HEAT STRESS AND MALE FERTILITY IN SWINE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1006658
• Project Start Date: Oct 1, 2015
• Project End Date: Sep 30, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
Animal Sciences

Non Technical Summary
Swine are an important agricultural species to Wisconsin and the US in general. Losses due to summer infertility and heat stress to boars are significant. The major objectives of this proposal are: 1) to investigate the mechanisms by which heat stress impacts the production of sperm in the boar testes and 2) determine if mitigation to heat stress impacts on sperm production can be accomplished by pharmacological means. We propose to use scrotal insulation as a model we have developed to induce defined and repeatable heat stress to the testes of the boar. In the first aim we will examine how heat stress to the boar testes causes disruption of the blood testis barrier (BTB) and the apical ectoplasmic specialization junctions leading to loss of meiotic spermatocytes and spermatids using immuno-histochemistry, electron microscopy and western blot analysis. The time course of BTB recovery will be examined in testes of boars at various points after heat stress. The final aim is to determine if mitigation to heat stress impacts on sperm production can be accomplished by pharmacological means with FSH and PG600 (eCG +HCG) to increase cAMP in Sertoli cells and/or testosterone production by Leydig cells and promote BTB formation.

Animal Health Component

40%

Research Effort Categories

Basic

40%

Applied

40%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3510	1020	100%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1020 - Physiology;

Keywords

swine

male fertility

heat stress

spermatogenesis

Goals / Objectives
The goals of the research we propose will utilize our controlled scrotal insulation model in swine to: 1) investigate the mechanisms by which heat stress impacts the production of sperm in the boar testes and 2) determine if mitigation to heat stress impacts on sperm production can be accomplished by pharmacological means.The specific objectives are:1. To determine how heat stress to the boar testes causes disruption of the blood testis barrier (BTB) and the apical ectoplasmic specialization junctions leading to loss of meiotic spermatocytes and spermatids. Testes of heat stress boars will be infused with a tracer that does not pass the BTB and then imunohistological examination of testes section examined following euthanasia to determine if the BTB is compromised or not.2. To determine if disruption of the BTB is rapidly reversed following removal of scrotal insulation by following the integrity of the BTB following scrotal insulation via techniques used for objective 1.3. To determine if PG600 (eCG + HCG) or FSH can prevent the heat stress (scrotal insulation) damage to spermatogenesis and production of abnormal sperm.

Project Methods
We propose to use our 48 hr. scrotal insulation model to induce damage to spermatogenesis. In Experiment 1, at the end of insulation, boars are anesthetized, portions of testis infused into the interstitum with a tracer of EZ-Link Sulfo-NHS-LC_Biotin, then animals will be euthanized, testes removed and tissue prepared for analysis. The areas perfused with the Biotin conjugate will be prepared and evaluated for an intact BTB. We propose to use immunofluorescence (confocal microscopy or with thin sections using standard epiflourescence) and electron microscopy. In addition we could label antibodies with colloidal gold and perform the protein localizations with an electron microscopy approach. We will obtain antibodies known to cross react with the porcine and directed against BTB components of occludin, zonula occuludin-1(ZO-1), claudin-11. We can also use other antibodies directed against other proteins as determined necessary from the results, perhaps cadherin, desmoglein, Jams, and connexin. Also it will be of interest with the same approach to investigate the proteins of the apical ectoplasmic specializations such as VE-cadherin, Nectin's and Jams. In addition to the above, protein quantization can be done with Western blotting.While multiple assays can be performed on separate sections taken from the testes, at least 5 insulated and 5 control boars will be evaluated per protein. Again, this may include multiple different proteins evaluated on a particular sample. Statistical analysis will be performed via SAS using a one-way ANOVA for all quantitative results. Under the current HATCH (Yr 3), we are investigating the real-time PCR expression of mRNA from the genes responsible for the above described proteins so that will not be required for this proposal.In experiment 2, we will repeat procedures from experiment 1 that were found to be useful in identify disruptions to junctional complexes. The boars in an individual replicate will be scrotal insulated for 48 hr. and then sacrificed at 0, 2, 4 or 7 days following sack removal. An additional control boar will be included as well that is sham insulated and sacrificed at sack removal. This implies 5 boars per replicate and we will perform at least 3 replicates. If recovery has not occurred by 7 days then we will reassess the time points for additional replicates.In experiment 3, for each replicate we will scrotal insulate 4 boars, 1 control sham treated boar, 1 boar with drug intervention, 1 boar treated day -1 to 0 with 100 mg Follitropin (FSH) given every 12 hours, 1 boar given PG600 at 5ml (400 IU eCG, 200 IU HCG, standard dose)at day -1. The administration of FSH and PG600 before insulation is to ensure any transcription that might occur has sufficient time for translation. Also FSH has a shorter half-life than PG600 and so multiple injections are being used. At the end of insulation, boars will be euthanized and evaluated with testes morphometry of fixed and stained cross sections as we are currently doing with the existing HATCH or using methods described in experiment 1. The experiment will be replicated 5 times.

Progress 10/01/15 to 09/30/18

Outputs
Target Audience:The target audience reached seminar for International Farmers Aid ASSN (2018 international undergraduate students training in animal farm production, NCERA57 annual meeting (2018, researchers in swine reproduction in the US), American Association of Animal Science annual meeting (2018, researchers, educators and industry representatives in swine production).Research at meetings was discussed in terms of how climate and heat impacts fertility of boars and mechanisms of how heat impacts spermatogenesis. Undergraduate students at UW-Madison were reached through a case scenario entitled "Impact of the male on meat production: A case scenario in swine" focused on the effects on heat stress on male reproduction utilizing actual data collected in the project. Students are asked to analyze the data, interpret results, and explain the impact of heat stress on the boar and their ability to impregnate a female. Changes/Problems: As stated in the previous progress reports sham sacks were shown to slightly increase testicular temperature and control boars no longer have any type of sack applied. Further, boars did not need to be anesthetized to administer EZ-LinkSulfo- NHS-Biotin as blood clots were apparent in the testes after administration and harvest. We did not seedrastic changes to the BTB as was expected in objective 1 and so could not pursue objective 2. We did address objective 3 to analyze the efficiency of PG600 to minimize the effects of heat stress on spermatozoa in the ejaculate. The PG600 treatment appears to complete eliminate the impacts of short term heat stress application. The new problem is at what time should PG600 be applied and if either HCG or eCG, the combonents of PG600, are the active ingredients. What opportunities for training and professional development has the project provided? One graduate student became proficient in insertion and maintenance of ibutton thermometers below the scrotal skin, biotin injections, and histological analysis of BTB presence. Another graduate student learned how to perform in vitro fertilization through UW-Madison. A third graduate student learned how to train boars for collection and analyze the ejaculate via total sperm output, motility, morphology, and fourier harmonic analysis. How have the results been disseminated to communities of interest? Results have been disseminated to the swine industry via a publication in Molecular Reproduction and Development, the American Association of Swine Veterinarians and through presentations and attendance at meetings. Undergraduate students have been reached through lectures and case studies on the impacts of heat stress to the boar testes and lectures to international students on swine. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Weutilized our controlled scrotal insulation model in swine to: 1) investigate the mechanisms by which heat stress impacts the production of sperm in the boar testes and 2) determine if mitigation to heat stress impacts on sperm production can be accomplished by pharmacological means. In the project, our model has developed monitoring systems via ibutton insertion below the scrotal skin of boars to monitor testicualar temperatures during scrotal insulation in control and insulated boars demonstrating a 1.9 - 3.1°C change during insulation and approaching body temperature. Sham insulated boars had slight increases in scrotal temperatures and so have been replaced with uninsulated boars but measurement via ibutton technology of scrotal temperatures to provide a comparison with insulated boars. It was hypothesized a breakdown in the blood-testis barrier was the primary molecular mechanism impacting spermatogenesis during heat stress, but this has not been supported by examination of the junctional integrity with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific, Waltham, MA, USA) in scrotal insulated boar testes (Berndtson & Parrish, unpublished).Work is on-going on other mechanisms of heat stress damage.The mitigation of heat stress was investigated using PG600 treatment of boars prior to scrotal insulation.PG600 is a combination of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (HCG) which have Follicular stimulating hormone (FSH) and luteinizing hormone (LH) activity.Both FSH and LH (via testosterone) could impact spermatogenesis, in particular meiosis and Sertoli cells.We have completed 2 replicates with 3 boars/replicate and scrotal insulation induced the expected changes to sperm nuclear shape between days 19 - 33 post insulation and this was prevented in boars receiving PG600 prior to insulation.No effect of PG600 was found on control boars without scrotal insulation. This was a significant finding of interest to several swine genetics companies.

Publications

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audiences reached include: the American Association of Swine Veterinarians (2017, symposium presentation; Boar stud managers and veterinarians); International Conference on Swine Reproduction (2017, syposium presentation, boar stud managers and researchers in pig reproduciton); seminar for International Farmers Aid ASSN (2017, international underfgraduate students training in animal farm production); NCERA57 annual meeting (2017, researchers in swine reproduction in US). Research at meetings was discussed in terms of how climate and heat impacts fertility of boars and mechanisms of how heat impacts spermatogenesis. Undergraduate students at UW-Madison were reached through a case scenario entitled "Impact of the male on meat production: A case scenario in swine" focused on the effects on heat stress on male reproduction utilizing actual data collected in the project. Students are asked to analyze the data, interpret results, and explain the impact of heat stress on the boar and their ability to impregnate a female. Changes/Problems:As stated in the previous progress report sham sacks were shown to slightly increase testicular temperature and control boars no longer have any type of sack applied. Further, boars did not need to be anesthetized to administer EZ-LinkSulfo-NHS-Biotin as blood clots were apparent in the testes after administration and harvest. Currently, as samples are being processed for objective 1 we are not seeing drastic changes to the BTB as expected. We have moved onto objective 3 to analyze the efficiency of PG600 to minimize the effects of heat stress on spermatozoa in the ejaculate due to these results and the availability of boars. What opportunities for training and professional development has the project provided?One graduate student became proficient in insertion and maintenance of ibutton thermometers below the scrotal skin, biotin injections, and histological analysis of BTB presence. Another graduate student learned how to perform in vitro fertilization through UW-Madison. A third graduate student learned how to train boars for collection and analyze the ejaculate via total sperm output, motility, morphology, and fourier harmonic analysis. How have the results been disseminated to communities of interest?Results have been disseminated to the swine industry via a publication in Molecular Reproduction and Development, the American Association of Swine Veterinarians and through presentations and attendance at meetings. Undergraduate students have been reached through lectures and case studies on the impacts of heat stress to the boar testes and lectures to international students on swine. What do you plan to do during the next reporting period to accomplish the goals? Start objective 3. Boars have begun training for collection and materials are being prepared to begin data collection for objective 3 before the end of January. Confirm that heat stress is happening in the boars from objective 1 regardless of an intact BTB. Finish analysis of objective 1 samples including statistics.

Impacts
What was accomplished under these goals? Objective 1: We have continued to successfully insert ibutton thermometers into the scrotum of the boars to receive more accurate measurements of internal scrotal temperature without signs of infection or rejection. No control boars have sacks applied due to slight increases in internal scrotal temperature with sham sacks. Images of the BTB of control and insulated boars utilizing biotin have been obtained. Moving forward more photos will be collected of the BTB barrier of control and insulated boars as cryosectioned samples on slides come back. Due to the current BTB results from objective 1, we will move onto objective 3, using pharmacological means to mitigate heat stress. Only PG600 will be used to mitigate heat stress, not FSH as previously proposed. FSH has a shorter half-life than PG600 and PG600 has both LH and FSH like qualities, making it a better candidate to mitigate heat stress in boars. Boars will be trained and data collection will start before the end of January 2018.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Parrish JJ. Willenburg KL, Gibbs KM, Yagoda KB, Krautkarmer MM, Loether TM, Melo FCSA. 2017. Scrotal insulation and sperm production in the boar. Molecular Reproduction and Development 84:969-978.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Remple LA, Krautkramer MM, Loether TM, Parrish JJ, Miles JR. 2018. Season of collection and sperm head shape impacts expression of CARHSP and FTL from motile-rich boar sperm. 7:1-6.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Parrish JJ. 2017. Effect of heat stress on semen quality; recommendations for keeping AI boars. Proceedings of the 48th Meeting of the American Association of Swine Veterinarians. Pp. 18-19.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audiences reached include the American Association of Animal Science annual meeting, 2016; seminar for International Farmers Aid ASSN; National Hog Farmer Research Review. Research was discussed in terms of how it applies to agriculture, swine reproduction, heat stress on boars and impacts of climate change. Undergraduate students at UW-Madison were reached through the use of a case scenario on impact of heat stress on male reproduction: an impact on swine, which utiilized some of the actual data collected in the project. and asked students to analyze the data, interpret results and explain impact of heat stress on the boar and ability to impregnate females. Changes/Problems:Sham sacks, previously used on control boars, were shown through internal scrotal thermometers to increase scrotal temperature. We decided that all control boars used in this study will not wear a sack of any kind. Previouslyit was believed boars would need to be anesthetized to injectEZ-LinkSulfo-NHS-Biotin 30 minutes before harvest. However, we have shown that the injection site can be seen through blood clots in the testes meaning anesthesiais unneccessary. What opportunities for training and professional development has the project provided?One graduate studentattended the ImageJ User and Development Conference to better understand and use ImageJ for image analysis. One graduate student attended the Core Techniques in Protein and Genetic Engineering course through the BioPharmaceutical Technology Center Institute to gain knowledge and laboratory skills in core protein and genetic analysis methods. One graduate student learned how to perform the biotin injections and histological analysis of the BTB presence or absence. A graduate student and lab manager learned how to insert an ibutton to measure temperature below the surface of the scrotum, close the incision and maintain the ibutton in place for several days without infection. How have the results been disseminated to communities of interest?Impacts of the temperature monitoring have been disseminated to undergraduate students through lectures and case studies on heat impact to the boar testis; to the swine industry through publication in an industry summary review. What do you plan to do during the next reporting period to accomplish the goals?We plan on working to complete objective 1. We plan to continue to induce heat stress on boars and assess BTB integrity utilizing the biotin stain. Depending on availability of boars and success of insulations for objective 1, we may also start objective 2, determining if the disruption of the BTB is rapidly reversed following insulation removal.

Impacts
What was accomplished under these goals? This study consists of 3 objectives. Objective 1, to determine how heat stress to the boar testes causes disruption of the blood testis barrier (BTB) and the apical ectoplasmic specialization junctions leading to loss of meiotic spermatocytes and spermatids is proving to be successful. We were able to insert ibutton thermometers into the scrotum of the boar to receive more accurate measurements of internal scrotal temperature, without signs of infection or rejection. This change in thermometer insertion procedure from previous studies showed that sham sacks, made of nylon and canvas,previously used on control boars actually increased the temperature of the scrotum. Moving forward no sham sack will be used on control boars.After some trial and error we were able tosuccessfully usethe EZ-Link Sulfo-NHS-Biotin stain to identify the integrity of the BTB. We have obtained several photos of control boars with intact BTB stained with biotin.

Publications

• Type: Other Status: Published Year Published: 2016 Citation: J.J. Parrish, K.M. Gibbs, T.M. Loether, K. Yagoda, F. Melo. 2016. 2015 Research Review: Heat impacts boar fertility. National Hog Farmer Jan 5, 2016. http://nationalhogfarmer.com/reproduction/2015-research-review-heat-impacts-boar-fertility.