Development of techniques for freezing boar semen

Abstract

The objective of the present study was to investigate the feasibility of boar semen cryopreservation in Thailand and to investigate factors that could affect the boar semen quality after cryopreservation. Fifteen purebred boars (5 Landrace, 5 Yorkshire and 5 Duroc) from 2 commercial herds in Nakorn-prathom province were used in the experiment. Three ejaculates from each boar were collected with over one-week interval using gloved-hand technique. After collection, the semen was diluted with isothermal Beltsville thawing solution (BTS) extender. Diluted semen was held at 15 °C for 2 h and centrifuged. The supernatant was discarded and the semen precipitant was re-suspended with lactose-egg yolk (LEY) extender. The diluted semen was cool down to 5°C within 90 min. Two parts of semen were mixed with one part of LEY extender with 9% glycerol and 1.5% Equex-STM®. Thereafter, the processed semen was loaded into 0.5 mL straws. The straws were placed in liquid nitrogen (LN2) vapor approximately 3 cm above the level of the LN2 for 20 min and then were plunged into LN2. Thawing was achieved by immersing the straws in water at 50 °C for 12 second. After thawing, the semen was diluted (1:4) with an extender consisting of 95% BTS and 5% LEY extender. The extended thawed semen was incubated in a 38 °C water-bath for 30 min before evaluating the semen quality after thawing. Sperm concentration, individual motility, sperm viability, percentage of normal apical ridge (NAR), sperm plasma membrane function (sHost) and sperm plasma membrane integrity (SYBR) were evaluated. The semen qualities before and after thawing were compared for each boar using paired t-test. Pearson’s correlation was used to evaluate the correlation among all sperm parameters that were measured. Data on individual motility, viability, NAR, sHost positive spermatozoa and SYBR after thawing were analyzed using the General Linear Model (GLM) procedure of the SAS. On average, the sperm concentration of pre-diluted fresh semen was 529.7x106 spz/mL and the sperm concentration of frozen thawed semen was 811x106 spz/mL. The individual motility, the sperm viability, the NAR, the sHost and the SYBR of boar spermatozoa after frozen-thawed were 28%, 36.2%, 26.4%, 18.5% and 30.9%, respectively. All of the sperms parameters measured significantly decreased after frozen and thawed i.e., individual motility decreased by 44.2%, sperm viability decreased by 37.9% and NAR deceased by 59.5%. The individual motility of frozen-thawed spermatozoa was significantly correlated with the sperm viability (P<0.001), the NAR (P<0.02), the sHOST (P<0.001) and the membrane integrity (SYBR) (P<0.001). The higher concentration of frozen-thawed semen resulted in the lower membrane integrity (r=-0.3, P=0.04). Breed of boars and the individual boar within the same breed significantly influenced most of the sperm parameters after being frozen-thawed. The sperm viability of frozen-thawed semen in Duroc and Landrace boars was significantly higher than Yorkshire boars (P<0.05). The membrane integrity of frozen-thawed semen in Landrace boar was significantly higher than Yorkshire boars (P<0.05). In conclusions, the semen cryopreservation of boar could be performed successfully in Thailand with an average concentration of spermatozoa in frozen thawed semen of 811x106 spz/mL, the individual motility of 28% and the sperm viability of 36%. Breed of boars and the individual boar within the same breed significantly influenced most of the sperm parameters after being frozen-thawed.