Progress 09/20/19 to 05/31/21
Outputs
Target Audience:The objective of this study was to characterize the protein expression of sperm acrosome associated 3 (SPACA3) in theequine ovary. Formalin-fixed and paraffin-embedded ovarian sections from 16 horses were processed for routineimmunohistochemistry for SPACA3. Representative images were digitally captured at 400 x magnification. In all mares,SPACA3 was expressed in granulosa cells of all stages of follicles. Expression of SPACA3 in all equine follicular stagessuggests that this may be a permanent immunosterilant target for the management of feral horse herds. Additional research is needed to determine if horses can produce a robust humoral response to a SPACA3 vaccine to induce sustained infertility. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Brynley Cozzi (undergraduate student majoring in Animal Science) performed most of the experiments under supervision andpresented the results at the scientific meeting. How have the results been disseminated to communities of interest?The results of this research were presented virtually at the 2020 Society for Theriogenology conference and have beenpublished in the journal Clinical Theriogenology. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Both domesticated mares (n = 8) and feral mares (n = 8) were used in this study. Domesticated mares (3 - 14 years old) werepastured on a ranch located in Stayton, Oregon. Each pasture contained a 4.9 x 7.3 meter, 3-sided shelter. Feral mares (3years old) were maintained at the Warm Springs herd management area located in Harney county, Oregon. The procedureswere conducted under a protocol approved by the institutional animal care and use committee of Oregon State University(protocol #3924).Ovaries were obtained via a standing colpotomy performed by an experienced veterinarian. Briefly, feed was withheld for 36hours prior to surgery. For sedation and analgesia, detomidine hydrochloride (0.02 - 0.04 mg/kg), butorphanol tartrate (0.01 -0.04 mg/kg), and xylazine hydrochloride (0.9 - 1.2 mg/kg) were given intravenously. Surgical preparation included wrappingthe tail with gauze and tying it up to keep it out of the surgical field, transrectal palpation to manually evacuate the feces, scrubbing the perineal area with chlorhexidine (Vet Solutions, Inc., Bedford, TX), and flushing the vagina with very dilutepovidone iodine. An incision in the anterior dorsolateral wall of the vagina was made, 2% lidocaine was injected into eachovarian pedicle for local analgesia, and the ovaries were removed using a chain ecrasure. The vaginal incision was left to healby second intention. For postsurgical analgesia, flunixin meglumine was given intravenously (1.2 -1.5 mg/kg) andbuprenorphine hydrochloride (10 mg) was given subcutaneously. Additionally, each mare received intramuscularly the long-acting antibiotic ceftiofur crystalline-free acid (6.6 mg/kg).Ovaries were hemi-sectioned, fixed in 10% neutral buffered formalin, and paraffin embedded. Sections were serially sectioned(4 μm) on charged slides for routine immunohistochemistry for SPACA3. Slides from all mares were processed in 1experiment to avoid variations. Briefly, slides were deparaffinized in xylene and rehydrated in a graded ethanol series (100,75, and 50%). Antigen retrieval was conducted by incubating sections in a citrate buffer, Target Retrieval Solution #S1699(Dako North America Inc., Carpinteria, CA) in a microwave for 10 minutes and cooled for 20 minutes. Slides were thenwashed in buffer (Dako North America Inc., Wash buffer #S3006) and tissue-specific endogenous peroxidases were inhibitedby incubating slides in 3% hydrogen peroxide. Slides were washed again and nonspecific binding was blocked with serum-free protein (Protein block serum-free ready to use, #X0909, Dako North America Inc.) for 20 minutes at room temperature.Subsequently, slides were tapped off and the primary antibody (#21137-1-AP, Proteintech, Rosemont, CA) diluted 1:200(background reducing components, #S3022, Dako North America Inc.) was applied to sections for 105 minutes at roomtemperature. A fusion protein with the following sequence was used to produce primary antibody.Primary antibody reactivity was confirmed by the manufacturer in the mouse testis and was also confirmed in a preliminaryexperiment in the equine testis. Specificity of immunostaining was verified by replacing the primary antibody with negativecontrol rabbit serum (negative control rabbit IgG, #NC495H, Biocare Medical, Pacheco, CA). Slides were washed in bufferand a secondary antibody (One step horseradish peroxidase-conjugated polymer antirabbit IgG, #IH-8064-OSU-15, ImmunoBiosCience, Mukilteo, WA) was applied to sections for 30 minutes at room temperature. The secondary antibody was washedoff and NovaRED (#SK4800, Vector Laboratories Inc., Burlingame, CA) was applied to sections for 5 minutes at roomtemperature. Sections were then counter stained in hematoxylin, dehydrated in a graded series of ethanol (50, 75, and100%), moved through a series of 3 xylene baths and cover slipped.Slides were evaluated by a single observer at 400 x magnification with a bright-field microscope (Leica DM4000B, LeicaMicrosystems Inc. Buffalo Grove, IL). Representative images from each ovary were electronically captured using a digitalcamera (QImaging, QICAM 12-BIT, #QIC-F-M-12-C, Surrey, BC, Canada) and image capture software (QCapturePro,Surrey). The cellular expression on SPACA3 was recorded for primordial, primary, secondary, and tertiary follicles.SPACA3 was localized to the sperm acrosomes in the equine testis (positive control), and to the pregranulosa cells ofprimordial follicles, and to the granulosa cells of primary, secondary, and tertiary follicles of all equine ovaries examined.There was no positive staining in any other cell type. In addition, slides stained with the universal negative did not have anypositive staining.
Publications
|
Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The objective of this study was to characterize the protein expression of sperm acrosome associated 3 (SPACA3) in the equine ovary. Formalin-fixed and paraffin-embedded ovarian sections from 16 horses were processed for routine immunohistochemistry for SPACA3. Representative images were digitally captured at 400 x magnification. In all mares, SPACA3 was expressed in granulosa cells of all stages of follicles. Expression of SPACA3 in all equine follicular stages suggests that this may be a permanent immunosterilant target for the management of feral horse herds. Additional research is needed to determine if horses can produce a robust humoral response to a SPACA3 vaccine to induce sustained infertility. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Brynley Cozzi (undergraduate student majoring in Animal Science) performed most of the experiments under supervision and presented the results at the scientific meeting. How have the results been disseminated to communities of interest?The results of this research were presented virtually at the 2020 Society for Theriogenology conference and have been published in the journal Clinical Theriogenology. What do you plan to do during the next reporting period to accomplish the goals?The project has been completed.
Impacts
What was accomplished under these goals?
Both domesticated mares (n = 8) and feral mares (n = 8) were used in this study. Domesticated mares (3 - 14 years old) were pastured on a ranch located in Stayton, Oregon. Each pasture contained a 4.9 x 7.3 meter, 3-sided shelter. Feral mares (3 years old) were maintained at the Warm Springs herd management area located in Harney county, Oregon. The procedures were conducted under a protocol approved by the institutional animal care and use committee of Oregon State University (protocol #3924). Ovaries were obtained via a standing colpotomy performed by an experienced veterinarian. Briefly, feed was withheld for 36 hours prior to surgery. For sedation and analgesia, detomidine hydrochloride (0.02 - 0.04 mg/kg), butorphanol tartrate (0.01 - 0.04 mg/kg), and xylazine hydrochloride (0.9 - 1.2 mg/kg) were given intravenously. Surgical preparation included wrapping the tail with gauze and tying it up to keep it out of the surgical field, transrectal palpation to manually evacuate the feces, scrubbing the perineal area with chlorhexidine (Vet Solutions, Inc., Bedford, TX), and flushing the vagina with very dilute povidone iodine. An incision in the anterior dorsolateral wall of the vagina was made, 2% lidocaine was injected into each ovarian pedicle for local analgesia, and the ovaries were removed using a chain ecrasure. The vaginal incision was left to heal by second intention. For postsurgical analgesia, flunixin meglumine was given intravenously (1.2 -1.5 mg/kg) and buprenorphine hydrochloride (10 mg)was given subcutaneously. Additionally, each mare received intramuscularly the long-acting antibiotic ceftiofur crystalline-free acid (6.6 mg/kg). Ovaries were hemi-sectioned, fixed in 10% neutral buffered formalin, and paraffin embedded. Sections were serially sectioned (4 μm) on charged slides for routine immunohistochemistry for SPACA3. Slides from all mares were processed in 1 experiment to avoid variations. Briefly, slides were deparaffinized in xylene and rehydrated in a graded ethanol series (100, 75, and 50%). Antigen retrieval was conducted by incubating sections in a citrate buffer, Target Retrieval Solution #S1699 (Dako North America Inc., Carpinteria, CA) in a microwave for 10 minutes and cooled for 20 minutes. Slides were then washed in buffer (Dako North America Inc., Wash buffer #S3006) and tissue-specific endogenous peroxidases were inhibited by incubating slides in 3% hydrogen peroxide. Slides were washed again and nonspecific binding was blocked with serum-free protein (Protein block serum-free ready to use, #X0909, Dako North America Inc.) for 20 minutes at room temperature. Subsequently, slides were tapped off and the primary antibody (#21137-1-AP, Proteintech, Rosemont, CA) diluted 1:200 (background reducing components, #S3022, Dako North America Inc.) was applied to sections for 105 minutes at room temperature. A fusion protein with the following sequence was used to produce primary antibody. Primary antibody reactivity was confirmed by the manufacturer in the mouse testis and was also confirmed in a preliminary experiment in the equine testis. Specificity of immunostaining was verified by replacing the primary antibody with negative control rabbit serum (negative control rabbit IgG, #NC495H, Biocare Medical, Pacheco, CA). Slides were washed in buffer and a secondary antibody (One step horseradish peroxidase-conjugated polymer antirabbit IgG, #IH-8064-OSU-15, Immuno BiosCience, Mukilteo, WA) was applied to sections for 30 minutes at room temperature. The secondary antibody was washed off and NovaRED (#SK4800, Vector Laboratories Inc., Burlingame, CA) was applied to sections for 5 minutes at room temperature. Sections were then counter stained in hematoxylin, dehydrated in a graded series of ethanol (50, 75, and 100%), moved through a series of 3 xylene baths and cover slipped. Slides were evaluated by a single observer at 400 x magnification with a bright-field microscope (Leica DM4000B, Leica Microsystems Inc. Buffalo Grove, IL). Representative images from each ovary were electronically captured using a digital camera (QImaging, QICAM 12-BIT, #QIC-F-M-12-C, Surrey, BC, Canada) and image capture software (QCapturePro, Surrey). The cellular expression on SPACA3 was recorded for primordial, primary, secondary, and tertiary follicles. SPACA3 was localizedto the sperm acrosomes in the equine testis (positive control), and to the pregranulosa cells of primordial follicles, and to the granulosa cells of primary, secondary, and tertiary follicles of all equine ovaries examined. There was no positive staining in any other cell type. In addition, slides stained with the universal negative did not have any positive staining.
Publications
|
Progress 09/20/19 to 09/30/19
Outputs
Target Audience:Those with interest and stake in animal welfare and care,especially those responsible for the management ofwild horses and burros. Changes/Problems:We were unable to collect ovaries from20 wild horse mares due to availability of animals but we were able to collect normal ovaries from 8 wild horse mares. We were able to collect normal ovaries from 8 domestic horse mares and were able to compare SPRASA expression. There was no difference in SPRASA expression in wild horse and domestic horse ovaries. In addition, we were able to collect ovaries with tumors (specifically granulosa cell tumors) from 5 mares. This was important because it led to an unexpected finding that SPRASA expression is increased in tumor tissue compared to normal tissue. What opportunities for training and professional development has the project provided?Brynley Cozzi (senior undergraduate student majoring in Animal Science at Oregon State University) was trained to perform the immunochemistry experiments and the image analysis. Brynley was assisted by Zahra Kiesler (immunohistochemistry experiments) and Ann Ramsey (image analysis), who are also undergraduate students majoring in Animal Science at Oregon State University that benefitted from training from this funding. Brynley wrote a scientific abstract that was submitted to the Society for Theriogenology. Her abstract was selected as one of the top 8 (out of over 80) to be presented during the "Competitive Category" of oral presentations typically limited to graduate students and residents. During the competition in July 2020, Brynley's presentation was awarded 4th place and received a cash prize of $200, which is an amazing achievement considering all of the other presenters were veterinarians in advanced training programs. In September 2020, Brynley submitted a scientific article to the Journal of Veterinary Diagnostic Investigation. The manuscript that she wrote is currently under review. How have the results been disseminated to communities of interest?The results have been presented in both written (abstract) and verbal formats to veterinarians focusing on animal reproduction (theriogenology). What do you plan to do during the next reporting period to accomplish the goals?We plan to make any edits necessary for revision of the submitted manuscript so that it will be published before the next reporting period.
Impacts
What was accomplished under these goals?
Tissue sections of normal ovaries from domesticated horses (n=8) and from wild horses (n=8) as well as ovaries with granulosa cell tumors from domesticated horses (n=5) were subjected toroutine immunohistochemistry against SPRASA.SPRASA was expressed in all normal ovaries from domesticated mares and wild mares. SPRASA was expressed in granulosa cells of primordial, primary, secondary, and tertiary follicles. However, SPRASA expression was more intense in granulosa cells oftertiary follicles compared primordial follicles. There was no difference in SPRASA follicular expression between domesticated mares and wild mares. However, granulosa cells from tumors in domestic mares expressed SPRASA with greater intensity than granulosa cells from mares withnormal ovarian follicles.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
COZZI, B., HABEEB, H. and KUTZLER, M. 2020. Sperm protein reactive with antisperm antibody is immunoexpressed in equine primordial, primary, secondary, and tertiary follicles. Clin. Theriogenology. 12(3):362.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
COZZI, B. and KUTZLER, M. Sperm Acrosome Associated 3 (SPACA3) protein is immunoexpressed in equine primordial, primary, secondary, and tertiary follicles. J. Vet. Diagn. Invest.
|
|