Effect of Hydroxytyrosol Supplementation in Semen Extender During on Preservation of Boar Semen

EFFECT OF HYDROXYTYROSOL SUPPLEMENTATION IN SEMEN EXTENDER DURING ON PRESERVATION OF BOAR SEMEN

BY

TSHABUSE ZWIVHUYA

201533312

A RESEARCH PROPOSAL SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICTURE (ANIMAL PRODUCTION) IN THE DEPARTMENT OF AGRICULTURAL ECONOMICS AND ANIMAL PRODUCTION, SCHOOL OF AGRICULTURAL AND ENVIRONMENTAL SCIENCE, FACULTY OF SCIENCE AND AGRICULTURE, UNIVERSITY OF LIMPOPO, SOUTH AFRICA.

SUPERVISOR: Professor JW Ngambi

CO-SUPERVISOR: Dr CM Pilane

July , 2016

1. PROBLEM STATEMENT

Worldwide, artificial insemination (AI) is an important tool in pig production for genetic improvement (Mapeka et al., 2012). One of the most important steps of AI is the semen preservation, liquid storage or freezing. Prolonged storage of semen might lead to reduced fertility, thus in most artificial insemination of swine conducted, fresh diluted semen is preserved for 1-5 days, at about 15-20 ◦C (Allai et al., 2015; Castellano et al., 2010). It has been shown that processes involved during   cryopreservation of boar semen result in cold-shock, leading to excessive reactive oxygen species formation, which causes  oxidative stress that has an influence on sperm quality (Allai et al., 2015; Kaeoket et al., 2012; Kaeoket., 2012). Pilane et al., (2016) indicated on their report of a study that ROS can compromise boar sperm velocity parameters, demonstrating that the use of antioxidants in any boar semen processing is essential during cryopreservation. Due to this problem, it is imperative to improve cryopreservation of boar semen for conservation of the species genetic material for future breeding and research purposes through the use of antioxidants.

1. RATIONALE OR MOTIVATION

Boar semen cryopreservation greatly facilitates the distribution of valuable and desirable genes because the frozen semen can be transported over a long distance and a stock of frozen semen enables the use of a boar that is dead or ill (Chanapiwat et al.,2011). Many studies have been conducted which focused on the supplementation of freezing extenders with a variety of antioxidants (e.g., vitamin E, vitamin C,L-cysteine, glutathione, taurine, pyruvate, superoxide dismutase (SOD), catalase) in attempt to minimize the detrimental effects of reactive oxygen species (ROS), which occurs during the storage process (Chanapiwat et al., 2009; Kaeoket et al., 2012).

Antioxidants are agents which break the oxidative stress chain reaction, thereby reducing the oxidative stress (Bansal and Bilaspuri, 2010). It has been reported that phenolic contents of olive oil contribute to the protection against lipid oxidative damage in humans in a dose-dependent manner (Khymenets et al., 2010). Hydroxytyrosol (HT) is one such phenolic which is a highly potent antioxidant originating in plants, and is most abundant in olives (Oleaeuropaea) (Achmon and Fishman, 2014). A variety of antioxidants have been tested to either scavenge reactive oxygen species (ROS) directly or counter the effects of ROS toxicity in the semen of a variety of mammalian and avian species (Roca et al., 2004). Among them, HT, has been widely used as a food additive in the diet of these species (Achmon and Fishman, 2014).

HT has been proven to positively affect antioxidant defense system of cells, in favour of cell integrity and resistance to stressful situations (Goya et al., 2007). HT has also been successfully tested to minimize DNA strand damage in cells and reduce intracellular ROS formation (Guo et al., 2010). Recent intervention clinical trials have provided evidence that HT contributes to the protection against lipid oxidative damage in humans in a dose-dependent manner (Khymenetes et al., 2010). In spite of these promising results, the use of HT in semen extenders is not commonplace, and no results are available that evaluate the potential effect of HT on preservation of boar semen. The aim of the study is to evaluate the possible protective effect of HT on sperm survivability and quality during preservation and cryopreservation of boar semen.

Objectives

1. To determine the effect of inclusion of optimum concentration of hydroxytyrosol (HT) with and without hydrogen peroxide (H2O2) in semen extender on the quality (Motility, viability & morphology, acrosome integrity) of boar semen stored 17ºC at different time periods (0, 3, 6, 24, 48 & 72 hours).
2. To determine the supplemental effect of hydroxytyrosol with and without H2O2 in lactose-egg yolk extender on the quality (Motility, viability & morphology, acrosome integrity) of frozen thawed boar semen.
3. To determine HT effects on the fertilizing ability of post thaw semen using in vitro fertilization method.

3. METHODOLOGY AND ANALYTICAL PROCEDURES

3.1 Animals

The experiment will be conducted in South Africa, in the Gauteng province at the Agricultural Research Council, Animal Production institute, Irene. Eight mature boars of proven good quality, ranging from 1-3 years of age, will be used in the study. The boars will be housed in individual cages for routine semen collection (Mapeka et al., 2012).Commercial diet will be fed daily, 1kg per boar and water will be available ad libitum.

3.2 Semen collection, handling and processing

3.2.1 Liquid preservation

Semen will be collected twice weekly from each boar by the gloved hand technique (Roca et al., 2004). The sperm-rich fraction will be  collected using a thermo flask containing warm water (39 ºC) and a glass beaker covered with a gauze filter to separate the gel fraction from the sperm-rich fraction (Roca et al., 2004). Within an hour of collection, semen will be transported to the laboratory for microscopic evaluation. The collected semen will be pooled and divided into a total of four 10mL centrifuge tubes, i.e. one tube per treatment (T (Tris), TH2O2 (Tris with hydrogen peroxide), TH2O2 + HT (Tris with hydrogen peroxide and hydroxytyrosol), THT (Tris with hydroxytyrosol. Thus, each semen samples will be diluted with Tris extender and supplemented with their respective treatments. After dilution, the different treatments will then be stored at 17°C incubator. Semen quality parameters (Motility, viability, acrosome integrity) will be assessed at different hours (0, 3, 6, 24, 48 & 72hrs) of preservation.