at -5, 0, 10, 15, 20, 30, 40, 60, 80, 100, 120, 140, 160, 180 min after the challenge. CS and I were assayed by RIA and G by colorimetry. CS, G, I, insulinogenic index and their interactions with time and temperament were analyzed by GLM for repeated measures. Peak I concentration, time to peak, G disappearance and time to half life were analyzed using GLM procedures. During cannulation there was a significant effect of temperament on G (P=.0517) and I (P=.0496), but not for CS (P > .05). There was no temperament by time interaction influencing G, I, or CS during cannulation. During the challenge temperament by time interactions affected CS (P=.0295), G (P=.0004), and I (P=.011). Glucose concentrations were significantly higher in T heifers (P=.0485) at half life (P=.0485), but time to half life was similar (P=.7789) between temperaments. Peak I concentrations (mIU/mL) for the C and T heifers were 27.52 ±12.96 and 62.54±12.96 respectively. Insulinogenic index was not affected by temperament nor was there a temperament by time interaction. These data indicate that temperament has an impact on CS secretion following cannulation stress which subsequently results in elevated G and I concentrations. Temperament clearly modifies metabolic regulatory responses to a metabolic challenge in heifers.

J.E. Larson, J.D. Davis, K.G. Gebremedhin, C.N Lee

Heat stress is a major inhibitor of production in livestock operations, causing severe economic loss. The objective of this study was to determine whether spraying the udder with water, with or without fans blowing air onto the udder, cools the body as effectively as spraying water on the back of the animal with or without fans blowing air on the back. Twelve pregnant, lactating Holstein cows were used over 4 d with 4 applications of each treatment each d. Treatments included wetting of the back with a fan (B+F, n = 24), and without a fan (B+NF, n = 72) blowing air on the back; wetting of the udder, with a fan (U+F, n = 24) or without a fan (U+NF, n = 72) blowing air on the udder. The back or udder of each animal was sprayed with water for 1 min, and in appropriate treatment groups, air from identical fans was blown on the wetted area for the duration of the treatment and measurement time periods. Rectal temperature, respiration rate, and surface skin temperature of the back and udder were collected 10 min after treatment application. Data were analyzed using the Mixed Procedure of SAS with the replication during each day analyzed as a repeated measurement and Black globe humidity index (BGHI) used as a covariate. Mean BGHI and Temperature Humidity Index for the period were 80.3 ± 0.3 and 81 ± 0.3, respectively. Rectal temperatures and respiration rates were not different (P > 0.05) among treatments. The LSMeans and SEM of rectal temperatures were 39.8 ± 0.1, 39.6 ± 0.2, 39.6 ± 0.1, and 39.6 ± 0.2°C and the respiration rates were 110.6 ± 1.6, 106.1 ± 2.9, 109.0 ± 2.9 and 109.0 ± 1.6 breaths/min for cows treated with B+NF, B+F, U+NF, and U+F treatments, respectively. Skin surface temperatures of the back were similar among treatments. Interestingly, cows that received B+F had a cooler udder surface temperature (38.1 ± 0.3°C; P ≤ 0.05) compared to all other treatments: 38.5 ± 0.2, 38.7 ± 0.3, and 38.5 ± 0.2°C for cows receiving U+NF, U+F, and B+NF, respectively. In conclusion, efforts to abate heat stress by spraying the udder with water either with or without a fan is as effective as spraying the back with water.

K.E. Pfeiffer, J.A. Binversie, J.D. Rhinehart, and J.E. Larson

The objective of this study was to evaluate the biostimulatory effect of bull exposure on the expression of estrus and pregnancy rate to AI in cattle. Beef heifers (n=86) and cows (n=193) during two consecutive yr were allocated to one of three treatments: 1) no bull exposure (CON; n=95), 2) exposure to a bull with a surgically deviated penis for 21 d prior to AI (SB; n=88), or 3) exposure to a vasectomized bull for 21 d prior to AI (VB; n=96). The SB treatment provided the physical presence of the bull but prevented intromission whereas the VB treatment allowed for intromission and deposition of seminal plasma but not spermatozoa. The estrous cycles of all females were synchronized using the Hybrid-Synch+CIDR protocol (GnRH+CIDR insertion-7 d-CIDR removal+PGF2α, visual detection of estrus 3 × daily with AI 12 h later for 82 h, and clean-up TAI+GnRH at 82 h). Blood samples were collected on d -17 and -7 relative to the initial injection of GnRH and analyzed for concentrations of progesterone to determine cyclicity status at the initiation of the experiment (at least one sample > 1 ng/mL). Pregnancy was detected by transrectal ultrasonography on d 35 post-AI. At the onset of the experiment, 75.7% of heifers and 86.1% of cows were cycling. The percentages of females that displayed estrus after CIDR removal were increased (P<0.001) in Year 1 (52.3%) compared to Year 2 (23.1%) as well as increased (P<0.05) in nulliparous (52.3%) compared to primiparous and multiparous females (26.0 and 31.5%, respectively). The percentages of females that displayed estrus were similar (P=0.15) among treatments (31.6, 39.8, and 39.6% for CON, SB, and VB, respectively). Pregnancy rates were increased (P<0.01) in Year 2 (55.8%) compared to Year 1 (42.4%) and were increased (P<0.05) in females treated with CON and SB (49.5 and 59.1%, respectively) compared to females treated with VB (40.6%). In conclusion, a similar percentage of females among treatments displayed estrus during the 82 h detection period but pregnancy rates were decreased in females exposed to a vasectomized bull compared to those exposed to either no bull or a bull presence only.

J.A. Binversie, K.E. Pfeiffer, J.E. Larson

The objectives of this study were to determine whether conception, ovulation or presynchronization rates were altered, or follicle and CL characteristics were altered after modifying the Double-Ovsynch (DO) protocol to include human chorionic gonadotropin (hCG) compared to the DO protocol. Holstein (n=146) and Jersey (n=37) cows were blocked by parity and randomly assigned to one of two treatments. Cows received either an injection of 100 µg of GnRH (n=91) or 2000 IU of hCG (n=92) at the initiation of the Pre-Ovsynch (PO) portion of the DO protocol (PO: GnRH/hCG-7d-PGF_2α-3d-GnRH). After 7 d, cows started the Breeding-Ovsynch (BO) portion of the DO protocol (BO: GnRH-7d-PGF_2α-56h-GnRH-16h-TAI with sex-sorted semen). Transrectal ultrasonography and blood samples were used to assess ovarian structures, ovulation, pregnancy diagnosis (at d 32 post TAI), and circulating concentrations of progesterone (P4). Conception rates were similar for cows treated with GnRH or hCG (32.2 and 25.0%; P>0.1). Ovulation rates at the onset of PO were increased in cows treated with hCG compared to GnRH (77.2 and 62.2%; P<0.05). Concentrations of P4 7 d post hCG/GnRH treatment for cows that ovulated were greater in cows treated with hCG compared to those treated with GnRH (LSMeans ± SEM; 5.1±0.3 and 3.8±0.4 ng/ml; P<0.05). The size of the largest follicle 7 d post hCG/GnRH treatment for cows that had ovulated was smaller in cows treated with hCG compared to cows treated with GnRH (12.4±0.5 and 13.8±0.6 mm; P<0.05). Luteal regression (P4<1.0 ng/ml) from the injection of PGF_2α of PO did not differ between GnRH and hCG treated cows (67.0 and 60.9%; P>0.1). Although more cows ovulated to hCG, a greater proportion of these cows tended to fail to have undergone luteolysis by 3 d post PGF_2α compared to cows that had ovulated to GnRH (29.6 and 16.1%; P=0.09). Therefore, the overall percentage of cows which were synchronized to the PO did not differ between GnRH and hCG treated cows (61.5 and 52.2%; P>0.1). In conclusion, no improvement was achieved by replacing the first injection of GnRH in the DO protocol with hCG.

J.D. Rhinehart, A.M. Arnett, L.H. Anderson, W.D. Whittier, J.E. Larson, W.R. Burris, J.B. Elmore, D.T. Dean, and J.M. DeJarnette

Cryopreserved bovine sperm that have been sex-sorted by flow cytometry have been documented to achieve conception rates of 70 to 90% of that obtained using unsorted conventional semen in dairy heifers and cows. Limited trials have investigated conception rates of this product in commercial beef herds. Though dairy and beef heifers have similar fertility, dairy cows are considered to be less reproductively efficient compared to beef cows. Two trials were conducted to investigate fertility differences of sex-sorted and conventional semen when used in beef cows and heifers. For both trials, beef cows and heifers were synchronized by a modified 5-day CO-Synch+CIDR protocol. Females received an injection of GnRH and Controlled Internal Drug Release device on D 0. On D 5, the CIDR was removed and an injection of dinoprost was administered. A second dinoprost injection followed 12 h later. For trial 1, females were inseminated with either sex-sorted (SS) or conventional (CON) semen 12 h after the first display of standing estrus until 80 h after the initial dinoprost injection, at which time all remaining females were mass inseminated and a GnRH injection was administered. For trial 2, females were mass inseminated with either SS or CON semen at an average of 80 h after the first dinoprost injection. For trial 1, conception rate of both cows and heifers bred to observed estrus was lower (P < 0.05) to SS semen (30%, 31/103 and 30%, 16/53; respectively) than CON semen (62%, 61/98 and 68%, 38/56; respectively). Conception rate to CON and SS semen was similar between cows and heifers. For trial 2, conception rate to CON semen was lower (P < 0.05) for heifers (31%, 39/124) than cows (43%, 169/369). Conception rate to SS semen was similar for cows and heifers (33%, 78/240 and 36%, 24/66; respectively). In trial 2 conception rates for both cows and heifers were similar between CON and SS. In conclusion, results from trial 1 indicated an overall decreased fertility to SS semen while results from trial 2 did not. Relatively low conception to CON semen in trial 2 suggests that lower overall fertility might not be exacerbated by SS semen use in beef cows and heifers.

A.N. Loyd, D.G. Riley, D.A. Neuendorff, A.W. Lewis, R.C. Vann, T.H. Welsh Jr., and R.D. Randel

Brahman (n=771) and F₁ Brahman x Hereford (n=56) calves born over 9 years (2002 to 2010) from 33 sires and 355 dams at the Texas AgriLife Research Center in Overton were utilized to evaluate the genetic and non-genetic factors influencing calf temperament. Calves were evaluated for pen score (PS), exit velocity (EV) and temperament score (TS) 28 d prior to weaning, at weaning, 28 d post-weaning, 56 d post-weaning, and as yearlings. Mixed models were used to determine non-genetic factors influencing PS, EV and TS at each sampling day and also as repeated records analyses. Contemporary group (calves of the same sex and weaned together) and dam age group were fixed effects, calf age at weaning and proportion Brahman were linear covariates, and sire and calf nested within sire were random effects. Exit velocity, PS and TS differed across contemporary groups (P< 0.05) and by day (P< 0.0001) but not by proportion Brahman (P = 0.63). Dam age group was significant for PS (P< 0.03) but not for TS (P = 0.07) or EV (P 0.17). Solutions for calf age at weaning were 0.0048, 0.0033 and 0.0040 for PS, EV and TS, respectively (P< 0.02; average SE = 0.001). Table 1 presents means for temperament traits by day. Animal models were subsequently used to estimate heritability of PS, EV and TS at weaning: 0.48, 0.29 and 0.43, respectively (average SE = 0.08). From repeated records analyses, the proportion of phenotypic variance due to permanent environmental effects were 0.31, 0.43 and 0.25 (average SE = 0.02) and heritability estimates were 0.44, 0.28 and 0.41 (average SE = 0.07) for PS, EV and TS, respectively. These results suggest temperament is moderately to highly heritable and is influenced by contemporary group, dam age, calf age at weaning, and permanent environmental effects.

J.M. Greene, C.W. Dunnaway, S.D. Bowers, B. Grillis, B.J. Rude, J.M. Feugang, P.L. Ryan

Arginine is a conditionally essential amino acid for mature mammals, yet it is considered to be essential for young, developing mammals. Regarded as one of the most versatile amino acids, arginine serves as a precursor for many molecules and has been reported to improve the reproductive performance of rats and pigs. While the full mechanism is still unclear, it has been proposed that arginine may improve reproductive performance by altering angiogenesis during pregnancy. To this end, we sought to determine if the beneficial effects of arginine are associated with an alteration of fetoplacental vascular endothelial growth factor receptor-2 (Vegfr2) translational activity. Homozygous FVB/N – Tg(Vegfr2-luc) – Xen (Vegfr2-luc) males, possessing a transgene comprised of a cloned murine VEGFR2 promoter upstream to firefly luciferase gene, were purchased from Caliper Life Sciences, Inc. (Hopkinton, MA, USA). Eighteen wild-type FVB/N females were bred to Vegfr2-luc males so that only the fetoplacental tissues would possess the luciferase transgene. Once bred, females received either a control diet (Con), the control diet supplemented with 2.0% (wt:wt) L-arginine (+Arg), or the control diet supplemented with 4.1% (wt:wt) alanine (+Ala) to serve as an isonitrogenous control for the +Arg diet (n = 6 per diet). Body weights and total feed intake were recorded during gestation. Pregnant mice were imaged daily from d12 to d18 of gestation to detect luciferase activity as an indicator of Vegfr2 translational activity using the IVIS 100 imaging system (Caliper Life Sciences, Inc.). Number of pups and litter weight were recorded at birth, and the number of placental attachments was determined three weeks postpartum. Data were analyzed using ANOVA. Means were separated using Fisher's LSD and were considered to be significantly different at a value of P < 0.05 and tended to differ at a value of P < 0.10. Total feed intake did not differ (P = 0.78); however, animals receiving the +Arg diet consumed more arginine (P < 0.05). Arginine supplementation increased weight gain during the latter third of gestation (d 12 to d 18), total litter size, number of pups born alive, number of placental attachments sites, litter birth weight, and litter weight of pups born alive (P < 0.05). Conversely, individual birth weights of pups born alive was less (P < 0.05) in pups born to arginine supplemented dams. There were no diet by day interactions (P > 0.10) for fetoplacental Vegfr2 gene activity; however, arginine supplementation tended (P = 0.07) to increase the average total fetoplacental Vegfr2 gene activity, and when comparing only the animals consuming the two isonitrogenous diets, arginine supplementation increased (P = 0.04) the average total Vegfr2 gene activity in fetoplacental tissues. When the data was corrected for fetoplacental mass, the average fetoplacental Vegfr2 gene activity did not differ (P = 0.62); however, arginine supplementation did produce an earlier rise in fetoplacental Vegfr2 gene activity when the data was separated by treatment group. These results further demonstrate the beneficial effect that arginine supplementation has on mammalian reproduction and illustrate that arginine supplementation causes an early rise in fetoplacental Vegfr2 translational activity during the latter third of gestation. This suggests that arginine supplementation may enhance mammalian reproduction by influencing fetoplacental angiogenesis.

J.M. Greene, C.W. Dunnaway, S.D. Bowers, B.J. Rude, P.L. Ryan

Background: Vascular endothelial growth factor-2 (VEGFR2) plays a pivotal role in angiogenesis by eliciting vascular endothelial cell growth when bound to VEGF, a powerful pro-angiogenic ligand. While VEGF and VEGFR2 are expressed throughout gestation, the latter third of gestation in mice is especially characterized by a marked increase in fetoplacental angiogenesis. Accordingly, the objective of this study was to determine the feasibility of monitoring fetoplacental VEGFR2 gene activity non-invasively using a Vegfr2-luc transgenic mouse and bioluminescent imaging.
Methods: Imaging parameters were optimized using two wild-type (WT) females, bearing Vegfr2-luc fetuses. Once imaging parameters were established, six WT females, bred to Vegfr2-luc males, were imaged from d12 to d18 of gestation to determine the usefulness of the Vegfr2-luc mouse as model for studying fetoplacental VEGFR2 activity during pregnancy. Additionally, resultant neonates were imaged at week one, two, and three post-partum to monitor Vegf2-luc expression during post-natal development.
Results: Fetoplacental VEGFR2 expression was detected as light emissions beginning on d12 of gestation and increased throughout the imaging period. A decline in fetoplacental Vegfr2-luc expression was associated with a poor pregnancy outcome in one pregnancy, indicating that this approach has potential use in monitoring pregnancy well being. Additionally, neonatal Vegfr2-luc expression was detected at d7, d14 and d21 post-partum but declined with time.
Conclusion: The results indicate that fetoplacental and neonatal Vegfr2-luc gene expression can be monitored in utero using bioluminescent imaging, allowing fetoplacental and neonatal angiogenic activity to be monitored quatitatively and longitudinally. Accordingly, this approach allows the potential to evaluate certain pro-and anti-angiogenic factors encountered in utero and how they might impact neonatal angiogenesis within the same animal.

P.L. Ryan, D.L. Christiansen, R.M. Hopper, F.K. Walters, K. Moulton, J. Curbelo, J.M. Greene, S.T. Willard

Uterine and placental infections are the leading cause of abortion, still birth and preterm delivery in the mare. While uterine and placental infections in women have been studied extensively, a comprehensive examination of the pathogenic processes leading to this unsatisfactory pregnancy outcome in the mare has yet to be explored. Most information in the literature relating to late term pregnancy loss in mares is based on retrospective studies of clinical cases submitted for necropsy. Here we report the development and application of a novel approach, using transgenically modified bacteria transformed with lux genes of Xenorhabadus luminescens or Photorhabdus luminescences origin and biophotonic imaging, being utilized to better understand pathogen-induced preterm birth in late-term pregnant mares. This technology employs highly sensitive bioluminescence imaging camera systems to localize and monitor pathogen progression during tissue invasion by measuring the bioluminescent signatures emitted by the lux-modified pathogens. This method has an important advantage in that it allows for the potential tracking of pathogens in vivo in real time and over time hitherto not possible to do so. While the application of this technology is in its infancy in domestic animal models, investigators were successful in identifying the fetal lungs, sinuses, nares, urinary and gastrointestinal systems as primary tissues for pathogen invasion following experimental infection of pregnant mares with lux-modified Escherichia coli. Importantly, pathogens were not detected in other vital organs such as the liver, brain and cardiac system. Such precision in localizing sites of pathogen invasion provides potential application for this novel imaging technology coupled with lux-modified organisms may aid in the development of more targeted therapeutic interventions for pathogen-related diseases in the equine and other domestic species.

H.L. Sánchez-Rodríguez, R.C. Vann, E. Baravik-Munsell, S.T. Willard, P.L. Ryan

B-mode and Duplex Doppler ultrasound were used to determine the effects of acetylsalicylic acid (ASA) on uterine arteries diameter, external iliac artery Resistance Index (RI), and subsequent pregnancy rate in open, cycling Angus crossbred cows [4.27 ± 1.22 yr old; 568.76 ± 46.56 kg BW (mean ± SD)]. Acetylsalicylic acid (2,500 mg, ASA; n = 19) was administered twice daily in the feed from d -9.5 to 45 (d 0 = AI date). Control cows (CN; n = 19) received 5 g/d of dry molasses in flakes (placebo) in the feed during the same period. Dimensions of both uterine arteries were recorded once daily during d -10.5, -2.5, 0, 3, 6, 10, 16, 20, 25, 32 in a subsample of 16 cows (8/treatment). Pregnancies were confirmed at d 45 post AI. Jugular vein blood samples were collected after each ultrasound sampling for serum progesterone, and plasma prostaglandin F_2α and prostaglandin E₂ analyses. No difference (P = 0.31) in the diameter was observed between right and left side uterine arteries within a cow. In general, cows receiving ASA had larger diameter of uterine arteries than cows receiving the placebo (4.67 ± 0.04 and 4.54 ± 0.04 mm, respectively; P = 0.01). Uterine arterial diameters were larger in the ASA than in CN cows during sampling d -2.5 (4.61 ± 0.12 vs. 4.23 ± 0.12 mm; P = 0.03) and d 0 (4.52 ± 0.12 vs. 4.10 ± 0.10 mm; P = 0.04). During sampling d 10, ASA cows tend to have larger uterine arterial diameter values than did CN cows (4.86 ± 0.18 and 4.41 ± 0.16 mm, respectively; P = 0.06). Acetylsalicylic acid-treated cows achieved a pregnancy rate (P = 0.18) of 73.7% (14/19) in comparison with 52.6% (10/19) for CN cows. In a subsample of 12 cows (6/treatment), the RI in the right external iliac artery was recorded. The RI values were higher (P = 0.04) in the ASA-treated compared with the CN cows (0.78 ± 0.02 and 0.72 ± 0.02, respectively).These preliminary findings demonstrate that treatment with acetylsalicylic acid improves uterine arterial blood perfusion in beef cows and thus, may be an economical means of enhancing reproductive efficiency in postpartum cows.

H.L. Sánchez-Rodríguez, R.C. Vann, E. Baravik-Munsell, S.T. Willard, P.L. Ryan

The relationship between temperament, blood flow and dimension of the external jugular vein, and body temperature was assessed in Angus crossbred calves [223.4 ± 33.2 kg BW; 262.72 ± 24.94 d old (mean ± SD)] during December, 2010. An average between exit velocity and pen score was used to classify the calves according to their temperament (calm, intermediate, and temperamental). Calves (n = 91) were weighed and the hair of the neck over the jugular vein was clipped. Pulsatility Index (PI) and area of the lumen of the external jugular vein were measured via Duplex Doppler and B mode ultrasound, respectively. The luminal area of the jugular vein was standardized by body weight. Rectal and superficial temperatures of the neck region (over the hair and over the skin) were also recorded. Blood samples were collected for future plasma cortisol analysis. There was a tendency for higher PI values (P = 0.09) in temperamental than in calm calves [1.78 ± 0.16, 1.87 ± 0.15, and 2.28 ± 0.18 for the calm (n = 31), intermediate (n = 32), and temperamental (n = 28) groups, respectively]. Luminal areas of the jugular vein were not affected by temperament (P = 0.65; 0.254 ± 0.016, 0.242 ± 0.015, and 0.263 ± 0.018 mm 2/kg of BW for the calm, intermediate, and temperamental groups, respectively). Rectal temperatures were greatest (P = 0.01) in temperamental than in calm and intermediate calves (39.38 ± 0.13, 38.92 ± 0.12, and 38.90 ± 0.11°C, respectively). There was no effect of temperament on the superficial temperature of the hair (P = 0.10; 24.08 ± 0.65, 26.00 ± 0.61, and 25.15 ± 0.72°C) or the skin (P = 0.84; 33.23 ± 0.55, 33.59 ± 0.51, and 33.17 ± 0.61°C) in the neck region for the calm, intermediate, and temperamental calves, respectively. In this study there was a relationship between temperament and some important indicators of the animals' physiological status (internal body temperature and Pulsatility Index). The effect of these physiological changes can influence the performance of beef cattle and therefore these markers may be beneficial in developing better tools for selection of beef cattle.

J.C. Rodríguez Muñoz, J.M. Feugang, S.T. Willard, P.L. Ryan

This study was conducted to investigate the role of porcine relaxin (pRLX) on the motility characteristics of boar spermatozoa during storage (˜21°C) using a CASA system (HTM-IVOS Hamilton-Thorne Biosciences, Version 12.3. Beverly, MA, USA). This motility assessment was divided in two experiments. The first one consisted of a series of physiological relaxin concentrations (0, 25, 50 and 100ng/mL), and experiments were conducted during three consecutive days (day 1(sperm collection), 2 and 3). The second experiment consisted of a high relaxin concentration (0 and 500ng/mL), and experiments were conducted on day 5 of storage. Samples were incubated with relaxin for 60 minutes at 37°C using the same batch of semen. Second, the establishment of specific relaxin receptors on boar spermatozoa (RXFP1 and RXFP2) throughout an immunofluorescence approach. Third, determination at a molecular level of relaxin actions in the intraspermatic cAMP content using an ELISA approach. For all experiments, diluted semen from several boars was centrifuged, and Nidacon products (Mölndal, Sweden) were used to purify spermatozoa. Motile spermatozoa were selected through a discontinuous percoll gradient, washed twice, counted and diluted with the BoviExtend to a final concentration of 75x10⁶ spermatozoa/mL. Three microliters of each treatment group were loaded into 4-chamber Leja® slides for CASA analysis. Four independent replicates were used and pRLX effects assessed in triplicate for each treatment. For the relaxin receptors study, washed spermatozoa were smeared on glass slides, and the presence of RXFP1 and RXFP2 receptors were identified by addition of primary antibodies followed by addition of a secondary antibody (FITC), and DAPI. For the intraspermatic cAMP content study, a concentration of 7 x 10⁶ spermatozoa/well was used and a commercial kit employed for such purpose. All data were analyzed using ANOVA-2, followed by Fisher's LSD test. The threshold of significance was P<0.05. With regards to the motility assessment (experiment 1), we found that relaxin affected the proportion of motile, progressive and rapid spermatozoa. For velocities, straight line velocity (VSL) was affected by relaxin. Similar effects were observed in progression ratios, both straightness, and Linearity. When employing a high relaxin dose (500ng/mL) (experiment 2), progressive and rapid spermatozoa were affected. With regards to the velocities, average path velocity (VAP), straight line velocity (VSL) and curvi-linear velocity (VCL) were affected as well. Linearity was the only progression ratio affected positively by the hormone. The relaxin receptors study demonstrated the presence of RXFP1 and RXFP2 receptors in the head, neck and midpiece of tail in boar spermatozoa. The addition of different pRLX concentrations did not impact the intraspermatic content of cAMP in boar spermatozoa during storage. Overall, our study indicates a beneficial effect of relaxin on motility of boar spermatozoa during storage. Relaxin positively affects STR and LIN, which are indices of rapid sperm movements. The present study also determined the presence of relaxin receptors (RXFP1 and RXFP2). These results suggest that probably relaxin could be promoting such increase in sperm motility acting through its specific receptors; however, we did not find any increase in the intraspermatic cAMP content. Further research in needed to confirm if relaxin is promoting any intraspermatic cAMP modification.

A.G. Youngblood, B.J. Rude, J.D. Davis, D.L. Christiansen, C. Mochal1, P.M. Ward, P.L. Ryan

Twelve mares (BW= 540 ± 46.8 kg) were used for three 14 d trials comparing granulated paper-clay mix (GP) to pine pellets (PP) and wood shavings (WS) as a bedding for horses. After each trial, mares were re-randomized for each subsequent trial. During d 1 through 5 stalls were cleaned daily of feces only. On d 6 stalls were re-bedded with clean bedding and feces and saturated bedding (wet spots) were removed daily through d 14. If needed, clean bedding was added to maintain depth of bedding. Additional GP and WS were needed, but not PP. Due to apparent decreased absorptive capacity, WS were added; however, GP was added mostly because of loss of bedding during the removal of feces and wet spots. Horse and stall (AM and PM) cleanliness scores were assigned daily (1 to 5; 1=clean and 5=heavily soiled). Horses were subjected to a nasal swab (d 5 and 14) and tracheal wash (d 14) during each trial. Tracheal washes were scored from 1 to 5 (1=none and 5=chronic) for cytology, aerobic bacteria and fungal growth. Piled bedding was used to acquire NH3 and pH (5 d and 14 d). Individual horse was considered the experimental unit, and data was analyzed as a complete random design, using the GLM procedures of SAS. Horse cleanliness was not different (P > 0.10) among bedding types. For A.M. and P.M. stall cleanliness, PP (2.79, 3.12; respectively) was cleaner (P = 0.0014) than both GP (3.30, 3.74; respectively) and WS (3.09, 3.47; respectively). Initial pH was greater (P = 0.0001) for both GP and WS compared to PP and all bedding types increased with use. No differences (P > 0.10) were found for ammonia on d 5 (between 3.3 and 88.5 ppm) or d 14 (between 7.3 and 42.7 ppm). Amount of bacteria (cfu) found in the nasal cavity was not different (P > 0 .05) among bedding types on d 5 (between 107,413 and 148,575 cfu) or d 14 (between 152,500 and 191,775 cfu). No differences (P > 0.05) were found for cytology and aerobic bacteria scores within the tracheal wash. However, WS (1.67) and PP (1.67) had less (P = 0.0474) fungal growth than GP (2.17). Results indicate the use of GP as a bedding material for horses has potential.

J.M. Feugang, K. Pendarvis, M. Crenshaw, S.T. Willard, P.L. Ryan

Cryopreservation is a tool of choice for seedstock constitution of genetic superior males. Its successful application in swine artificial insemination industries is limited due to the poor freezability of boar semen. Indeed, there is a subset of boars that can be successfully frozen-thawed for artificial insemination, while another group appears highly cryosusceptible, and therefore unusable for long-term semen storage. The reasons for such differences are unknown, and the full characterization of the protein composition of boar spermatozoa will help determine potential cryosensitive proteins. Here, we performed high-throughput proteomic analyses of boar spermatozoa and compared the proteome profiles of "good" and "poor" freezer boars. Eight commercial proven fertile boars were selected upon conception rates after artificial inseminations using fresh semen. Semen of three independent ejaculations were collected from four "good" and four "poor" freezer boars and frozen in 5ml-straws for the study. Frozen-thawed semen were diluted in the thawing solution and centrifuged through a discontinuous percoll gradient (90/45) to remove seminal plasma, freezing extender, somatic and dead sperm cells. Purified motile spermatozoa were washed three times with a cold-PBS and pooled in pellets of 3x10⁸ spermatozoa per boar. Protein samples were digested with trypsin and prepared for LC-MS/MS analysis. Peptides yielding probability scores lower than 0.05 were subjected to protein identification, and the significance of differentially expressed protein were fixed at p<0.05. Over 3,000 proteins were identified in each group of spermatozoa. Proportions of 63% and 61% total proteins were exclusively detected in "good" and "poor" freezer boars, respectively. Many of the identified proteins were related to different cellular compartments and important molecular mechanisms related to sperm function, such as cell death regulation, macromolecule metabolism and energy related pathways. Approximately 5% of total proteins, representing 163 to 182 individual proteins, were detected at higher levels in both semen groups. Half of these highly abundant proteins were differentially expressed between "good" and "poor" freezer boars. Only eight appeared partially annotated and eleven were predicted. The remaining list of fully annotated proteins included candidates such as transferrin, albumin, and fascin3 which were significantly (p<0.05) abundant in "good" freezer, and outer dense fiber (ODF) 2, protamine (PRM) 2, and calmodulin (CALM) 1 in "poor" freezer boars. Overall, the results indicate that boar spermatozoa contain large amount of proteins whose susceptibility to cryopreservation and implication to sperm function are still to be characterized. Our findings are particularly important for (i) the search of potential biomarkers of semen freezability, and (ii) improvement of semen freezing-thawing extenders for boars, and other species with similar issues.

A. Borazjani, B. Weed, S. Patnaik, J.M. Feugang, D.L. Christiansen, S. Elder, P.L. Ryan, J. Liao

Objectives: To biomechanically characterize and compare human, porcine, equine, and ovine fetal membranes.
Study Design: Non-contact metrology was used for topographical analysis. Uniaxial tensile testing was performed to resolve ultimate tensile load, ultimate tensile stress, tensile modulus, extensibility, ductility, and toughness. Puncture force and biaxial failure strength were determined via biaxial puncture testing. Microstructure and tortuosity were histologically analyzed.
Results: Equine and human membranes sustained larger magnitude loading; however, ovine and porcine fetal membranes exhibit stronger material properties. Both biaxial puncture and uniaxial testings found that human and equine groups accommodated largest loads but lowest stresses. Equine membrane had richest vasculature and surface tortuosity was highest in porcine membranes.
Conclusion: The anatomy of placentation and length of species' gestation show distinct relationships to biomechanical properties of fetal membrane. Unlike other species, human fetal membranes do not compensate for material weakness with a thicker membrane. This finding may explain the high incidence of PROM in humans.

H.T. Boland, S.T. Willard, K. Umemura, G. Scaglia, J.A. Parish, T.F. Best

Two-stage weaning can potentially reduce stress associated with abrupt weaning of calves. British crossbred beef cattle (n=96 cow-calf pairs)were used to evaluate three weaning methods: "one-size fits all" nose-clips (ONE), adjustable size nose-clips (ADJ), and fence-line weaning (FL). In a fourth control treatment group (CTRL) calves remained in pastures with their dams. Nose-clips were placed on ONE and ADJ calves on d -4 and FL calves were placed in pastures adjacent to their dams on d -4. All calves were completely separated from cows the morning of d 0. Calves wore bite counters and IceTag™ sensors in order to evaluate pre-separation grazing behavior and locomotor activity. Three trial periods were conducted in sequential weeks in Fall 2010. There were two replicate groups (4 cowcalf pairs per group) of each treatment per wk and each group grazed an area of 2.0 ha. Pasture assignment was randomized for treatment each week to minimize effects of differently shaped paddocks on behavior. A standard stride length of 65 cm was used to estimate distance traveled. Data were analyzed using PROC MIXED of SAS. There was no effect of weaning method on number of bites and steps, distance traveled, and time spent standing or lying per day (P>0.05). On d -4, bites/d were less while steps/d and distance traveled/d were greater compared with d -3, -2, and -1 (P<0.05). This was likely due to cattle being handled the morning of d -4 to start the experimental treatments and being introduced to new pastures. Unseasonably high ambient temperatures during wk 1 may have led to an effect of wk (P<0.05) on bites/d with values being less in wk 1 than wk 2. Average high temperatures during wk 1, 2, and 3 were 36, 27, and 25 °C, respectively. Time spent standing was greater, and consequently time spent lying was less in wk 1 compared to wk 3 (P<0.05). On d -4 FL calves spent more time standing (P<0.05) than CTRL calves, but only tended (P=0.07) to stand more than ONE calves. The data indicate that use of these gradual weaning methods did not greatly alter behavior of calves during the first stage of the weaning process compared with calves that continued to nurse.