Progress 08/14/15 to 09/30/16
Outputs
Target Audience:Acupuncture is already accepted by producers in dairy cows as a complementary treatment for abomasal displacement (Lee et al., 2007; Jang et al., 2003) and to provide analgesia during laparatomies (Kim et al., 2004; White et al., 1985). In addition, acupuncture is accepted by producers as a complementary treatment for a variety of reproductive conditions including delayed uterine involution (Korematsu et al., 1993), repeat breeding (Lin et al 2002), Mycoplasma sp. infections (Yan et al., 2008), and other disorders (Chan et al., 2001; Habacher et al., 2006; Holiday, 1989; Turnbull, 1986). To date, there have been no published studies investigating the use of acupuncture as a complementary treatment for retained placentas in dairy cows. However, acupuncture has been used as a complementary treatment for the condition of retained placenta in humans (Bobik and Habek, 2012). Through both paracrine and autocrine regulation, acupuncture treatment stimulates production and release of catecholamines, estrogens and growth factors (Kawakita et al., 2006; Langevin et al., 2001; Stener-Victorin and Wu, 2010). Through these mechanisms, we hypothesized that acupuncture will increase MMP2 and MMP9 caruncular expression and thereby result in a more rapid separation of the cotyledon from the caruncle (i.e. faster release of the placenta). The objectives of this study were to determine the effects of acupuncture in dairy cows on caruncular MMP2 and MMP9 tissue concentrations and immunolocalization at 0, 2, and 4 hours after calving. Changes/Problems:In addition to the change previously described in the Progress Report (i.e. changing from pro-MMP2 to MMP9), we encountered serious difficulties and delays with the gel zympgraphy assay. After careful consultation with our collaborator (Dr. Fred Menino), the decision was made to measure tissue MMP2 and MMP9 concentrations using an ELISA. This was a wise decision and the results from this experiment supported those from the immunohistochemistry experiment. What opportunities for training and professional development has the project provided?Five undergraduate students from my laboratory were responsible for watching the cows in the study for calving. Only one student had previously worked around cattle so this opportunity provided important hands-on agricultural experiences for urban undergraduate students. In addition, two of these undergraduate students performed all of the laboratory experiments. Neither of these students had previously worked in a research laboratory. They both learned basic laboratory methods as well as complex techniques related to their specific projects. One student (Lauren Gentle) write a scientific abstract and then gave two poster presentation of her research findings. The other student (Katrina Hiebel) wrote a considerable amount of the manuscript that was submitted for publication. How have the results been disseminated to communities of interest?Two posters of the findings were presented in May and June of this year. Publication of the manuscript will further disseminate the results. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Immediately after a natural (spontaneous, term) calving, each cow was restrained in a stanchion and the vulva and perineal area was aseptically prepared. A caruncle was obtained from the body of the uterus using a placentome attainment device (McNeel et al., 2013). This technique has been used for research purposes extensively by its creator and found to not result in a loss of subsequent fertility (personal communication). Following caruncle collection, half of the tissue was fixed in 10% neutral buffered formalin and the other half was flash frozen in liquid nitrogen and stored at -80°C until processed. Caruncle sample collection was repeated two additional times at 2 and 4 hours after calving (for a total of three caruncles collected). This study was approved by the Oregon State University Institutional Care and Animal Use Committee (ACUP # 4570). The acupuncture treatment was administered to cows (n=6) following each caruncle collection (at 0 and 2 hours after calving). Untreated controls (n=9) were kept in a stanchion for 15 minutes without any stimulation. While standing in a stanchion, acupuncture needles (30 X 0.30 mm; No. 8, J type; Seirin Corporation, Shizuoka, Japan) with a guide tube were placed into cows and left in place for 15-20 minutes. Acupuncture was applied to six points: bladder (BL) 31, 32, and 34 (Figure 1), Baihui (lumbosacral space, DU 20), Guanyuanshu (UB 26), and governing vessel (GV)-1. Baihui, BL 31-34 and UB 26 were selected as these points relax the cervix and stimulate uterine contractions (Lin et al., 2001). Baihui and GV-1 are also the local points to move the Qi (energy) and resolve stagnation (Xie and Preast, 2007). In addition, the Baihui acupuncture point is known as the "point of 100 meetings" where all the Yang meridians merge and has been used as a complementary treatment in dairy cows for anestrus, cystic ovaries, retained or cystic corpus lutea, silent estrus, pseudopregnancy, inflammation of the reproductive tract, uterine prolapse, prevention of abortion, or to induce parturition (Lin and Panzer Jr., 1992). The formalin-fixed caruncles were paraffin embedded, sectioned to 6 μm, and mounted on poly-l-lysine-coated slides. MMP2 and MMP9 expression in the caruncle was then determined using routine immunohistochemistry. Briefly, slides were deparaffinized in xylene, rehydrated in graded ethanol series (100%, 75%, 50%), and then subjected to either enzymatic or heat-induced epitope retrieval for MMP2 or MMP9, respectively. For MMP2, proteinase K (#S3020, Dako) was applied to slides for 5 minutes. For MMP9, slides were boiled in sodium citrate (#S1700, Dako) for 10 minutes in a Nordicware® tender cooker in the microwave and then cooled to room temperature for 20 minutes. Endogenous peroxidase activity was inactivated with 3% hydrogen peroxide and nonspecific binding was blocked with Protein Block serum (#X0909, Dako). MMP2 (#LS-B2799, LifeSpan Biosciences) was applied at a 1:1000 dilution and MMP9 (#LS-A9461, LifeSpan Biosciences) at a 1:100 dilution. Negative controls from each tissue were treated with a universal negative antibody (#N1699, Dako). Slides were then reacted with One Step Horse Radish Peroxidase-Conjugated Polymer Anti-Rabbit IgG (#IH-8064-custom-OrSU, ImmunoBioScience) followed by Nova Red Peroxidase substrate (#SK4800, Vector Laboratories). Slides were counter-stained with hematoxylin (#S3302, Dako), dehydrated, and mounted. Utilizing bright-field microscopy, images were digitally captured at 400X magnification. At 50X magnification, immunoexpression of MMP2 and MMP9 was scored (0-3) across four quadrants of the caruncle by a single observer. Frozen caruncle tissue samples were cut into 0.5 cm3 pieces and combined with about 1.5 mL of solubilization buffer (0.5M NaCl, 0.001M EDTA, 0.05M Tris-HCl pH 8.1, 1% Triton X-100, 0.2% SDS). Samples were homogenized on ice using a Biospec tissue tearer (model 985-370) and then centrifuged at 10,000 x g for 10 minutes at 4°C. The supernatant was collected and the total protein concentration was determined using the Stanbio LiquiColor kit (#0345, Stanbio Laboratory) per manufacturer's instructions on a 96-well plate reader at 550 nm. Homogenized protein samples were then stored at -80°C until analyzed. MMP2 and MMP9 concentrations (pg/mL) were calculated from caruncular homogenates run in triplicate using commercial assays specific for bovine (Nori® Bovine MMP2 and MMP9 ELISA, Genorise Scientific, Inc.) in accordance to the manufacturer's instructions. Each 96-well plate was read at 450 nm with a wavelength correction set to 540 nm. For MMP2, the assay sensitivity was 32 pg/mL with an intra- and inter-assay CV of 5% and 9%, respectively. For MMP9, the assay sensitivity was 8 pg/mL with an intra- and inter-assay CV of 6% and 9%, respectively. Computer software (GraphPad Prism 7.0, GraphPad Software) was used for all statistical comparisons. The average value (score or concentration) for each time point for each individual was used to calculate the mean ± SEM for each treatment group.Tukey's multiple comparison test was used to test for significant differences between the three time points as well as between the control and acupuncture treatment groups. Differences at values p<0.05 were considered statistically significant. RESULTS Immunoexpression of MMP2 and MMP9 (data not shown) was predominately localized to the epithelial and subepithelial stromal cells of the caruncles in both treatment groups. MMP2 immunoexpression did not differ between control and acupuncture treated cows at calving or two hours after calving (after the first treatment). However, MMP2 immunoexpression was lower four hours post calving in the control cows but not in the acupuncture treated cows. Previous studies have shown that MMP2 immunoexpression decreases quickly after parturition, so this finding in the control cows in the current study was expected. It is important to note that cows treated with acupuncture had prolonged MMP2 expression of after calving. When the MMP2 immunoexpression between these two groups was directly compared, there was a trend toward an increase in the acupuncture treated cows compared to controls (p=0.06). Similar to MMP2, MMP9 immunoexpression did not differ between control and acupuncture treated cows at calving or two hours after calving (after the first treatment). However, MMP9 immunoexpression was lower four hours post calving in the control cows but not in the acupuncture treated cows. MMP2 concentration in caruncle tissue homogenates did not differ between control and acupuncture treated cows at any of the time points. However, MMP2 tissue concentration was lower two hours after calving in the control cows but not in the acupuncture treated cows. In fact, there was a trend (p=0.09) for cows treated with acupuncture to have higher MMP2 tissue concentration two hours after calving (after the first treatment) compared to the time of calving. Similar to MMP2, MMP9 tissue concentration did not differ between control and acupuncture treated cows at any of the time points. In both treatment groups, MMP9 tissue concentrations was lower at calving compared to two and four hours after calving. These results are similar to those of Takagi and coworkers (2007) who found that MMP9 mRNA expression was lower at parturition compared to six hours postpartum.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Gentle L, Kutzler M. Matrix metalloproteinase type-2 (MMP2) and type-9 (MMP9) immunoexpression in bovine caruncular tissue at the time of calving, 2 hours post-calving, and 4 hours post-calving. Presented at the Celebrating Undergraduate Excellence event on May 13, 2016 (Corvallis, OR) and the Northwest Reproduction Sciences Symposium on June 2, 2016 (Philomath, OR).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
Hiebel K, Gentle L, Kutzler M. Acupuncture increases matrix metalloproteinase type-2 immunoexpression and tissue concentration in bovine caruncles after calving. Journal of Animal Science
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Progress 08/14/15 to 09/30/15
Outputs
Target Audience:In cattle, a retained placenta is defined as the failure to pass all or part of the placenta from the uterus within 12 hours of calving (Roberts, 1971). The herd incidence of retained placentas on dairies ranges from 8.3%-28.1% (Han and Kim, 2005). Retained placentas in dairy cattle result in serious animal health and welfare problems, but also have significant economic impacts. Projected milk loss due to placental retention is 1,657 pounds per cow (over 305 days in milk) (Dubuc et al., 2011). The condition has a direct effect on the reproductive performance of cattle, increasing the calving-to-conception interval by up to 51 days and almost doubling the average number of services per pregnancy (Gröhn and Rajala-Schultz, 2000; LeBlanc, 2008). Research carried out within the past 20 years suggests that there is a physiologic basis for retained placentas but the treatment and prevention of this condition continues to elude dairy producers and veterinarians. There have been dozens of different treatments for retained placentas in cattle described. Most treatments for placental retention in cattle result in drug withdrawal times prior to slaughter that vary depending upon the antibiotic, anti-inflammatory, or hormone used (Peters and Laven, 1996; Beagley et al., 2010. Pyörälä S et al., 2014). In addition, many treatments have limited efficacy and may actually be harmful to the cow. Changes/Problems:We found in the first four cows under study that the cervix was closed by six hours making it impossible to collect the last sample. As a result, the timing of sample collection was adjusted to 0, 2, and 4 hours after collection. In addition, the pro-MMP2 antibody purchased for immunolocalization did not work on the bovine tissue. MMP9 was substituted for pro-MMP2, which works well in our laboratory. What opportunities for training and professional development has the project provided?The project is providing valuable experience for undergraduate students with an interest in going into veterinary college or graduate school. These students will be watching cows calve as well as helping with the laboratory experiments. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?I plan to complete the sample collection and laboratory experiments, analyze the results and write a manuscript. In addition, I plan to present these finding at regional and national meetings.
Impacts
What was accomplished under these goals?
In the past six weeks since the projected was funded, samples were collected from two control and two acupuncture-treated cows. In these four cows, the cervix was closed by six hours making it impossible to collect the last sample. As a result, the timing of sample collection was adjusted to 0, 2, and 4 hours after collection. In addition, the pro-MMP2 antibody purchased for immunolocalization did not work on the bovine tissue. MMP9 was substituted for pro-MMP2, which works well in our laboratory.
Publications
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