Longitudinal fecal hormone analysis for monitoring reproductive activity in the female polar bear (Ursus maritimus)

The objective was to identify suitable enzyme immunoassays to monitor gonadal and placental function in the female polar bear. Immunoreactive progesterone, progesterone metabolite (PdG), estrogen, and androgen metabolite (T) concentrations were measured in fecal samples collected over 24 mo from captive female bears (N = 20). Whereas fecal extracts produced displacement curves parallel to the standard curve for each respective steroid, T and PdG more accurately reflected reproductive events. Concentrations of fecal T increased (P < 0.05) during the breeding season, and brief spikes were associated with estrus and mating. A postovulatory increase in PdG was not always detected, but sustained baseline T after mating appeared consistent with ovulation. Parturient bears excreted higher PdG concentrations (P < 0.05) during expected time of embryo implantation in Fall, and a late gestational rise in fecal T occurred 30 days prepartum. Many nonparturient bears also had a PdG rise in the Fall, suggesting they experienced either pregnancy loss or a pseudopregnancy. Differentiating pregnant and pseudopregnant states was not achieved using fecal PdG alone, but when combined with fecal T, comprehensive diagnoses could be made. Nonparturient bears demonstrated elevated (P < 0.05) fecal T during summer months, whereas parturient bears did not. In summary, noninvasive hormone monitoring techniques were established for the female polar bear. Although this study was directed at facilitating management and breeding efforts of captive polar bears, the methods could be applied to studies of reproductive function in wild populations.     

Non-invasive monitoring of the estrous cycle in captive crab-eating foxes (Cerdocyon thous)

In this study, four crab-eating fox females (Cerdocyon thous) maintained at the Federal University of Mato Grosso Zoo, Cuiabá, Brazil, were investigated for 16 mo, using transabdominal ultrasonography and measurement of estradiol and progesterone concentrations in blood plasma and feces. Blood collection and ultrasonography were performed once a month, whereas fecal collections were performed three times a week. During the experimental period, there was an annual estrous cycle in all females, with the reproductive season lasting from winter to spring, and three became pregnant. Transabdominal ultrasonography was inconclusive for characterization of estrus cycles phase, but was effective for early detection of pregnancy, pregnancy monitoring, and for evaluating postpartum uterine involution. There were similarities between C. thous female's reproductive aspect and bitches, with similar pregnancy data, although uterine involution was faster in C. thous. Peak serum concentrations of P4 and E2 were (mean ± SD) 14.58 ± 5.8 ng/ml and 31.62 ± 53.54 pg/ml, respectively, whereas mean fecal peaks of P4 and E2 were 2.37 ± 1.42 ng/g and 157.95 ± 82.63 pg/g, respectively. All pregnant females had serum and fecal P4 concentrations reaching maximum values (16.5 ± 4.0 ng/ml and 2.7 ± 0.4 ng/g, respectively) from 10 to 30 d of gestation; those values subsequently declined, reaching baseline at parturition (5.0 ± 4.0 and 0.7 ± 0.4 ng/g, respectively). Peaks of E2 occurred throughout the year, and were absent only during apparent lactational anestrus.     

Fecal endocrine monitoring of reproduction in female snow leopards (Uncia uncia)

Although the snow leopard (Uncia uncia) is a common endangered felid species in zoos, little is known about the complex endocrine interactions controlling ovarian function and conception in this species. The goal of this work was to characterize ovarian activity throughout the estrous cycle, nonpregnant luteal phase (pseudopregnancy), and gestation in female snow leopards. This goal was accomplished using an enzyme immunoassay to measure fecal concentrations of estrogen metabolites (E) and progesterone metabolites (P). Fecal samples were collected from 12 female snow leopards (ages 18 months to 18 years) during one to three breeding seasons. In each breeding season, the majority of females (78%, 88%, and 100%, respectively) began to exhibit ovarian activity in December or January. The estrous cycle, defined by the first day of estrus (E ≥ 2 × basal concentration) to the first day of the subsequent estrus, was 12.7 ± 0.6 days (n = 145 cycles). Estrus lasted 4.3 ± 0.4 days with mean concentrations of fecal E during the follicular phase (1661 ± 139 ng/g feces) increasing 3.2-fold above basal concentrations (515 ± 32 ng/g feces). No spontaneous ovulations were observed in any of the cycling females. Nonpregnant luteal phases were observed in eight females that bred but did not become pregnant. The length of the nonpregnant luteal phase ranged from 11 to 72 days (45.7 ± 5.7 days; n = 10) with mean concentrations of fecal P during the luteal phase (12.46 ± 1.7 μg/g feces) increasing 6.2-fold above basal concentrations of P (2.01 ± 0.2 μg/g feces). Three of the females in the study became pregnant and gave birth after a gestation of 93 (n = 2) and 95 (n = 1) days. Fecal P concentrations during pregnancy increased to 11.64 ± 1.3 μg/g feces, or 5.8-fold above basal concentrations. The results of this study provide a comprehensive characterization of reproductive endocrinology in snow leopards, and confirm that fecal hormone monitoring is an effective way to monitor female snow leopards throughout the breeding season.     

Comparative analysis of fecal microbiota and intestinal microbial metabolic activity in captive polar bears

The composition of the intestinal microbiota depends on gut physiology and diet. Ursidae possess a simple gastrointestinal system composed of a stomach, small intestine, and indistinct hindgut. This study determined the composition and stability of fecal microbiota of 3 captive polar bears by group-specific quantitative PCR and PCR-DGGE (denaturing gradient gel electrophoresis) using the 16S rRNA gene as target. Intestinal metabolic activity was determined by analysis of short-chain fatty acids in feces. For comparison, other Carnivora and mammals were included in this study. Total bacterial abundance was approximately log 8.5 DNA gene copies·(g feces)-1 in all 3 polar bears. Fecal polar bear microbiota was dominated by the facultative anaerobes Enterobacteriaceae and enterococci, and the Clostridium cluster I. The detection of the Clostridium perfringens α-toxin gene verified the presence of C.?perfringens. Composition of the fecal bacterial population was stable on a genus level; according to results obtained by PCR-DGGE, dominant bacterial species fluctuated. The total short-chain fatty acid content of Carnivora and other mammals analysed was comparable; lactate was detected in feces of all carnivora but present only in trace amounts in other mammals. In comparison, the fecal microbiota and metabolic activity of captive polar bears mostly resembled the closely related grizzly and black bears.     

The effect of selenium enrichment on baker's yeast proteome

The use of regular yeast (RY) and selenium-enriched yeast (SEY) as dietary supplement is of interest because the Nutritional Prevention of Cancer (NPC) trial revealed that SEY but not RY decreased the incidence of prostate cancer (PC). Using two-dimensional difference in gel electrophoresis (2D-DIGE)-tandem mass spectrometry (MS/MS) approach, we performed proteomic analysis of RY and SEY to identify proteins that are differentially expressed as a result of selenium enrichment. 2D-DIGE revealed 96 candidate protein spots that were differentially expressed (p ≤ 0.05) between SEY and RY. The 96 spots were selected, sequenced by LC/MS/MS and 37 proteins were unequivocally identified. The 37 identified proteins were verified with ProteinProphet software and mapped to existing Gene Ontology categories. Furthermore, the expression profile of 5 of the proteins with validated or putative roles in the carcinogenesis process, and for which antibodies against human forms of the proteins are available commercially was verified by western analysis. This study provides evidence for the first time that SEY contains higher levels of Pyruvate Kinase, HSP70, and Elongation factor 2 and lower levels of Eukaryotic Translation Initiation Factor 5A-2 and Triosephosphate Isomerase than those found in RY.     

Characterization of reproductive cycles and adrenal activity in the black‐footed ferret (Mustela nigripes) by fecal hormone analysis

The reproductive cycle of the black‐footed ferret (Mustela nigripes) was characterized by enzyme immunoassay (EIA) analysis of ovarian fecal steroids (estradiol, progestins) in 29 females over two consecutive breeding seasons. Estrous status was determined by measuring the vulva size and examining the percentage of superficial cells in vaginal lavages. Mean fecal estradiol concentrations were correlated with vulval area (r = 0.370, P < 0.0001) and the percentage of superficial cells (r = 0.380, P < 0.0001). Ovulation resulted in a rise in fecal progestin concentrations 5 days after breeding that differed (P < 0.05) between pregnant (n = 14) and pseudopregnant (n = 12) females during the late luteal phase (days 12–40), with concentrations remaining higher in pregnant animals. Gestation length was 41.3 ± 0.7 days with 3.6 ± 0.4 kits produced per female. Litter size correlated significantly (P < 0.05) with fecal estradiol, but not progestins during the 12 to 40 days after breeding. Females failing to breed (n = 3) remained in estrus for 31 ± 6.2 days before ovulation induction with human chorionic gonadotropin. Adrenal activity in male (n = 4) and female (n = 6) black‐footed ferrets was monitored by quantifying fecal corticoid metabolites after a series of manipulations (physical restraint, intramuscular saline, intramuscular gel adrenocorticotropic hormone (ACTH), intramuscular liquid ACTH). A significant (P < 0.0001) increase in fecal corticoids above the pre‐treatment baseline occurred 20 to 44 hours after restraint (five of 10 animals), saline (six of nine), gel ACTH (seven of 10), and liquid ACTH (nine of 10) treatments. Immunoreactivity of high‐performance liquid chromatography–separated fecal elutes was compared using antibodies against cortisol and corticosterone. The cortisol EIA demonstrated immunoreactivity that co‐eluted with ³H‐cortisol, whereas a corticosterone radioimmunoassay detected a metabolite peak that co‐eluted with ³H‐corticosterone in addition to a slightly less polar and one considerably more polar peak. Despite recognizing different metabolites, both assays produced similar temporal profiles of corticoid excretion after manipulation. This study provides new information on the black‐footed ferret regarding differences in fecal steroid excretion patterns between pregnancy and pseudopregnancy and the potential application of fecal corticoid metabolite monitoring for evaluating responses to stressors associated with practices used in breeding management. Zoo Biol 20:517–536, 2001. © 2002 Wiley‐Liss, Inc.     

Differential proteomics analysis of female and male adults of Angiostrongylus cantonensis

In this study, we identified the differentially expressed proteins of female and male adults of Angiostrongylus cantonensis through differential proteomics. We extracted and purified total proteins from male and female adults, separated proteins by two-dimensional difference gel electrophoresis (2D-DIGE) in pH 4-7, analyzed the gel images by DeCyder 7.0 software, and sacrificed the infected rats to count the number of male and female adults. It was found 28 protein spots that were differentially expressed; seven protein spots were then identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Five proteins were up-regulated and two proteins down-regulated in male adults compared with female adults. Three of the five up-regulated proteins with known functions ascribed to them were identified as galectin-1, proteasome alpha subunit and peroxiredoxin. The two down-regulated proteins were identified as indoleamine dioxygenase like-myoglobin and galectin. Furthermore, the female was significantly greater than male adults (P<0.01) in the rats. The findings demonstrate the differences in protein expression profiles and the ability to survive in the final host between female and male adults of A. cantonensis, and may provide a theoretical basis to study their developmental biology further.     

Pregnancy diagnosis in sows: direct ELISA for estrone in feces and its prospects for an on-farm test, in comparison to ultrasonography

The usefulness of fecal estrone (E1) measurement as a tool for pregnancy diagnosis was investigated. Concentrations of E1 were measured in feces from pregnant and nonpregnant sows by a direct ELISA without extraction. Highly significant differences in E1 concentrations were found in feces from nonpregnant and pregnant sows (P = 0.016 to < 0.001). Pregnancy diagnosis on Days 26 to 32 after insemination, based both on fecal E1 concentrations as measured by ELISA and ultrasonography using a 5.0 MHz linear-array transducer, was performed in a group of 496 gilts and sows. The fecal E1 test had a sensitivity (correct diagnosis of pregnancy) of 96.5% and a specificity (correct diagnosis of nonpregnancy) of 93.6%, using 3.65 ng E1/g feces as a cut-off value. For ultrasonographic pregnancy diagnosis the test sensitivity and specificity were 99.3 and 92.5%, respectively. Although an increase of fecal E1 concentrations was noticed for increasing litter sizes, the results indicated that these concentrations could not be used to predict litter size. It is concluded that the distribution of fecal E1 concentrations in both nonpregnant and pregnant sows offers a suitable basis for the development of a simple, sow-side pregnancy test.     

Pregnancy diagnosis based on the fecal progesterone concentration in beef and dairy heifers and beef cows

The present study was undertaken to examine whether pregnancy diagnosis was possible by measuring fecal progesterone concentrations in beef and dairy heifers and beef cows. Rectal fecal samples collected on days 18–24 after insemination or days 11–17 after embryo transfer were mixed with methanol and shaken for preparation of a fecal solution. After centrifugation, the supernatant was extracted with petroleum ether followed by an enzyme immunoassay for progesterone. All pregnant animals showed fecal progesterone concentrations greater than 50 ng/g of fecal material on days 18–24 after AI or estrus. In non-pregnant animals, however, the fecal progesterone concentrations ranged widely from 5 to 180 ng/g of fecal material. In non-pregnant cattle, the percentage of cattle with <50 ng progesterone/g of fecal material compared with the total number was 37–60% on days 18–20, whereas the percentages increased more than 70% to a maximum of 78.1% on day 23. When 50 ng/g was considered as the cut-off value, the sensitivity and specificity of positive pregnancy tests were less than 70% on days 21–24, and 100% for negative pregnancy tests on days 18–24. There were significant differences in the mean fecal progesterone concentrations between pregnant and non-pregnant cattle on days 19–24. These results suggest that feces can be utilized to substitute for plasma and milk to measure progesterone for the purpose of pregnancy diagnosis in heifers and cows.     

Seasonal changes in ovarian steroid hormone concentrations in the large hairy armadillo (Chaetophractus villosus) and the crying armadillo (Chaetophractus vellerosus)

Knowledge of armadillo reproductive physiology is essential for developing ex situ and in situ assisted reproductive techniques for propagating and/or controlling populations of these animals. The present study included assessment of fecal sex steroids by radioimmunoassay, determining reproductive status via monitoring ovarian activity (in the wild) and therefore reproductive status, in wild females of the large hairy armadillo (Chaetophractus villosus) and the crying armadillo (Chaetophractus vellerosus) in the southern hemisphere. Plasma and fresh fecal progesterone concentrations were not significantly correlated in either species. However, in both species, there was a significant positive correlation between plasma progesterone and dry fecal progesterone concentrations (r = 0.82, P < 0.05 and r = 0.60, P < 0.05, respectively). Dry fecal progesterone and estradiol concentrations were measured in one captive C. villosus (average baseline progesterone and estradiol concentrations 28.72 ± 11.75 ng/g dry feces and 3.04 ± 0.80 ng/g dry feces, respectively) and one captive C. vellerosus (average baseline progesterone and estradiol concentrations 14.05 ± 3.03 ng/g dry feces and 3.46 ± 1.20 ng/g dry feces, respectively) to detect hormonal peaks over 1 y; these occurred from late fall to early summer. Feces from wild C. villosus and C. vellerosus were also collected over 1 y to determine progesterone peaks, which occurred in winter and spring in both species (with no peaks during the summer or fall). Accordingly, C. villosus and C. vellerosus had a seasonal reproductive pattern. The significant correlations between dry fecal and plasma progesterone concentrations validated this method for monitoring reproductive status in these species.     

Fecal progesterone metabolites and ovarian activity in cycling and pregnant mountain gazelles (Gazella gazella)

Fecal reproductive progestagen monitoring in the mountain gazelle (Gazella gazella) provided a non-invasive method for tracking reproductive cycling, estimating age of sexual maturity and diagnosing pregnancy in this species of gazelle. Fresh fecal samples were collected from eight female mountain gazelle (Gazella gazella) for a period of two months. Two of the animals were pregnant while the other six were not. Using the progestagen profile the luteal phase, interluteal (follicular) phase and estrous cycle in adult female gazelles were determined to be 12.5 ± 1.2, 5.9 ± 0.59 and 18.8 ± 0.98 days respectively. Significant inter-animal differences in fecal progestagen concentration were observed in both the luteal and follicular phases. Significant differences were observed in the levels of fecal progestagen between cycling females and females in late pregnancy. Low concentrations of fecal progestagen in females aged less than 18 months old indicated that sexual maturity in captivity is not attained before that age.     

Alteration of DBP Levels in CSF of Patients with MS by Proteomics Analysis

Objective The diagnosis of multiple sclerosis (MS) is still challenging recently due to the lack of a specific diagnostic test. Proteomics analysis was applied to biomarkers discovery and their pathways study. Methods First, the proteins of CSF from MS patients and control group were analyzed individually with 2D-DIGE technology (two-dimensional difference gel electrophoresis). Then, protein spots were found out with DeCyder6.0 software which showed different expression levels in the gel images between the two groups. The information regarding these proteins was collected based on MALDI-TOF/MS and related database searches. Lastly, interaction between these proteins was further analyzed by using Metacore software. Results There were 13 proteins that showed more than 1.5-fold difference in expression levels between the two groups. Furthermore, the identification made by MALDI-TOF/MS revealed that one of the most significant differential proteins was DBP (vitamin D-binding protein), which decreased in the experimental group. This result was confirmed by ELISA (P < 0.01). Moreover, network between the 13 proteins were partially got, which showed some biological interactions. Conclusion These results support a correlation between the level of DBP and MS. DBP may be a potential useful biomarker for diagnosis or a medicine target for treatment of MS.     

LCM纯化的鼻咽癌间质和正常鼻咽间质的定量蛋白质组学研究

间质在肿瘤发生发展中的作用越来越受到重视．为寻找与鼻咽癌( nasopharyngeal carcinoma,NPC )发生发展相关的特异性间质蛋白,采用激光捕获显微切割技术( laser capture microdissection,LCM )纯化鼻咽癌间质和正常鼻咽黏膜间质,荧光差异双向凝胶电泳( fluorescent two-dimensional difference gel electrophoresis 2-D,DIGE )结合质谱技术分离鉴定间质相关蛋白．Western blot及免疫组织化学技术验证了其中3个差异蛋白(CapG、L-plastin和S100A9),证实了2D-DIGE结果的可靠性．建立了LCM 纯化的鼻咽癌间质和正常鼻咽间质的荧光差异蛋白表达图谱,高通量筛选与肿瘤发生相关的间质蛋白,共得到34个有统计学意义的蛋白质点,质谱鉴定得到20个差异蛋白．研究结果提示：这些差异表达的蛋白质将有助于阐明鼻咽癌细胞和周围间质的关系．对间质蛋白功能的进一步研究,将有助于解析间质在肿瘤发生中的作用机制,并为从间质途径寻找肿瘤治疗靶标提供新思路．     

Non-invasive detection and monitoring of estrus,pregnancy and the postpartum period in pygmy loris (Nycticebus pygmaeus) using fecal estrogen metabolites

Estrone-conjugates (E₁C) were measured in the feces of six female pygmy lorises (Nycticebus pygmaeus) during estrus (n = 12), pregnancy (n = 4) and the postpartum period (n = 3). Noninvasive feces collection permitted frequent sampling throughout estrus and pregnancy, without disturbance of animals. The estrous period was defined as an increase in fecal E₁C levels above an average of 70 ng/g feces with peaks above 100 ng/g feces obtained in consecutive fecal samples collected over a 6- to 11-day period between the end of July and the first third of October. Comparison of the periovulatory profile of E₁C and the stage of labial opening of the vagina revealed a high agreement (P < 0.001). In all pregnant females, an E₁C rise was found approximately 47 days postestrus, the source of which may be the growing fetal placental unit. Estimated gestation lengths ranged between 187 and 198 days (n = 4). Am. J. Primatol. 41:103–115, 1997. © 1997 Wiley-Liss, Inc.     

Comparison of fecal storage methods for steroid analysis in black rhinoceroses (Diceros bicornis)

Many field studies and conservation programs for wildlife species include noninvasive endocrine monitoring of gonadal function. Freezing fecal samples immediately after collection until further analysis is often not a viable option for researchers in remote areas. Phase 1 of this study was designed to compare different methods of preserving fecal samples over several time periods (30, 90, or 180 days) in order to determine which method provided the most accurate and reliable technique for measuring fecal progestagens. Fecal samples were collected from two female black rhinoceroses (Diceros bicornis) housed at Disney's Animal Kingdom, Lake Buena Vista, FL. We compared three storage methods: 1) storing fecal samples without processing or preservatives (untreated), 2) storing an aliquot of fecal sample in 80% methanol (MeOH), and 3) drying the fecal sample in a solar box cooker prior to storage. Control samples (day 0) were collected and extracted, and then stored at ?20°C until they were analyzed. Phase 2 of the study was designed to examine the effects of long‐term storage (up to 180 days) on fecal progestagen profiles that reflect reproductive activity (pregnancy and estrous cycles). In samples obtained from a pregnant female and stored for 30 days, there were no significant differences in fecal progestagen concentrations between the three treatment conditions. However, the mean concentrations of progestagens (± SE) in untreated samples increased significantly from 8.3 ± 0.3 µg/g wet weight feces at day 0 to 17.7 ± 5.1 µg/g feces at day 90, and 17.8 ± 4.7 µg/g feces at day 180. Samples that were collected from a pregnant female and stored in 80% MeOH or dried in the solar box correlated with controls (r=0.86 and 0.87, respectively; P<0.05) at day 180. In contrast, samples that were stored without preservatives for 180 days did not correlate with controls (r=0.35, P>0.05). Progestagen concentrations from samples of the estrous cycling female showed similar results. In conclusion, fecal samples dried in a solar box cooker or stored in 80% MeOH maintained absolute and relative progestagen concentrations for at least 180 days when they were stored outdoors and exposed to the climatic conditions of central Florida. Both methods can have significant applications for the study of reproductive events in areas where access to electricity is limited. Zoo Biol 23:291–300, 2004. © 2004 Wiley‐Liss, Inc.     

Effects of salazosulfapyridine on the profile of cell surface proteins,revealed by biotinylation of cell surface proteins and 2-dimentional electrophoresis

ObjectiveWe investigated effects of salazosulfapyridine (SASP) on the protein profile of cell surface (CS)-proteins of SW982, a human synovial sarcoma cell line, using biotinylation of CS-proteins and 2-dimensional fluorescence difference gel electrophoresis (2D-DIGE).MethodsSW982 cells were treated with SASP and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5ASA). Then the cells were treated with a membrane-impermeable biotinylating reagent. Biotinylated CS-proteins were isolated using NeutrAvidin-bound beads. CS-proteins affected by the drugs were detected by 2D-DIGE and subjected to mass spectrometry.ResultsBy the 2D-DIGE analysis, in total 576 spots were detected, 29 out of which showed more than ±1.5-fold different intensity in the SASP-, SP-, and 5ASA-treated cells, compared to non-treated cells (p < 0.05). Interestingly, 7 out of the 29 spots changed their intensity only by SASP and 17 spots changed their intensity only by SP. We identified 9 protein from 15 out of the 29 spots, most of which were evidenced to exist on the cell surface by flow cytometry.ConclusionWe found novel effects of SASP and its metabolites on SW982 cells by the combination of biotinylation of cell surface proteins and 2D-DIGE analysis. These data would help understanding of anti-rheumatic actions of SASP. Furthermore, the combination would be a useful method for the analysis of CS-proteins in various conditions.     

Observations of mortality associated with extended open-water swimming by polar bears in the Alaskan Beaufort Sea

During aerial surveys in September 1987–2003, a total of 315 live polar bears were observed with 12 (3.8%) animals in open water, defined for purposes of this analysis as marine waters >2 km north of the Alaska Beaufort Sea coastline or associated barrier islands. No polar bear carcasses were observed. During aerial surveys in early September, 2004, 55 polar bears (Ursus maritimus) were seen, 51 were alive and of those 10 (19.9%) were in open water. In addition, four polar bear carcasses were seen floating in open water and had, presumably, drowned. Average distance from land and pack ice edge for live polar bears swimming in open water in 2004 (n=10) were 8.3±3.0 and 177.4±5.1 km, respectively. We speculate that mortalities due to offshore swimming during late-ice (or mild ice) years may be an important and unaccounted source of natural mortality given energetic demands placed on individual bears engaged in long-distance swimming. We further suggest that drowning-related deaths of polar bears may increase in the future if the observed trend of regression of pack ice and/or longer open water periods continues.     

Luteal blood flow increases during the first three weeks of pregnancy in lactating dairy cows

The objectives of this experiment were to characterize luteal blood flow in pregnant and non-pregnant cows and to determine its value for early pregnancy diagnosis. Lactating dairy cows (n = 54), 5.2 ± 0.2 y old (mean ± SEM), average parity 2.4 ± 0.2, and ≥ 6 wk postpartum at the start of the study, were used. The corpus luteum (CL) was examined with transrectal color Doppler ultrasonography (10.0-MHz linear-array transducer) on Days 3, 6, 9, 11, 13, 15, 18, and 21 of the estrus cycle (estrus = Day 0). Artificially inseminated cows (n = 40) were retrospectively classified as pregnant (embryonic heartbeat on Day 25; n = 18), nonpregnant (interestrus interval 15 to 21 d, n = 18), or having an apparent early embryonic loss (interestrus interval >25 d, n = 4). There was a group by time interaction (P < 0.001) for luteal blood flow from Days 3 to 18; it was approximately 1.10 ± 0.08 cm² (mean ± SEM) on Day 3, and increased to approximately 2.00 ± 0.08 cm² on Day 13 (similar among groups). Thereafter, luteal blood flow was numerically (albeit not significantly) greater in pregnant cows, remained constant in those with apparent embryonic loss, and declined (not significantly) between Days 15 and 18 in nonpregnant cows. Luteal blood flow was greater in pregnant than in nonpregnant (P < 0.05) and nonbred cows (P < 0.05, n = 14) on Day 15 (2.50 ± 0.16, 2.01 ± 0.16, and 2.00 ± 0.18 cm², respectively) and on Day 18 (2.40 ± 0.19, 1.45 ± 0.19, and 0.95 ± 0.21 cm²). In cows with apparent early embryonic loss, luteal blood flow was 2.00 ± 0.34 and 2.05 ± 0.39 cm² on Days 15 and 18, which was less (not significantly) than in pregnant cows, but greater (P < 0.05) than in nonbred cows on Day 18. Although mean luteal blood flow was significantly greater in pregnant than nonpregnant (and nonbred) cows on Days 15 and 18, due to substantial variation among cows, it was not an appropriate diagnostic tool for pregnancy status.     

Direct ELISA for estrone measurement in the feces of sows: prospects for rapid, sow-side pregnancy diagnosis

Both free and conjugated fecal estrogens were surveyed in sows by means of RIA's after extraction and column chromatography. Six different RIA's were performed using the same fecal suspension. Based on differences in concentration in feces from 6 pregnant and 4 nonpregnant sows, estrone (E1) was selected for the development of a homologous, competitive ELISA for pregnancy diagnosis purposes. Polyclonal antibodies were raised against 6-keto-E1-carboxymethyloxime coupled to BSA. Biotinylated E1 was used as the competitive agent and was detected with a streptavidin-peroxidase conjugate. Tetramethylbenzidine was used as chromogen. Validation of this direct, easy to perform ELISA showed satisfactory specificity, accuracy, precision and sensitivity. Comparison of the assay with a validated RIA (including extraction and column chromatography) resulted in a linear regression equation of ELISA = 0.88 RIA - 0.72 (r = 0.95; n = 24 fecal samples). The E1 values in feces from 11 pregnant sows during the first 40 d of gestation compared with values from 10 nonpregnant sows showed significant differences between Days 23 and 35 of pregnancy (P < 0.05). The highly significant difference in E1 concentrations (P < 0.0012) between Days 27 and 30 in particular offers promise for pregnancy diagnosis.     

A proteomic approach towards understanding crypoprotective action of Me2SO on the CHO cell proteome

Chinese hamster ovary (CHO) cell lines are the most widely used in vitro cells for research and production of recombinant proteins such as rhGH, tPA, and erythropoietin. We aimed to investigate changes in protein profiles after cryopreservation using 2D-DIGE MALDI-TOF MS and network pathway analysis. The proteome changes that occur in CHO cells between freshly prepared cells and cryopreserved cells with and without Me2SO were compared to determine the key proteins and pathways altered during recovery from cryopreservation. A total of 54 proteins were identified and successfully matched to 37 peptide mass fingerprints (PMF). 14 protein spots showed an increase while 23 showed decrease abundance in the Me2SO free group compared to the control. The proteins with increased abundance included vimentin, heat shock protein 60 kDa, mitochondrial, heat shock 70 kDa protein 9, protein disulfide-isomerase A3, voltage-dependent anion-selective channel protein 2. Those with a decrease in abundance were myotubularin, glutathione peroxidase, enolase, phospho glyceromutase, chloride intracellular channel protein 1. The main canonical functional pathway affected involved the unfolded protein response, aldosterone Signaling in Epithelial Cells, 14-3-3-mediated signaling. 2D-DIGE MALDI TOF mass spectrometry and network pathway analysis revealed the differential proteome expression of FreeStyle CHO cells after cryopreservation with and without 5% Me2SOto involve pathways related to post-translational modification, protein folding and cell death and survival (score = 56, 22 focus molecules). This study revealed, for the first time to our knowledge the proteins and their regulated pathways involved in the cryoprotective action of 5% Me2SO. The use of 5% Me2SO as a cryoprotectant maintained the CHO cell proteome in the cryopreserved cells, similar to that of fresh CHO cells.