PROVIDING A USEFUL TOOL FOR SWINE BIOSECURITY: IMPROVING PORCINE SPERM CRYOPRESERVATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0206944
• Project Start Date: Jan 28, 2006
• Project End Date: Jan 27, 2007
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF PENNSYLVANIA
(N/A)
PHILADELPHIA,PA 19104

Performing Department
SCHOOL OF VETERINARY MEDICINE

Non Technical Summary
A biosecurity tool to improve long-term storage of boar semen via cryopreservation is needed. Improving porcine semen cryopreservation, extending the fertilizable lifespan of the semen would facilitate biosecurity meansures aimed at preventing the spread of pathogens.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3599	1101	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3599 - Swine, general/other;

Field Of Science
1101 - Virology;

Keywords

swine biosecurity

porcine

porcine sperm

cryopreservation

artificial insemination

Goals / Objectives
Several important swine pathogens, including Porcine Reproductive and Respiratory Syndrome virus, pseudorabies virus, porcine parvovirus, and bacterial agents such as leptospira species, can be shed in the semen. Artificial insemination (AI) with cryopreserved or cooled semen is of central importance in modern swine production because it allows the selective use of valuable boar genetics. Because successful cryopreservation of boar sperm has been problematic, boar semen used for AI is currently stored by cooling rather than freezing, limiting its convenience, geographical area for use, and functional lifespan. The latter is of particular importance to biosecurity efforts since it limits the amount of time (approximately one week) available for pathogen screening. The goal of the proposed study is to provide a valuable biosecurity tool by improving long-term storage of boar semen via cryopreservation.

Project Methods
Our studies have determined that using cyclodextrins to deliver cholesterol to porcine sperm plasma membranes improves viability following cold shock or cryopreservation and inhibits sperm capacitation after incubation under conditions that would otherwise support this maturational event. The effects of cholesterol manipulation before cryopreservation on post-thaw sperm function have not been determined. The aim of this proposal is to determine the ability of porcine sperm frozen in the presence of 2-hydroxypropyl-beta-cyclodextrin (HBCD) to survive freeze-thaw and undergo capacitation. Confirming the functional capacity of porcine sperm cryopreserved in the presence of HBCD will validate this method for preserving sperm from valuable boars in agricultural, industrial, and research settings. Following the completion of this study, the next phase will be to conduct IVF studies using porcine sperm frozen with and without HBCD in order to determine the effect, if any, of this treatment on in vitro fertilization and embryo development

Progress 01/28/06 to 01/27/07

Outputs
Improving porcine semen cryopreservation such that it becomes a viable alternative to cooled semen would, by extending the fertilizable lifespan of the semen, facilitate biosecurity measures aimed at preventing the spread of pathogens. Our previous studies have indicated that freezing porcine sperm in semen extender improves cryosurvival. Incubation of porcine sperm with CD plus cholesterol 3-sulfate (CD/ChS), a treatment assumed to favor cholesterol uptake, inhibits porcine sperm capacitation and also protects porcine sperm from cryodamage. However, for this treatment to become applicable, it is necessary to demonstrate that sperm capacitation is not permanently inhibited. This is the objective of our study. A total of 6 experiments using different semen samples from 4 adult boars were performed. Sperm were incubated for 3h in capacitation medium Following incubation, duplicate CD/ChS samples were diluted and incubated for an additional 3h. Samples were removed at 3h and 6h for evaluation of sperm viability calcium ionophore-induced acrosome reaction (iAR) (Coomassie G-250 staining of paraformaldehyde-fixed samples, data represent differences between induced and uninduced samples), and antiphosphotyrosine immunoblotting of extracted sperm proteins followed by densimetry via image analysis (protein PY). Protein PY correlates with porcine sperm capacitation under standard conditions and is expressed as a percentage of positive control. As previously reported, 3h incubation with CD/ChS inhibits sperm capacitation as assessed by iAR and protein. In contrast, sperm capacitated for 3h with CD have significantly increased capacitation as assessed by by iAR and protein. Sperm that were incubated for 3h with CD/ChS and then an additional 3h following 1:5 dilution in CD/ChS still displayed inhibited capacitation at 6h. In contrast, incubation for 3h with CD/ChS followed by 1:5 dilution with CD resulted in sperm capacitation at 6h. These results demonstrate that the inhibitory effect of CD/ChS on in vitro porcine sperm capacitation is reversible. It is likely that CD used as a cryoprotectant during semen freezing could inhibit capacitation, but the results presented here suggest that any inhibition would be reversible. In order to explore this hypothesis, we have frozen semen from one boar and plan to freeze semen from a second boar at 0, 25 and 50 mM CD. This semen will be used for in vitro fertilization and for in vitro assays of viability and sperm capacitation.

Impacts
A valuable biosecurity tool is improving long-term storage of boar semen via freezing. Improving porcine semen freezing such that it becomes a viable alternative to cooled semen would, by extending the fertilizable lifespan of the semen, facilitate biosecurity measures aimed at preventing the spread of pathogens.

Publications

• Galantino-Homer,H., Zeng,W.X., Megee,S.O., Dallmeyer,M., Voelkl,D., and Dobrinski,I. (2006). Effects of beta-cyclodextrin and cholesterol on porcine sperm viability and capacitation status following cold shock or incubation. Mol. Reprod. Dev. 73, 638-650.