Spermatozoa cooled to 5°C one day after collection from porcine commercial semen doses retain sperm functionality with reduced bacterial load.

BACKGROUND: Commercial porcine semen is stored at 17°C, leading to a reduction of sperm quality and increase of bacterial growth. OBJECTIVES: To evaluate the effect of 5°C storage on porcine sperm functionality cooled one day after collection. MATERIALS AND METHODS: Semen doses (n = 40) were transported at 17°C and cooled at 5°C one day after collection. Spermatozoa were evaluated at Days 1, 4, and 7 for motility, viability, acrosome integrity, membrane stability, intracellular zinc, oxidative stress, and bacterial growth. RESULTS: Contaminated semen doses predominantly exhibited Serratia marcescens, with increasing bacterial load during 17°C storage. Under hypothermal storage, negative doses for bacteria growth at Day 1 remained negative, and bacterial load did not increase in bacterial contaminated samples. Motility was significantly reduced through 17°C storage, but at 5°C, motility was only reduced at Day 4. Samples with bacterial growth (35.0%, 14/40) had significantly reduced motility at 17°C, but motility was unaltered at 5°C. Plasma membrane and acrosome integrity without bacterial contamination were unaffected at 17°C, but were significantly reduced at 5°C on Day 7. Plasma membrane and acrosome integrity significantly decreased with bacterial contamination regardless of temperature. High mitochondrial activity in viable spermatozoa without bacteria was not altered by temperature, but was significantly reduced by bacterial contamination at 17°C. Membrane stability was significantly reduced at Day 4, but tended (p = 0.07) to be higher in samples without bacterial growth. Viable spermatozoa exhibiting high zinc were significantly reduced throughout storage regardless of temperature. Oxidative stress levels were not altered, but significantly increased with bacterial contamination at 17°C. DISCUSSION AND CONCLUSION: Porcine spermatozoa cooled to 5°C one day after collection retain functional attributes similar to spermatozoa stored at 17°C, but have a reduced bacterial load. Cooling extended boar semen to 5°C is feasible after transport to avoid modifying semen production.

A novel experimental design for boar sperm cryopreservation.

Cryopreservation of sperm is a routine technology in many livestock species, but not in swine. Frozen sperm must result in acceptable conception rates and produce 11 to 12 piglets/litter to be competitive with traditional cooled semen. The development of an extender that results in high post-thaw sperm quality and acceptable litter size requires the identification of factors that markedly affect post-thaw semen quality. The present study aims to first identify factors in boar sperm cryopreservation that significantly affect post-thaw sperm quality using an efficient, cost-effective, and relatively rapid approach. The Plackett-Burman experimental design is ideal for the screening of factors at their extreme, greatly reducing the amount of time and resources needed for a follow-up, full factorial design. Using commercial semen, a 9-factor, 12-run Plackett-Burman design was used on 10 boars split between 12 treatments. Through this method, glycerol concentration, cooling rate, antioxidant supplementation with GameteGuard (Membrane Protective Technologies, Inc. Fort Collins, CO), and straw size were identified as highly influential factors that affect post-thaw sperm quality. Extender type, starting osmolality, sodium dodecyl sulfate addition, and stepwise addition of glycerol were also influential for some but not all post-thaw sperm parameters (P < 0.05). Equilibration time in the straws before freezing was determined to have no impact on post-thaw sperm quality parameters. Using the Plackett-Burman design, it can be concluded that four of the nine factors warrant detailed investigation in full factorial experiments in the development of boar sperm cryopreservation extenders.

Freezing of sperm is a routine in many livestock species, but not in pigs, because it results in lower conception in small litter sizes. This study aims to identify factors in pig sperm freezing protocols that affect sperm quality using an efficient and relatively rapid approach. The Plackett­Burman experimental design is one such method used to rapidly screen factors and was the method used for this study. Using commercial pig semen, freezing factors were tested to determine their impact on sperm quality after freezing. Through this method, glycerol concentration (prevents cell damage), cooling rate (speed used to cool sperm to refrigerator temperature), antioxidants, and straw size (straws in which sperm are packaged for freezing) were identified as highly influential factors that affect sperm health. Extender type (the chemical base used to freeze sperm), starting osmolality (the concentration of salts and sugars in a solution), sodium dodecyl sulfate addition (detergent), and stepwise addition of glycerol (to prevent sperm damage) were also influential for some but not all sperm health measures tested. Using the Plackett­Burman design, it can be concluded that four of the nine factors warrant detailed investigation in the development of boar sperm freezing extenders.

Validation of a new multiparametric protocol to assess viability, acrosome integrity and mitochondrial activity in cooled and frozen thawed boar spermatozoa.

BACKGROUND: Motility, morphology, membrane integrity and DNA fragmentation are sperm characteristics routinely used to assess quality of boar spermatozoa. However, the evaluation of individual parameters has intrinsic restrictions in the estimation of potential fertility. Therefore, we aimed to validate a new multiparametric protocol to assess fertility potential through the evaluation of viability, acrosome integrity and mitochondrial activity within the same sperm population for cooled and frozen-thawed boar spermatozoa. METHOD: Three multicolor protocols to assess viability, acrosome integrity and/or mitochondrial activity were compared for agreement containing two dyes (HM-panel; Hoechst 33342, MitoTracker™ Deep Red), three dyes (3-panel; SYBR®14, propidium iodide and lectin PNA-Alexa™ 647) or four dyes (4-panel; Hoechst 33342, lectin PNA-Alexa™ 488, propidium iodide and MitoTracker™ Deep Red). Cooled (n = 132) and frozen-thawed (n = 254) samples of boar spermatozoa were assessed by flow cytometry. RESULTS: 4-Panel enabled the detection of several sperm subpopulations based on plasma membrane integrity, acrosome status and mitochondrial activity in cooled and frozen-thawed spermatozoa. No significant differences were observed between 3-panel and 4-panel for the percentage of live, live-acrosome intact, and dead-acrosome reacted spermatozoa. However, the percentage of acrosome-intact spermatozoa was significantly higher in cooled samples when stained by 3-panel than 4-panel. Percentages of sperm parameters between protocols were strongly correlated, and agreement analysis demonstrated that both assays resulted in similar values for both sperm sample type. CONCLUSION: Our results indicate that a four-color protocol is a practical, simple and reliable procedure to simultaneously evaluate boar sperm viability, acrosome integrity and mitochondrial activity under clinical conditions.

Effects of boar sperm antioxidant supplementation on fertility.

Use of cooled stored boar semen for artificial insemination accounts for 99% of inseminations in pork production sectors worldwide. The aim of the present study was to examine use of GameteGuard®-CP, a natural plant derived blend of antioxidants, supplemented in one commercially available semen extender on sperm quality, conception rate, farrowing rate, and litter size. Ejaculates from 16 commercial Duroc boars were used in a split-ejaculate single-sire artificial insemination study. Sperm were evaluated for motility, viability, acrosome integrity, membrane stability, mitochondrial membrane potential, DNA intactness, and total antioxidant reactivity on both day 1 and 4 subsequent to ejaculate collection. Sows were inseminated after a treatment regimen was imposed to synchronize time of ovulation (n = 1476) among females using sperm collected 4 days subsequent to ejaculate collection extended in AndroStar Plus® or GameteGuard®-CP supplemented extender. At day 4 post-ejaculate collection, there was maintenance of sperm viability, membrane stability, acrosome intactness, mitochondrial membrane potential, and DNA intactness (P < 0.01) when there was use of GameteGuard®-CP treatments. When control samples were used, there were lesser (P < 0.01) values from day 1-4 for all variables evaluated. Conception rate and farrowing rates were 7.4% and 9.7% greater, respectively (P < 0.05), but there was no difference as a result of GameteGuard®-CP-treatment in mean litter outcomes. The results in the present study indicate antioxidant supplementation using GameteGuard®-CP maintains sperm quality during cool sperm storage resulting in improved conception and farrowing rates when using semen stored in the cooled state for as long as 4 days.