Seasonal variation in equine follicular fluid proteome.

BACKGROUND: Proteomic studies of follicular fluid (FF) exist for several species, including the horse; however, the seasonal influence on FF proteome has not been explored in livestock. The application of high-throughput proteomics of FF in horse has the potential to identify seasonal variations of proteins involved in follicle and oocyte growth. METHODS: This study (i) profiles the proteomes of equine FF collected from dominant growing follicles during the spring anovulatory season (SAN), and spring (SOV), summer (SUM), and fall (FOV) ovulatory seasons; and (ii) identifies season-dependent regulatory networks and associated key proteins. RESULTS: Regardless of season, a total of 90 proteins were identified in FF, corresponding to 63, 72, 69, and 78 proteins detected in the SAN, SOV, SUM, and FOV seasons, respectively. Fifty-two proteins were common to all seasons, a total of 13 were unique to either season, and 25 were shared between two seasons or more. Protein-to-protein interaction (PPI) analysis indicated the likely critical roles of plasminogen in the SAN season, the prothrombin/plasminogen combination in SUM, and plasminogen/complement C3 in both SOV and FOV seasons. The apolipoprotein A1 appeared crucial in all seasons. The present findings show that FF proteome of SUM differs from other seasons, with FF having high fluidity (low viscosity). CONCLUSIONS: The balance between the FF contents in prothrombin, plasminogen, and coagulation factor XII proteins favoring FF fluidity may be crucial at the peak of the ovulatory season (SUM) and may explain the reported lower incidence of hemorrhagic anovulatory follicles during the SUM season.

Effect of cryopreservation techniques on proliferation and apoptosis of cultured equine ovarian tissue.

Preservation of cellular integrity and its mechanisms after ovarian tissue cryopreservation (OTC) and in vitro culture (IVC) procedures are crucial aspects for the success of preservation and recovery of female fertility. This study aimed to evaluate the effects of two cryopreservation methods (slow-freezing, SF, and vitrification, VIT) on the equine ovarian tissue after 1, 3, and 7 days of IVC by assessing: (i) preantral follicle morphology and distribution of follicle classes; (ii) protein expression of markers of cell proliferation for EGFR and Ki-67; (iii) markers of apoptosis for Bax and Bcl-2; and (iv) DNA fragmentation. Percentages of normal primordial follicles were similar (P > 0.05) among SF-control, VIT-control, and fresh control groups. After 7 days of culture, VIT-IVC7 had a greater (P < 0.05) total percentage of normal preantral follicles when compared with SF-IVC7, but both had a lower (P < 0.05) percentage than fresh IVC7 group. Prior to and after 7 days of culture, expression of EGFR and Ki-67 were similar (P > 0.05) among fresh, SF, and VIT groups. After 7 days of culture, VIT had higher (P < 0.05) Bax expression than the fresh and SF tissues, but Bcl-2 was similar (P > 0.05) among groups. Prior to IVC, TUNEL signals were similar (P > 0.05) among groups; however, VIT-IVC7 had greater (P < 0.05) TUNEL signals when compared with the fresh IVC7 group. In conclusion, findings demonstrated: (i) similar efficiency between SF and VIT compared with fresh control to preserve morphologically normal follicles; and (ii) similar tissue functionality and cell proliferation capability after equine OTC by either SF and VIT methods following IVC for 7 days. The results herein presented shed light on equine fertility preservation programs using OTC techniques.

A hormone priming regimen and hibernation affect oviposition in the boreal toad (Anaxyrus boreas boreas).

Declines of the southern Rocky Mountain population of boreal toad (Anaxyrus boreas boreas) have led to the establishment of a captive assurance population and reintroduction program, in an attempt to preserve and propagate this geographically isolated population. One of the unique adaptations of this species is its ability to survive in cold environments by undergoing long periods of hibernation. In captivity, hibernation can be avoided altogether, decreasing morbidity caused by compromised immune systems. However, it is not entirely clear how essential hibernation is to reproductive success. In this study, the effects of hibernation versus nonhibernation, and exogenous hormones on oviposition, were examined in boreal toad females in the absence of males. In the summers of 2011 and 2012, 20 females housed at Mississippi State University were treated with a double priming dose of hCG and various ovulatory doses of hCG and LH-releasing hormone analog but denied hibernation. Exogenous hormones, in the absence of hibernation, could not induce oviposition over two breeding seasons (2011-2012). In contrast, during the summer of 2012 and 2013, 17 of 22 females (77%) housed at the Native Aquatic Species Restoration Facility (Alamosa, CO, USA) oviposited after they were treated with two priming doses of hCG (3.7 IU/g each) and a single ovulation dose of hCG (13.5 IU/g) and LH-releasing hormone analog (0.4 µg/g) after hibernation. There was a significant difference in oviposition between females that were hibernated and received hormones (2012, P < 0.05 and 2013, P < 0.01) compared to hibernated control females. In 2013, 12 of 16 remaining Mississippi State University females from the same group used in 2011 and 2012 were hibernated for 1, 3, and 6 months, respectively and then treated with the same hormone regimen administered to females at the Native Aquatic Species Restoration Facility. Together, hibernation and hormone treatments significantly increased oviposition (P < 0.05), with 33% of females ovipositing. These results suggest that (1) hibernation is a key factor influencing oviposition that cannot be exclusively circumvented by exogenous hormones; (2) females do not require the presence of a male to oviposit after hormone treatments; and (3) longer hibernation periods are not beneficial for oviposition. The hormonal induction of oviposition in the absence of males and shorter hibernation periods could have important captive management implications for the boreal toad. Furthermore, the production of viable offspring by IVF where natural mating is limited could become an important tool for genetic management of this boreal toad captive population.

Relationship among eye and muzzle temperatures measured using digital infrared thermal imaging and vaginal and rectal temperatures in hair sheep and cattle.

Digital infrared thermal imaging (DITI) using a thermal camera has potential to be a useful tool for the production animal industry. Thermography has been used in both humans and a wide range of animal species to measure body temperature as a method to detect injury or inflammation. The objective of these experiments was to compare the temperature of the eye (EYE) or muzzle (MUZ) measured using DITI to vaginal (VT) and rectal temperature (RT) as measures of core body temperature in hair sheep and beef cattle. In Exp.1 EYE, VT and RT were measured in lactating, multiparous hair sheep ewes (St. Croix White, n = 10, and Dorper × St. Croix White, n = 10) in a non-febrile state 5 times over a 48-h period. Data loggers were used to measure VT and a digital veterinary thermometer was used to measure RT. There was a high correlation (P < 0.001) between VT and RT (r = 0.95), EYE and RT (r = 0.76) and EYE and VT (r = 0.77). In Exp. 2 EYE, MUZ, VT and RT were measured in multiparous, lactating ewes (St. Croix White, n = 2, and Barbados Blackbelly, n = 12) at -12, -1, 0, 1, 2, 3, 4, 6, 12, 24, 36, and 48 h after being administered lipopolysaccharide (LPS; n = 7; 0.2 µg/kg BW, i.v.) or saline (n = 7; 0.5 mL, i.v.). Data loggers were used to measure VT and a digital veterinary thermometer was used to measure RT. When data were combined across treatments (LPS and saline) there was a high correlation (P < 0.001) between VT and RT (r = 0.96), EYE and RT (r = 0.82), MUZ and RT (r = 0.72), and EYE and VT (r = 0.93). In Exp. 3 EYE, MUZ, VT and RT were measured in multiparous, non-lactating, pregnant Senepol cattle (n = 44) between 0900 and 1200 h on a single day. A digital veterinary thermometer was used to measure both VT and RT. There was a high correlation (P < 0.001) between VT and RT (r = 0.78), a moderate correlation (P < 0.001) between VT and EYE (r = 0.52), RT and EYE (r = 0.58) and EYE and MUZ (r = 0.48). There was no correlation (P > 0.10) between RT or VT and MUZ. The findings of these three studies indicate that temperature of the eye, measured using DITI, can be used as an indicator of core body temperature in hair sheep and beef cattle as an alternative to using vaginal or rectal temperature.

Genetic parameters of three methods of temperament evaluation of Brahman calves.

The objective of this study was to estimate the heritability of 3 measures of temperament in Brahman and Brahman-influenced calves (n = 1,209). Individual animal pen scores (PS) were determined by a trained observer who evaluated groups of 5 or 4 calves at a time for willingness to be approached by a human. Exit velocity (EV) was the rate (m/s) at which each calf exited a squeeze chute. Temperament score (TS) was calculated individually as (PS + EV)/2. Temperament was evaluated at 5 different times of record (28 d preweaning, weaning, 28 d postweaning, 56 d postweaning, and yearling). Contemporary groups (n = 34) comprised calves of the same sex born in the same season of the same year. There were an average of 36 calves per contemporary group and group size ranged from 3 to 78 calves. Average weaning age (186 d) ranged from 105 to 304 d. Calves were born from 2002 through 2012. Random effects included additive genetic and the permanent environmental variance. The fixed effects analyzed were age of dam, sex of calf, contemporary group, fraction of Brahman (2 levels: 1 and 0.5), age of calf at record, and weaning age. At weaning, the mean PS was 2.68 ± 0.1, the mean EV was 2.41 ± 0.1, and the mean TS was 2.48 ± 0.1. The PS was affected by fraction of Brahman (P = 0.034) and tended to be affected by age of dam (P = 0.06). The EV was affected by contemporary group (P < 0.001) and tended to be affected by weaning age (P = 0.074). Contemporary group affected TS (P < 0.001). All 3 methods of temperament evaluation were affected by time of record (P < 0.001). The regression coefficients for PS, EV, and TS were 0.0023 ± 0.0014, 0.0022 ± 0.0012, and 0.0015 ± 0.0012 m·s(-1)·d(-1) of age, respectively. Estimates of maternal genetic effects were always 0 and omitted from final models. Estimates of heritability were 0.27 ± 0.1, 0.49 ± 0.1, and 0.43 ± 0.1 for EV, PS, and TS, respectively. Estimates of permanent environmental variances as proportions of phenotypic variance were 0.33 ± 0.1, 0.23 ± 0.1, and 0.33 ± 0.1 for EV, PS, and TS, respectively. There appears to be sufficient additive genetic variance for selective improvement of temperament characteristics in Brahman cattle.

Plane of nutrition x tick burden interaction in cattle: effect on fecal composition.

Effective tick management on grazing animals is facilitated by accurate noninvasive detection methods. Fecal analysis provides information about animal health and nutrition. Diet affects fecal composition; stress may do likewise. The constituents in feces that may be affected by tick burdens and in turn affect near-infrared spectra have not been reported. Our objective was to examine the interaction between plane of nutrition and tick burden on fecal composition in cattle. Angus cross steers (n = 28; 194 ± 3.0 kg) were assigned to 1 of 4 treatments (n = 7 per group) in a 2 × 2 factorial arrangement: moderate (14.0 ± 1.0% CP and 60 ± 1.5% TDN) vs. low (9.0 ± 1.0% CP and 58 ± 1.5% TDN) plane of nutrition and control (no tick) vs. tick treatment [infestation of 300 pair of adult Lone Star ticks (Amblyomma americanum) per treated animal]. Fecal samples were collected at approximately 0700 h on d -7, 0, 7, 10, 14, 17, and 21 relative to tick infestation. Fecal constituents measured were DM, OM, pH, Lactobacillus spp., Escherchia coli, acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, IgA, and cortisol. Experimental day affected (P < 0.05) all constituents measured. Plane of nutrition affected (P < 0.05) DM, OM, VFA, and IgA. Tick treatment numerically (P = 0.13) reduced cortisol. A multivariate stepwise selection model containing cortisol and E. coli values on d 10 and d 14 accounted for 33% of the variation in daily adult female tick feeding counts across both planes of nutrition (P < 0.07). Within the moderate plane of nutrition, a model containing only cortisol on d 10 and d 14 described 59% of the variation in the number of feeding ticks (P < 0.02). Similarly, a model including cortisol, propionate, isovalerate, and DM at d 10 and d 14 d described 95% of the variation in total feeding ticks in the low plane of nutrition. Of the constituents measured, fecal cortisol offers the best possibility of noninvasively assessing stress by way of a single assay but the presence of ticks would still need to be confirmed visually. Although several constituents measured in this study should exist in sufficient quantity to directly affect near-infrared spectra, none stood out as a clear descriptor of prior observed differences in fecal spectra between tick-treated versus non-tick-treated animals. There were, however, groups of fecal constituents related to daily adult female tick feeding numbers (as a visual estimation of tick stress).

Horse species symposium: a novel approach to monitoring pathogen progression during uterine and placental infection in the mare using bioluminescence imaging technology and lux-modified bacteria.

Uterine and placental infections are the leading cause of abortion, stillbirth, and preterm delivery in the mare. Whereas 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 completed. 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, whereby transgenically modified bacteria transformed with lux genes of Xenorhabdus luminescens or Photorhabdus luminescens origin and biophotonic imaging are utilized to better understand pathogen-induced preterm birth in late-term pregnant mares. This technology uses 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, which was hitherto impossible. Although the application of this technology in domestic animals is in its infancy, investigators were successful in identifying the fetal lungs, sinuses, nares, urinary, and gastrointestinal systems as primary tissues for pathogen invasion after experimental infection of pregnant mares with lux-modified Escherichia coli. It is important that 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 approach in the development of more targeted therapeutic interventions for pathogen-related diseases in the equine and other domestic species.

Gestational, periparturient and preweaning growth traits of Holstein versus Gir x Holstein F1 crossbred dairy calves born to Holstein dams.

The objectives of this study were to assess the gestational, periparturient and preweaning growth traits of Holstein vs Gir x Holstein (G x H F1) cross-bred dairy calves. Holstein cows (n=64) pregnant with Holstein (H x H) calves (bulls, n=16; heifers, n=18) or G x H (bulls, n=8; heifers, n=22) calves were sampled from 60 days of gestation through calving for serum progesterone (P4) and in utero placentome sizes, and at parturition for calving ease scores (CES), calf vigour scores (CVS), and placental characteristics. Post-calving calf measures included birth weight (day 0) and body weight (kg), hip width (HW), body length (BL), wither height (WH), hip height (HH) and heart girth (HG) through 42 days of age. Serum IgG and calf vital function tests (respiration rate (RR), heart rate (HR), rectal temperature (RT) and fecal scores) were recorded on days 1, 3, 7 and 14 of age (a.m. and p.m.). Serum gestational P4 was higher (p < 0.05) for cows with H x H than G x H calves. Placentome measurements in utero were greater (p < 0.01) for H x H calves than for G x H calves, while at parturition placental characteristics did not differ (p > 0.10). Gestation length, CES and CVS also did not differ (p > 0.10) between H x H and G x H calves. Calf RR and HR were higher (p < 0.05) for H x H than G x H calves at both a.m. and p.m., while RT, fecal scores and serum IgG did not differ (p > 0.10) between H x H and G x H calves. Birth weight did not differ (p > 0.10) between H x H and G x H calves within sex. Moreover, bulls did not differ (p > 0.10) in any of the growth measures between H x H and G x H calves, whereas H x H heifers at days 28 and 42 of age were greater (p < 0.05) in all growth traits than G x H heifers. Nevertheless, the relative change in growth measures over time (days 0 to 42) did not differ (p > 0.10) between H x H and G x H calves. While subtle differences in gestational, early growth and vital function characteristics were observed, these data suggest very similar developmental profiles between H x H and G x H calves.

Administration of 6-methoxybenzoxazolinone (MBOA) does not augment ovulatory responses in St. Croix White ewes superovulated with PMSG.

The objective of this investigation was to examine the effects of 6-methoxy-benzoxazolinone (MBOA), a plant compound that resembles melatonin and alters ovarian function in rodents, in combination with PMSG on superovulatory responses in the cycling ewe. In Experiment I, St. Croix White ewes (n = 44) were synchronized (intra-vaginal progestin sponge) for 14days followed by hCG (750 IU) at 1 day after sponge removal (day 0). Ewes were assigned to one of six treatments administered on day -1: Control (no PMSG or MBOA; n = 7); PMSG (1000 IU i.m.; n = 7); Low MBOA (0.43 mg/kg i.m.; n = 7); High MBOA (1.15 mg/kg i.m.; n = 7); Low MBOA + PMSG (n = 8); High MBOA + PMSG (n = 8). In Experiment II, St. Croix White ewes (n = 24) were synchronized (progestin CIDR) for 14 days followed by hCG on day 1 after CIDR removal (day 0). Ewes were assigned to one of three treatments administered on day -1: Control (n = 8); PMSG (n = 8); Low MBOA+PMSG (n = 8). Laparoscopy was performed on day 9 to assess numbers of corpora lutea (CL) and visible follicles on each ovary. Blood samples were collected on day -13, -1, 0, 1, and days 6 or 7-12 for analysis of serum progesterone (P4) by RIA. Treatment groups receiving PMSG (alone or with MBOA) exhibited greater (P < 0.05) serum concentrations of P4 post-synchrony than Control and MBOA-only groups. Ovulation rate was lower (P < 0.05) for Control and MBOA-only treated ewes than ewes receiving PMSG. Ovulation rate in ewes treated with MBOA alone was similar (P > 0.10) to Controls, and PMSG treatment alone did not differ (P > 0.10) from MBOA + PMSG treatment. Ewes treated with PMSG alone did not differ (P > 0.10) in follicle number from High MBOA + PMSG treated ewes, however, Low MBOA + PMSG treated ewes had greater numbers of follicles at day 9 (P < 0.05) than the PMSG or High MBOA + PMSG groups in Experiment I; although, this was not replicated in Experiment II with numbers of follicles in the Low MBOA + PMSG group being similar (P > 0.10) to PMSG alone. In summary, the addition of MBOA in combination with PMSG as part of a synchronization-superovuation protocol in the ewe did not increase ovulation rate.

Plasma progesterone response following ACTH administration during mid-gestation in the pregnant Brahman heifer.

Previous reports of adrenal progesterone (P4) contributions during late gestation in cattle, and ACTH-induced P4 responses in the non-pregnant heifer, prompted a retrospective investigation to evaluate the plasma P4 response and the relative ratio of plasma cortisol (CT) to P4 following ACTH administration during mid-gestation in pregnant Brahman heifers. Twenty-three pregnant (139.0 +/- 5.0 days of gestation) Brahman heifers received one of the following treatments: 0 (saline; n = 5), 0.125 (n = 4), 0.25 (n = 5), 0.5 (n = 4), or 1.0 (n = 5)IU of ACTH per kg BW. Blood samples were collected at -15 and -0.5 (time 0), 15, 30, 45, 60, 75, 105, 135, 165, 195, and 255-min post-ACTH challenge. Plasma P4 and CT were quantified by RIA. Pre-ACTH P4 did not differ (P > 0.10) among ACTH treatment groups (pooled, 12.1 +/- 0.6 ng/mL). Among peak P4 values at 15-min post-ACTH infusion, control P4 (9.6 +/- 1.2 ng/mL) tended to be lower (P < 0.07) than 0.5 IU ACTH-treated heifers (13.3 +/- 1.1 ng/mL); and were lower (P < 0.02) than 0.25 and 1.0 IU ACTH-treated heifers (14.7 +/- 1.1 and 22.2 +/- 3.7 ng/mL, respectively). During the primary P4 response period (0 to 75-min post-ACTH), the area under the curve (AUC) was greater (P < 0.05) for 1.0 IU ACTH-treated heifers than all other groups. The CT:P4 ratios were lower (time x treatment, P < 0.01) for control heifers than all ACTH-treated heifers. Among ACTH-treated heifers, CT:P4 ratio response and CT:P4 ratio AUC were similar (P > 0.10) following ACTH challenge. In conclusion, acute increases in ACTH elevated plasma P4, likely of adrenal origin, in mid-gestation pregnant heifers, while the CT:P4 ratio (relative output) remained constant irrespective of ACTH dose (0.125-1.0 IU). Whether ACTH-induced increases in P4 in pregnant animals are of physiological significance (e.g., an accessory role in the maintenance of pregnancy during periods of acute stress) remains to be determined.

Artificial insemination following observational versus electronic methods of estrus detection in red deer hinds (Cervus elephus).

The objectives were to determine the efficacy of the HeatWatch (HW) electronic estrus detection system for monitoring behavioral estrus (including duration and intensity) in red deer hinds and to evaluate pregnancy rate to AI after detected estrus. Red deer hinds (Cervus elephus; n = 50) were allocated into two treatment groups: AI following synchronization (CIDR/PMSG) and observed estrus (induced estrus group: IE; n = 25) or AI following the detection of natural estrus (NE; n = 25) without hormonal treatment. Hinds were fitted with two HeatWatch (HW) electronic estrus detection transmitters, one above the tail (bottom) and one between the tuber coxae of the pelvic girdle (top), and visual observations for mounting activity began with the aid of young sterile red deer stags (18 months old) fitted with marking harnesses. Hinds in both groups were inseminated (10-12h after observed estrus) with frozen-thawed red deer semen using a transvaginal/cervical AI approach. Following a 26-day period of AI, hinds were placed with a mature fertile stag for an additional 30-day natural breeding period. Pregnancy diagnosis was performed 57 and 86 days after the start of AI. While the hinds were housed with the young stags, 82% were detected in estrus by visual appraisal of stag crayon marks, but only 32% of these were detected by HW. In contrast, in the hinds housed with the mature stag, 93% detected in estrus by crayon marks were also detected by HW. The top HW transmitter consistently recorded more mounts (P < 0.05) than the bottom transmitter. The pregnancy rate was numerically better in IE versus NE hinds (42% versus 29%, P > 0.10). In summary, there were no differences (P > 0.10) in the intensity (number) or duration of mounts (detected by HW) during estrus in IE versus NE hinds, and HW was most effective in detecting estrus in the presence of a heavier, mature stag versus a younger stag. When used in combination with transvaginal AI, an overall first-service pregnancy rate of 36.6% was achieved with AI of frozen-thawed semen in red deer hinds following detected estrus.

4-Hydroxytamoxifen differentially exerts estrogenic and antiestrogenic effects on discrete subpopulations of human breast cancer cells.

Functional heterogeneity within populations of breast cancer cells contribute to the seemingly paradoxical effects of antiestrogens and the development of antiestrogen "resistance." Our objectives were to determine the degree to which T-47D cells may respond inappropriately (positively) to the antiestrogen 4-hydroxytamoxifen (HOT) alone, and whether all cells that respond to the stimulatory effects of estradiol-17beta (E2) are inhibited by the addition of HOT. Single, living T-47D cells were transfected by microinjection with an estrogen response element (ERE)-driven luciferase reporter plasmid. Transfected cells were then treated with medium alone, HOT, E2 or a combination thereof on consecutive days, exposed to the substrate luciferin and subjected to quantification of photonic emissions reflective of ERE-stimulated activity. This analysis revealed a subpopulation of cells that exhibited increased ERE-driven photonic activity in response to HOT. In companion studies, E2-stimulated ERE activity was reversed (on average) with HOT addition. However, analysis of individual cells revealed that although HOT reduced photonic activity in the majority (89.2%) of E2-responsive cells, there was a small subset (10.8% of the population) that was stimulated by E2 + HOT cotreatment. Our data support the hypothesis that these cells possess an intrinsic "resistance" to antiestrogenic agents, and that this could contribute to the remodeling of tumor cell populations toward a "resistant" phenotype.

Simultaneous indirect activity measurements of GH and PRL genes in the same, living mammosomatotrope.

Dynamic intracellular processes in endocrine cells are usually controlled by the coordinated modulation of two or more functionally related genes. Attempts to gain a more complete understanding of these processes would be facilitated greatly by a method enabling activity measurements of two genes at the same time. Here we describe how we developed such a system and used it to determine indirectly whether individual, living pituitary cells could concurrently express both the growth hormone (GH) and prolactin (PRL) genes. Our results demonstrate that coexpression of these genes is indeed possible. Moreover, our findings provide a general paradigm for future "real-time" analysis of other interrelated genes involved in the regulation of endocrine processes.

Pregnancy detection and the effects of age, body weight, and previous reproductive performance on pregnancy status and weaning rates of farmed fallow deer (Dama dama).

Fallow does (n = 502) of different ages (mature, 2-yr-old, and yearling) were maintained with bucks for a 60-d breeding season to determine whether previous reproductive performance and changes in BW affect doe pregnancy rates and to compare the effectiveness of ultrasonography and serum pregnancy-specific protein B (PSPB) for the detection of pregnancy in fallow does. Ultrasonography was performed, blood samples collected, and BW recorded at buck removal (d 0) and at 30 and 90 d after buck removal. Lactational status (lactating = WET; nonlactating = DRY) were determined from farm records taken at weaning prior to each breeding season (autumn 1990 through autumn 1994). Ultrasonography and PSPB for determining pregnancy were in agreement 93% of the time. Overall pregnancy rates did not differ (P>.10) relative to age of the doe; the combined pregnancy rate was 92%. We also determined that 82.9% of does conceived early in the breeding season and that the incidence of embryonal-fetal mortality during the first 90 d after buck removal was 2.8%. In general, mature and 2-yr-old DRY does were heavier and had lower pregnancy rates than WET does. The overall weaning rate for all does was 77.9%. Loss in the number of fawns from pregnancy detection to weaning was equivalent to 14.8% for mature does, 24.7% for 2 yr old does, and 42.5% for yearling does. These data indicate that even though pregnancy rates were relatively high, further study is needed to determine the causes associated with subsequent fawn losses, particularly among yearling does. As a production tool, lactational WET/ DRY status testing was found to be an acceptable means for determining the reproductive potential of individual does within the herd. In addition, serum PSPB may be used in place of ultrasonography for pregnancy diagnosis in fallow deer as early as d 30 after buck removal.

TGF-alpha exerts biphasic effects on estrogen--and phytoestrogen-mediated gene expression in breast cancer cells.

Transforming growth factor-alpha (TGF-alpha) contributes to the progression of mammary carcinogenesis in part through synergistic augmentation of estradiol (E2) action. To investigate this further, we sought to determine (1) whether the duration of TGF-alpha treatment might influence the nature of the TGF-alpha/E2 interaction, and (2) whether TGF-alpha would behave in a similar manner when combined with phytoestrogens. To this end, we transfected T47-D breast cancer cells with an estrogen-responsive reporter and then treated the cells (for 4-48 h) with varying concentrations of TGF-alpha, E2, the antiestrogen 4-hydroxy-tamoxifen (HOT), and/or one of three phytoestrogens. Our findings revealed that TGF-alpha has short-term synergistic and long-term inhibitory effects on E2- and phytoestrogen-regulated gene expression. Furthermore, this secondary inhibition of E2 action by TGF-alpha was similar in magnitude to that imposed by HOT. These findings demonstrate a novel role for TGF-alpha and invite reevaluation of current models regarding TGF-alphas interactions with E2 in breast cancer cells. Our results also raise the possibility that phytoestrogens, which interact with TGF-alpha in a manner conceptually identical to that of E2, may subserve a regulatory function in breast cancer cells.

Hybridization between wapiti (Cervus elephus manitobensis) and sika deer (Cervus nippon): a comparison of two artificial insemination techniques.

The present study compared pregnancy rates of sika deer (Cervus nippon) hinds artificially inseminated with frozen-thawed wapiti (Cervus elephus manitobensis) semen by laparoscopic intrauterine or transvaginal/cervical artificial insemination (AI) techniques. Estrous cycles of 59 sika hinds were synchronized with one-half of a norgestomet ear implant. Fourteen days after implant insertion, norgestomet ear implants were removed and hinds received 50 IU of PG-600 intramuscularly, a combination of 50 IU PMSG and 25 IU hCG. Hinds were then randomly allotted for laparoscopic (n = 25) or transvaginal/cervical (n = 34) AI. AI of the hinds with frozen-thawed wapiti semen (40 x 10(6) spermatozoa) was time-fixed to occur at 55 hr for transvaginal/cervical AI and 65 hr for laparoscopic AI postnorgestomet implant removal. Semen deposition for hinds inseminated by transvaginal/cervical AI occurred as follows: vagina. 8.8% (3/34); os cervix, 67.6% (23/34); intracervical, 20.5% (7/34); and uterus, 2.9% (1/34). On day 42 post-AI, transrectal ultrasonography was performed to determine pregnancy rates. Pregnancy rates were not significantly different (P > 0.10) between laparoscopic (8/25, 32.0%) and transvaginal/cervical AI (9/34, 26.4%), with an overall conception rate of 28.8% (17/59). These results indicate that transvaginal/cervical AI techniques can be as effective as laparoscopic AI under some circumstances. Further refinement of the transvaginal/cervical AI technique to improve pregnancy rates might lead to more widespread use of this technique when laparoscopic AI is not possible.

Phytoestrogens have agonistic and combinatorial effects on estrogen-responsive gene expression in MCF-7 human breast cancer cells.

Phytoestrogens can exhibit agonistic actions on estrogen-dependent gene expression in breast cancer cells. Since several different phytoestrogens may be found within a single dietary plant source, we sought to investigate whether estrogen-dependent gene expression may be further influenced by the collective treatment of breast cancer cells with multiple phytoestrogens. Accordingly, we transfected MCF-7 breast cancer cells with estrogen-responsive reporters followed by treatment with one of four phytoestrogens (genistein, daidzein, formononetin, and equol) or a combination of these in the absence of estradiol. Our results demonstrated clear-cut agonistic effects of phytoestrogens on estrogen-dependent gene expression. Moreover, combinatorial treatment consistently stimulated reporter activity above that observed for individual phytoestrogens. Inasmuch as the phytoestrogens tested are frequently found together in food sources, these combinatorial responses may more accurately reflect the consequences of in vivo exposure.

Early pregnancy detection and the hormonal characterization of embryonic-fetal mortality in fallow deer (Dama dama).

The objectives of this investigation were to 1) determine serum concentrations of progesterone (P4), estrone sulfate (E1S) and pregnancy-specific protein B (PSPB) from estrus synchronization through mid-gestation in the fallow doe (Dama dama) and 2) characterize the hormonal profiles of does whose embryos or fetuses died in utero. Ten fallow does were synchronized for 14 d with an intravaginal P4-releasing device (CIDR) and were naturally mated after CIDR removal. Blood samples were collected at CIDR insertion, CIDR removal and at intervals through Day 203 post-CIDR removal for analysis of P4, E1S and PSPB by radioimmunoassay (RIA). Ultrasonography was performed on Days 49 and 69 post-CIDR removal. Serum P4 at the time of CIDR insertion was 4.8 +/- 0.6 ng/ml, and at CIDR withdrawal it was 6.2 +/- 0.3 ng/ml. Concentrations of E1S and PSPB were nondetectable at CIDR insertion. Serum E1S was highest at Day 93, and PSPB was first detectable in pregnant does at Days 27 to 30 post-CIDR withdrawal. Ultrasonography on Day 49 revealed that 6 does were pregnant, 2 were not pregnant and 2 others were diagnosed originally as early pregnant. At Day 69, ultrasonography revealed that 6 does (60%) were pregnant and 4 (40%) were not. A comparison of the ultrasonographic and hormonal data indicated that the 2 does diagnosed as early pregnant on Day 49 had conceived but had lost the pregnancy. A third doe which was pregnant on Day 69 lost the fetus later in gestation. Hormonal profiles of does whose embryo or fetus had died were characterized by erratic P4 and E1S profiles, with PSPB becoming undetectable in the 3 does by Days 49, 65 and 80 post-CIDR removal. These data 1) demonstrate the timing for the collection of serum samples for determining early pregnancy in fallow does using 3 hormonal methods and 2) characterize the hormonal profiles of 3 fallow does with embryonic-fetal loss.

Real-time monitoring of estrogen-regulated gene expression in single, living breast cancer cells: a new paradigm for the study of molecular dynamics.

Elucidation of the molecular mechanisms underlying the development and progression of breast cancer has been hindered by two considerations. First, mammary tumor cells exhibit a considerable degree of morphological and functional heterogeneity. Thus, the conventional strategy of measuring a population response may not provide an accurate reflection of the behavior of the functional unit: an individual cell. Second, important regulatory events are generally separated in time, yet the strategies we use to monitor them are usually static as opposed to dynamic. With these considerations in mind, it would appear that a system for studying this problem should ideally combine the resolving power of single-cell analysis with a dynamic paradigm for making multiple measurements of gene expression from the same, living cell. This report summarizes our efforts at developing, validating, and optimizing such a system for monitoring hormone-driven gene expression in T47-D human breast cancer cells.

Effects of dietary fat on follicular development and circulating concentrations of lipids, insulin, progesterone, estradiol-17 beta, 13,14-dihydro-15-keto-prostaglandin F(2 alpha), and growth hormone in estrous cyclic Brahman cows.

Brahman cows were used to evaluate dietary fat (3.74% [control] and 5.20% [treated]) effects on blood hormone and lipid concentrations, follicular populations, and in vitro steroidogenesis. Cows were fed and ovaries were monitored by ultrasound from d 1 of the first estrous cycle (EC) until the first follicle of the second EC reached 8 mm, at which time ovaries were harvested. Follicular fluid (FF) was collected from large- and medium-sized follicles and assayed for progesterone (P4), estradiol-17 beta (E2), testosterone, cholesterol, and triglycerides. The corpus luteum was removed, minced, treated with LH, and incubated for 4 h. Granulosa cells harvested from the largest follicle were treated with forskolin, LH, or FSH and incubated for 48 h. Blood was collected during the treatment period and plasma assayed for 13,14-dihydro-15-keto-prostaglandin F(2 alpha) (PGFM), growth hormone (GH), insulin, P4 and E2. Cholesterol and triglycerides were assayed only from blood samples collected every 7 d. Treated cows had greater (P < .01) plasma E2 during the first EC and greater (P < .01) P4 during the second EC than controls. Treated cows had elevated (P < .01) insulin following d 16 of treatment, but GH and triglycerides were similar (P > .10) in both treatment groups. Treated cows tended (P = .10) to have greater PGFM peak concentrations than controls. Plasma cholesterol was elevated (P < .01) in treated cows on d 7 of the first EC. Treated cows tended to have greater (P < .09) follicular populations during the ovulatory and first wave of the first and second EC. Treatment did not affect (P > .10) FF concentrations of P4, E2, testosterone, cholesterol, or triglyceride from either large- or medium-sized follicles. There were no differences (P > .10) in steroid hormones produced in vitro.