Semen Preservation Research Articles

Analysis of changes in the morphological structures of sperm during preservation of liquid boar semen in two different seasons of the year

Animal fertility is the result of a combination of genetic, physiological and environmental factors. Assessment of semen quality plays a key role in determining the reproductive performance of boars. The aim of the study was to determine the effect of two seasons of the year on the morphology, morphometry, cell membrane integrity and mitochondrial activity of sperm during storage of liquid boar semen at 17 °C. The study was carried out using 20 boars. Four ejaculates were collected from each boar in each of two seasons of the year. All tests were carried out five times: at 1, 24, 48, 96 and 168 h during storage. The results showed that sperm in ejaculates collected in summer are more sensitive to storage conditions than those from ejaculates obtained in winter. The percentage of sperm with morphological defects was shown to increase with the storage time of the diluted ejaculates, particularly between 96 and 168 h of preservation. In summer, the percentage of sperm with an intact cell membrane and the percentage with high mitochondrial membrane potential are much lower than in winter, at every hour of semen preservation. In the case of boars used for artificial insemination, it is worth taking into account the season when the semen is collected, especially if it is to be stored in liquid form. Assessment of sperm cell structures during storage of liquid semen should be implemented at insemination stations and should be carried out more often in summer.

Comparative analysis of different metabolites in semen of Guanzhong dairy goats with different motility rates

In order to explore the different metabolites of buck semen with different motility stored at 4 °C, the semen of bucks was collected by artificial vagina. The collected semen was divided into high motility group and low motility group after treatment, with 6 replicates set for each group. The semen metabolites of high motility group and low motility group were detected by Liquid Chromatography-Mass Spectrometry (LC-MS). The results showed that 101 different metabolites were detected in the high and low motility groups of bucks, of which 48 metabolites were significantly up-regulated (P < 0.05) and 53 metabolites were significantly down regulated (P < 0.05). Most of these metabolites belonged to lipids and lipid-like molecules, organic acids and their derivatives, and organic oxygen compounds, which were mainly related to energy metabolism. According to the functional enrichment analysis of the former differential metabolites in KEGG database, the top 20 most representative metabolic pathways were detected, among which the glycerophospholipid metabolic pathways changed significantly. From the perspective of metabolomics, this study revealed the differences of metabolites and characteristic compounds of semen with different motility of bucks under low temperature preservation, which provided a scientific basis for the preservation and utilization of semen of Guanzhong dairy goats in the future.

Monitoring for Equine Viral Arteritis has decreased its prevalence in European breeding sires

Equine viral arteritis (EVA) can induce a persistent carrier state in stallions and shedding of the virus with semen can spread EVA and cause abortions in seronegative pregnant mares. In the early 1990s, obligatory testing for EVA of stallions used for artificial insemination was implemented within the European Union (Council Directive 92/65/EEC). In addition, vaccination programs for young stallions and exclusion of carrier stallions from certain breed registry events were implemented in Warmblood sport horses. Information on the efficacy of these actions on the prevalence of EVA in reproductively active stallions are not yet available in Europe. This study retrospectively analyzed results of serological and virus antigen testing for EVA in sires (n=308, one sample set per sire, EVA-vaccination status unknown) of different age and breed referred to the Centre for Artificial Insemination of Vetmeduni Vienna for semen preservation between 2001 and 2021. For analysis, stallions were grouped by age (1-3, 4-10, 11-16, >16 years) and breed. The EVA antibody titer was determined by serum neutralization test and semen was analyzed for EVA virus by PCR and/or virus isolation test. Statistical analysis was performed by chi2-test, Kruskal-Wallis test or Mann-Whitney U-test using the IBM SPSS 28.0 statistics software. Of 308 stallions tested, 14.9% (n=46) were EVA seropositive and in 12 stallions EVAvirus was detected in semen (26% of seropositive and 3.8% of all tested stallions, respectively). The incidence of seropositive stallions (22% of sires tested during 2001-05 to 4.1% during 2016-21) and of stallions shedding EVA virus via semen (8.5% of all serologically tested stallions 2001-05 to 1.4% during 2016-21) decreased over time (p<0.05). No difference among breeds with regard to occurrence of seropositive stallions was detected. There was a tendency towards more Standardbred and Lipizzaner stallions (14% and 13%, respectively) shedding EVA virus in semen compared to all other breeds (<3%, p=0.074 among groups). Differences in the seroprevalence of EVA antibodies existed among stallions from different age groups (p<0.01) with the highest percentage of seropositive stallions in 11-16 year-stallions (45.7%). No differences among age groups with regard to virus shedding existed. The EVA antibody titer increased with age (p<0.01), reflecting a higher possibility of repeated virus challenge in older stallions. Antibody titer was not associated with carrier state. In conclusion, monitoring of stallions for EVA in combination with vaccination programs resulted in a decrease of the prevalence of seropositive and virus-shedding stallions during the past 20 years in a European stallion population and can therefore be considered successful.

The Effect of Adding Different Levels of Reduced Glutathione to Extender on the Quality of Cooled Ring-Necked Pheasant Semen.

Aim: Artificial propagation of ring-necked pheasant through semen preservation is of significance, as this species is facing enormous threats in its natural habitat. Semen preservation inevitably induces oxidative stress, and exogenous antioxidants need to be investigated for the preservation of ring-necked pheasant semen. Therefore, the current study was conducted to investigate the role of glutathione (GSH) in extender on the liquid storage of ring-necked pheasant semen. Materials and Methods: Semen was collected from 10 sexually mature males, evaluated for sperm motility, and pooled. Pooled semen was aliquoted for dilution with Beltsville poultry semen extender (1:5) at 37°C having GSH levels of 0.0 mM (Control), 0.2, 0.4, 0.6, and 0.8 mM. Extended semen was gradually cooled to 4°C and stored in a refrigerator (4°C) for 48 hours. Semen quality, that is, sperm motility, membrane integrity, viability, acrosomal integrity, and DNA integrity, was assessed at 0, 2, 6, 24, and 48 hours. Results: Sperm motility (%), plasma membrane integrity (%), viability (%), and acrosomal integrity (%) were recorded higher (p < 0.05), whereas DNA fragmentation (%) was recorded lower in extender supplemented with 0.4 mM GSH up to 48 hours of storage compared with 0.2, 0.6, and 0.8 mM GSH concentrations and control. Conclusion: It is concluded that 0.4 mM GSH in extender improves sperm quality parameters of ring-necked pheasant during liquid storage up to 48 hours at 4°C.

Review on Sustainable Cryopreservation and Nonfreezing Storage Solutions of European Honeybee Apis Mellifera Drone Semen

The European honeybee Apis mellifera is the main pollinator for most crops used for human consumption. However, a number of diseases, parasites, pesticides and other factors that generally result in the widely described colony collapse disorder weakens honeybee colonies. In order to maintain the existing honeybee germ lines and facilitate the creation of new disease-resistant lines, it is necessary to ensure consistent breeding work, which would also allow the long-term preservation of the unique germplasm lines. One of the most promising solutions for the preservation of honeybee germplasm is the storage of honeybee drone semen. In recent decades, there has been a renewed interest in the preservation of honeybee drone semen using both cryopreservation and non-freezing storage methods. This review looks at the latest developments in novel sperm storage technologies and compares them to well-known preservation solutions. Additionally, the most accessible and widely used solutions will be reviewed, taking into account the cost of the necessary equipment, the complexity of the methods, time consumption and resulting sperm quality.

Effects of curcumin supplementation on quality of porcine spermatozoa irradiated with ultraviolet-C at 228 nm during liquid preservation.

It is important to prevent microbial contamination during liquid preservation of semen in pigs. We examined the effects of curcumin supplementation on the quality of porcine spermatozoa irradiated with ultraviolet-C (UV-C) at 228 nm. UV-C is used to disinfect gases and solid surfaces. In the first experiment, porcine semen was preserved with 0, 10, 25, 50 or 100 μM curcumin under UV-C irradiation at 228 nm for 7 days at 15°C. The irradiation did not affect the motility and viability of preserved spermatozoa but decreased the percentage of plasma membrane integrity of spermatozoa. Curcumin supplementation at 25 μM significantly improved the plasma membrane and acrosome integrity of irradiated spermatozoa compared with spermatozoa preserved without curcumin (p < .05). In the second experiment, semen was preserved with or without 25 μM curcumin with UV-C irradiation at 228 or 260 nm for 3 days at 15°C. Curcumin supplementation increased the percentages of total motility, sperm viability and plasma membrane integrity of preserved spermatozoa at both irradiation wavelengths (p < .05). All quality parameters of 260 nm irradiated spermatozoa decreased compared to those of the other groups, irrespective of curcumin supplementation. The collective findings indicate that porcine spermatozoa can retain their viability even after continuous long-duration irradiation with 228 nm UV-C. Curcumin supplementation improves the quality of UV-C irradiated spermatozoa during semen preservation.

Antimicrobial activity of cell free supernatants from probiotics inhibits against pathogenic bacteria isolated from fresh boar semen

The use of antibiotics with semen extender appears to be a practical solution to minimise bacterial growth in fresh boar semen preservation. Unfortunately, the excessive use of antibiotics promotes antimicrobial resistance (AMR). This becomes a worldwide concern due to the antimicrobial resistance genes transmitted to animals, environment, and humans. Probiotics are one of the alternative methods to reduce antibiotic use. They could inhibit pathogenic bacteria by producing antimicrobial substances in cell free supernatants (CFS). Nevertheless, there is no comprehensive study undertaken on inhibitory activity against pathogenic bacteria isolated from boar semen origin. Our study investigated the efficacy of CFS produced from selected probiotics: Bacillus spp., Enterococcus spp., Weissella spp., Lactobacillus spp., and Pediococcus spp. inhibiting pathogenic bacteria isolated from fresh boar semen. Besides, the semen-origin pathogenic bacteria are subjected to identification, antimicrobial resistance genes detection, and antibiotic susceptibility test (AST). Pseudomonas aeruginosa, Escherichia coli, and Proteus mirabilis are the most common pathogens identified in boar semen with resistance to numerous antibiotics used in pig industry. The CFS with its antimicrobial peptides and/or bacteriocin constituent derived from selected probiotics could inhibit the growth of pathogenic bacteria carrying antimicrobial resistance genes (mcr-3 and int1 genes). The inhibition zones for Pseudomonas aeruginosa, Escherichia coli, and Proteus mirabilis provided more efficient results in the CFS derived from Lactobacillus spp. and Pediococcus spp. than those of the CFS produced from Enterococcus spp., Weissella spp. and Bacillus spp., respectively. It is worth noted that as the incubation time increased, the antibacterial activity decreased conversely. Our results on CFS with its antimicrobial peptides and/or bacteriocin constituent inhibits semen-origin pathogenic bacteria guide the direction as a promising alternative method used in the semen extender preservation of the pig industry.

COMPARISON OF COMMERCIAL DILUENTS AND TRACE ELEMENTS ON FRESH ALPACA SEMEN (LAMA PACOS)

The reproduction of alpacas is a research subject of interest in recent times since their condition and morphology do not cause an impact on the Andean-habitat territories. The collection of semen from male alpacas by artificial vagina is the methodology used for the best extraction of seminal material. However, conservation methods are not fully defined, therefore, analyzing the semen conservation on male alpacas with the inclusion of trace elements and commercial diluents were the subject of interest in the study. Seven male alpacas were taken, with reproductive ages between 3 - 5 yrs. with an average weight of 60 ± 1.64 kg of BW. Distributed in two random homogeneous experimental groups (SSO, male alpacas semen without trace elements, and SCO, male alpacas semen with trace elements) to analyze the macroscopic and microscopic characteristics of the commercial trace elements administration. In addition, were subjected to a dilution procedure with two types of registered commercial diluents (Triladyl® and AndroMed®) and a laboratory-generated diluent (Tris Yema + Glycerol ). The semen conservation process was analyzed and compared to the trace elements application and has no difference, even though there is heterogeneous research on semen conservation. The authors recommended making more scientific contributions to the inclusion of trace elements and diluents in the fresh semen of male alpacas.

The Effects of Water-Based Coconut Extenders on Semen Preservation : A review

For animal reproduction, artificial insemination has become a need. Semen preservation has been crucial in artificial insemination as it can determine the success of fertilization and pregnancy. Semen extenders have been critical in prolonging the usability of semen while also retaining its viability, motility, and integrity so that it can be used to fertilize female animals. Due to its nutritional and chemical qualities that can create an environment for spermatozoa to survive and thrive before insemination, coconut water has been the focus of various research addressing its effect on semen preservation. These studies have shown that coconut water has the potential to be an effective semen extender due to its ability to protect the semen during cooling and cryopreservation. Coconut water also includes nutrients (water, sugars, proteins, salts, and vitamins) to help spermatozoa thrive in a healthy environment. However, its performance lags that of commercial extenders, but it can be a less expensive alternative or replacement when commercial extenders are unavailable.

Identification of a suitable buffer with coconut-water for turkey semen preservation

Identification of a suitable buffer with coconut-water for turkey semen preservation

Standardization of fresh cow milk with sodium citrate buffer for turkey semen preservation

Standardization of fresh cow milk with sodium citrate buffer for turkey semen preservation

Effect of Some Plant-Based Substances on Microbial Content and Sperm Quality Parameters of Bull Semen.

The rapid emergence of antibacterial resistance requires alternatives to antibiotics to be found, including for semen preservation. One of the possible alternatives would be to use plant-based substances with known antimicrobial effects. The objective of this study was to test the antimicrobial effect of pomegranate powder, ginger, and curcumin extract in two concentrations on bull semen microbiota after exposure for <2 h and 24 h. An additional aim was to evaluate the effect of these substances on sperm quality parameters. The bacterial count in semen was low from the beginning; however, a reduction was present for all tested substances compared with control. A reduction in bacterial count in control samples was also observed with time. Curcumin at a concentration of 5%, reduced bacterial count by 32% and was the only substance that had a slight positive effect on sperm kinematics. The other substances were associated with a decline in sperm kinematics and viability. Neither concentration of curcumin had a deleterious effect on sperm viability parameters measured by flow cytometry. The results of this study indicate that curcumin extract at a concentration of 5% can reduce the bacterial count and does not have a negative influence on bull sperm quality.

Seminal plasma metabolomics analysis of differences in liquid preservation ability of boar sperm.

The preservation of semen is pivotal in animal reproduction to ensure successful fertilization and genetic improvement of livestock and poultry. However, investigating the underlying causes of differences in sperm liquid preservation ability and identifying relevant biomarkers remains a challenge. This study utilized liquid chromatography-mass spectrometry (LC-MS) to analyze the metabolite composition of seminal plasma (SP) from two groups with extreme differences in sperm liquid preservation ability. The two groups namely the good liquid preservation ability (GPA) and the poor preservation ability (PPA). The aim was to explore the relationship between metabolite composition in SP and sperm liquid preservation ability, and to identify candidate biomarkers associated with this ability of sperm. The results revealed the identification of 756 metabolites and 70 differentially expressed metabolites (DEM) in the SP from two groups of boar semen with differing liquid preservation abilities at 17 ℃. The majority of identified metabolites in the SP belonged to organic acids and derivatives as well as lipids and lipid-like molecules. The DEM in the SP primarily consisted of amino acids, peptides, and analogues. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis also demonstrated that the DEM are mainly concentrated in amino acid synthesis and metabolism related pathways (P < 0.05). Furthermore, eleven key metabolites were identified and six target amino acids were verified, and the results were consistent with the non-targeted metabolic analysis. These findings indicated that amino acids and their associated pathways play a potential role in determining boar sperm quality and liquid preservation ability. D-proline, arginine, L-citrulline, phenylalanine, leucine, DL-proline, DL-serine, and indole may serve as potential biomarkers for early assessment of boar sperm liquid preservation ability. The findings of this study are helpful to understand the causes and mechanisms of differences in liquid preservation ability of boar sperm, and provide valuable insights for improving semen quality assessment methods and developing novel extenders or protocols.

The effect of varying concentrations of chicken plasma egg yolk and glycerol on the viability of canine sperm following short and long-term preservation.

Plasma egg yolk (PEY), due to simple preparation and easier access, could be a suitable alternative to raw egg yolk for preserving canine semen. The present study investigated suitable concentrations of PEY and glycerol for preservation of canine semen. Semen was collected by digital manipulation (seven replicates from four dogs). Following initial raw semen evaluation, the semen was diluted in a tris-based extender supplemented with varying concentrations of chicken PEY (0, 20, and 40% v/v) and glycerol (3%; v/v). After cooling the specimen to 4°C within 1 h, the specimens were diluted with an equal volume of freezing extender consisting of similar concentrations of chicken PEY and 0 and 7% glycerol to reach the final glycerol concentration of 1.5 and 5% for short-term storage of canine semen. Samples with different concentrations of PEY and 5% glycerol were frozen. The sperm viability parameters including total motility, progressive forward motility, plasma membrane integrity, and live percentage of sperm were assessed following short and long-term storage. Sperm viability parameters of semen extended in an extender supplemented with 20 or 40% chicken PEY with either 1.5 or 5% glycerol remained superior until 72 h after semen collection compared to the specimen that did not receive any PEY (P<0.05). Post-thaw sperm viability was also greater in samples extended in extender supplemented with either 20 or 40% PEY compared to 0% PEY. Tris-based extender supplemented with either 20% chicken PEY could be suitable for short and long-term preservation of canine semen.

The Effect of Adding Antioxidants on Cooled Zarabi Buck Semen During Different Seasons

Semen preservation by cooling or freezing is an essential artificial insemination (AI) step. AI is affected by many factors, such as semen quality. The aim of this study was to evaluate the effect of adding different concentrations of different antioxidants such as selenium, zinc oxide and Vitamin C to the diluted buck semen during cooling in both summer and winter. Eight mature healthy Zaraibi bucks were used in the study. semen was collected twice weekly during summer 2021 (august and September) and winter 2022 (January and February). Semen samples were collected by an electro- ejaculator device. Samples of good quality semen were pooled and diluted by extender and divided according to experimental groups: Group 1 Vitamin C: 50 mg /100 ml, 1oo mg /100 ml, 150 mg /100 ml. Group 2 Zinc oxide: 0.5 mg /100 ml, 1 mg /100 ml, 1.5 mg /100 ml. Group 3 Selenium: 100 μL/100 ml, 200 μL / 100, 300 μL / 100. After cooling by 1 hr. semen parameters were examined as motility, livability, acrosome integrity and cell membrane integrity and at 8 hours from cooling then every 8 hr till 64 hr. Seminal antioxidant activities as SOD and catalase were estimated at 0 hr, 24 hr and 48 hr. The results indicated that selenium 200 μL / 100 ml diluent has a favorable effect on cooled diluted buck semen during summer and winter more than selenium 100 μL and 300 μL. Selenium is better than Zinc oxide and Vitamin C for improving the semen quality as with minimum cost. In conclusion adding selenium as antioxidant to buck semen during cooling preservation with 200 μL concentration per 100 diluents is preferred for buck semen during cooling preservation.

Determination of Alternative Diluents for Preservation of Duroc Boar Semen Through Modification of Various Semen Diluents

The quality of porcine semen deteriorates very rapidly during the semen preservation process. The aim of this study is to find alternative diluents for Duroc semen preservation by changing different semen diluents. Two Duroc pigs, ± 3 years old, were trained twice weekly for semen collection. Immediately after collection, sperm were examined macroscopically and microscopically. Semen with sperm motility ≥ 70%, sperm concentration ≥200 x 106 cells/ml, and abnormality ≤20%) was diluted with six diluents, namely: Beltsville Thawing Solution® (BTS), Durasperm, Citrate Egg Yolk (CEY), Tris Egg Yolk (TEY), CEY + Olive Oil (CEYO) and TEY + Olive Oil (TEYO) in the ratio of one part semen and four parts diluent. The liquid sperm was stored at 18-20 oC and examined every eight hours for 80 hours. The result shows that sperm motility, viability and plasma membrane integrity of Duroc semen with CEYO and TEYO are comparable to BTS. No significant difference was found in sperm abnormalities. The results suggest that citrate egg yolk and Tris egg yolk in combination with olive oil are suitable as alternative diluents for the preservation of porcine sperm. Further studies are needed to determine the most suitable olive oil concentration for the preservation of porcine sperm of different breeds.

Preservation of Konkan Kanyal buck semen at refrigeration temperature

Preservation of Konkan Kanyal buck semen at refrigeration temperature

Review of Sustainable Cryopreservation and Above-Freezing Storage Solutions of European Honey Bee Apis mellifera Drone Semen

Abstract The European honey bee Apis mellifera is the main pollinator for most crops used for human consumption. However, a number of diseases, parasites, pesticides and other factors that generally result in the widely described colony collapse disorder weakens honey bee colonies. In order to maintain the existing honey bee germ lines and facilitate the creation of new disease-resistant lines, it is necessary to ensure consistent breeding work, which would also allow the long-term preservation of the unique germplasm lines. One of the most promising solutions for the preservation of honey bee germplasm is the storage of honey bee drone semen. In recent decades, there has been a renewed interest in the preservation of honey bee drone semen using both cryopreservation and above-freezing storage methods. This review summarizes the latest developments in novel sperm storage technologies and their assessment by sperm quality and queen fertility indicators. Additionally, this article analyses the advantages and disadvantages of the reviewed solutions and outlines directions in which additional research would be needed to develop effective, available and affordable drone semen storage solutions. To the best of the authors' knowledge this is the first review of cryopreservation and above-freezing storage solutions of European honey bee Apis mellifera drone semen in the scientific literature.

Effect of genotypes on reaction time, refractory period, semen index and liquid stored semen quality of breeding bulls in Bangladesh

The present study was conducted with the aim to reveal the effect of genotypes on reaction time, refractory period, semen index and liquid stored (at 4oC) semen quality of breeding bulls in Bangladesh. Semen was collected twice a week from Holstein Friesian, Sahiwal and Brahman breeding bulls. Reaction time and refractory period were examined for the breeding bulls. Semen volume, sperm concentration and total spermatozoa/ejaculate were measured immediately after collection. It was revealed that genotype had a significant (P&lt;0.01) effect on semen volume, sperm concentration, total sperm output and refractory period but not in reaction time. From the point of view of semen index, semen quality of Holstein Friesian breeding bulls was superior to Sahiwal and Brahnman breeding bulls. Progressive motility, live and normal spermatozoa of fresh semen did not differ significantly but after dilution progressive motility differed significantly (P&lt;0.05) in different genotypes of breeding bulls. During preservation time (0 to 120 hours), progressive motility, normal and live spermatozoa changed significantly (P&lt;0.05) in each genotype with the progress of time. During 0 hour, 72 hours and 120 hours of preservation, progressive motility was found in Holstein Friesian (73.11±1.12%, 53.11±5.14%, 13.40±2.53%), Sahiwal (78.21±1.68%, 64.96±4.60%, 13.22±1.42%), and Brahman (75.21±1.68%, 54.86±4.40%, 16.96±4.42%), respectively. On the other hand, non-return rate was found insignificant (P&gt;0.05), where higher fertility was observed in Holstein Friesian bull (67.2%) followed by Sahiwal (63.7%) and Brahman bull (57.38%). In a nutshell, Holstein Friesian bull has better fresh, diluted and preserved semen quality than the other two genotypes and after the 3 days of preservation, semen quality in respect of progressive motility, normal and live spermatozoa drastically deteriorated. Therefore, it is recommended that preserved semen of different genotypes should be used for AI within 3 days of liquid semen preservation at 4oC.

Frozen Semen Quality of Kolbroek Boars Concerning Glutathione Concentrations

Glutathione(GSH) is an antioxidant that is being studied for its ability to improvecellular responses to semen preservation challenges. The research goal aimed toevaluate the impact of introducing different glutathioneconcentrations to boar semen freezing extender (0, 1, 5, and 10 mM) onthe Kolbroek boar sperm's quality after being frozen and thawed. For 2 h, thecollected sperm-rich fraction was chilled at 17°C. Following the 1 h ofequilibration, fraction B: Control (egg yolk 20% + BTS 72% + glycerol 8%), 1 mM(egg yolk 20% + BTS 70% + glycerol 8% + GSH 2%), 5 mM (egg yolk 20% + BTS 62 + 8%glycerol + GSH 10%) and 10 mM (egg yolk 20% + BTS 52% + glycerol 8% + GSH 20%)was added into the semen samples. Following that, 0.5 mL straws with thediluted, cooled semen samples were submerged in liquid nitrogen (LN2)vapor and then stored inside the LN2 tank (-196°C). After thawing(37°C), parameters of boar sperm motility, velocity, morphology and viability,membrane permeability, and Malondialdehyde content were assessed and recorded.Using the analysis of variance, the data were examined. The results revealed adifference in total motility (%) on fresh semen as compared to post-thawedcontrol and GSH treatments. A significant difference was revealed in spermprogressive motility (%) in fresh semen (27.07±4.5). Significant differenceswere recorded in live sperm morphology and viability on fresh semen (81.8±2.8%)compared to control, 1, 5, and 10 mM. In the10 mM diluted semen (78.5±6.8), the proportion of sperm with an intact plasmamembrane integrity was highest (P<0.05), while the percentage in the1 mM diluted semen (73.0±2.7) was lower. Malondialdehyde levels were lowest (P<0.05)in the group receiving 5 mM treatment. In conclusion, 5 mM of GSH is therequired amount to be added to the freezing extender while cryopreserving semenfrom Kolbroek boars.