Milk Yolk Research Articles

Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen

Background and Aim: The Kacang goat (Capra hircus) is an indigenous livestock species in Indonesia that is at risk of extinction due to cross-breeding. Artificial insemination (AI) techniques are expected to increase the population of these goats. This study aimed to determine the addition of epigallocatechin-3-gallate chitosan nanoparticles (EGCG CNPs) to skim milk–egg yolk (SM–EY) extender to obtain the best possible quality of post-thawed Kacang buck semen for AI. Materials and Methods: Fresh Kacang buck semen was diluted in SM–EY without or with the addition of 0.5, 1.0, 1.5, or 2.0 µg of EGCG CNPs/mL extender. Extended semen was packaged in French mini straws, frooze, and stored in liquid nitrogen at −196℃ for 24 hours. Six replicates from each treatment group were thawed for catalase, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, malondialdehyde (MDA), sperm intact plasma membrane (IPM), viability and motility analyses. Results: Post-thawed semen that was previously frozen without EGCG CNPs in the extender (control group) exhibited the lowest levels of catalase, DPPH, sperm living cells, sperm motility, MPI, and the highest levels of MDA. The addition of EGCG CNPs in the T3 and T4 groups was increased (p < 0.05) post-thawed catalase, DPPH, living cells, and sperm motility and decreased (p < 0.05) MDA levels than those of the T0 group. Meanwhile, sperm MPI was higher (p < 0.05) in the T4 group than the sperm MPI of the T0 group. Conclusion: This study was the first in using of EGCG CNPs in the SM–EY extender, in which adding 1.5 or 2.0 μg/mL of EGCG CNPs in this extender increased the antioxidant capacity and post-thawed quality of Kacang buck semen.

Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen

Background and Aim: The Kacang goat (Capra hircus) is an indigenous livestock species in Indonesia that is at risk of extinction due to cross-breeding. Artificial insemination (AI) techniques are expected to increase the population of these goats. This study aimed to determine the addition of epigallocatechin-3-gallate chitosan nanoparticles (EGCG CNPs) to skim milk–egg yolk (SM–EY) extender to obtain the best possible quality of post-thawed Kacang buck semen for AI. Materials and Methods: Fresh Kacang buck semen was diluted in SM–EY without or with the addition of 0.5, 1.0, 1.5, or 2.0 µg of EGCG CNPs/mL extender. Extended semen was packaged in French mini straws, frooze, and stored in liquid nitrogen at −196℃ for 24 hours. Six replicates from each treatment group were thawed for catalase, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, malondialdehyde (MDA), sperm intact plasma membrane (IPM), viability and motility analyses. Results: Post-thawed semen that was previously frozen without EGCG CNPs in the extender (control group) exhibited the lowest levels of catalase, DPPH, sperm viability, sperm motility, IPM, and the highest levels of MDA. However, the addition of EGCG CNPs at doses of 1.5 µg/mL extender increased post-thawed catalase, DPPH, sperm IPM, viability, and sperm motility and decreased MDA levels (p < 0.05) than those of control group. Conclusion: This study was the first in which EGCG CNPs were used in SM–EY extender, and the addition of only 1.0 µg/mL of EGCG CNPs in this extender increased the antioxidant capacity and post-thawed quality of Kacang buck semen.

Green Tea Extract in the Extender Improved the Post-Thawed Semen Quality and Decreased Amino Acid Mutation of Kacang Buck Sperm.

This study was the first to combine the addition of antioxidants to a skim milk-egg yolk (SM-EY) extender and different equilibration periods to obtain higher quality post-thawed Kacang buck semen. This study aimed to determine the effects of green tea extract (GTE) on the quality of frozen Kacang goat sperm equilibrated for one and two hours. The pool of Kacang buck ejaculate was equally divided into four portions and was diluted in an SM-EY extender that contained four doses of 0, 0.05, 0.10, and 0.15 mg of GTE/100 mL for T0, T1, T2, and T3 groups, respectively. The aliquots were treated for an equilibration period of 1-2 h before further processing as frozen semen. Post-thawed semen quality was evaluated for sperm quality. The Sanger method was used for DNA sequencing, and the amino acid sequence was read using MEGA v.7.0. The post-thawed semen of the T2 group that was equilibrated for one hour had the highest semen quality. Pre-freezing motility had the highest determination coefficient compared to post-thawed sperm motility. This study is the first to report amino acid mutation due to freeze-thawing. The frequency of amino acid mutations revealed that T2 was the least mutated amino acid. Glycine, valine, leucine, serine, and asparagine strongly correlated to post-thawed sperm motility. It can be concluded that a combination of 0.1 mg GTE/100 mL extender as an antioxidant and one-hour equilibration period resulted in the best post-thawed Kacang buck semen quality.

Nanowater enhances cryoprotective properties of glycerol-containing extenders used for ram semen freezing: A preliminary study spanning laboratory testing

Abstract It has been suggested that nanowater (NW-water declustered in the cold plasma generator and characterized by a low freezing point and high diffusivity) could improve ram semen quality after freezing in glycerol-containing extenders. Eighteen ejaculates from six Olkuska rams were divided into six equal portions each, and then diluted (800×106 spermatozoa/ml) and frozen in the fructose-skimmed milk-egg yolk Kareta extenders containing 3% or 7% of glycerol. The extenders were prepared with deionized water (DW-3% and DW-7%) or NW declustered for 15 min (NW15’) or 30 min (NW30’). Post-thaw sperm motility, proportions of sperm defects and percentages of apoptotic, necrotic, and live spermatozoa were determined. The proportion of spermatozoa with midpiece defects was lower (P<0.05) in NW15’-3% compared with DW-3%. Sperm progressive motility was greater (P<0.05) for spermatozoa cryopreserved in both NW30’ (NW30’-3%/7%) extenders compared with their respective controls (DW30’-3%/7%). The proportion of necrotic spermatozoa 1 h after thawing was lower (P<0.05) in NW30’-7% compared with DW-7%, whereas the proportion of live cells detected immediately and 1 h after thawing was greater (P<0.05) in NW30’-7% than in DW-7%. In summary, NW enhanced cryoprotective effects of glycerol-containing extenders with an increase in sperm viability being greater with 7% than 3% of glycerol. Different declustering times appear to alter NW properties. These observations merit future studies of the utility of NW for semen cryopreservation in rams and other mammalian species. The specific mechanisms whereby NW ameliorates the quality of frozen-thawed ram spermatozoa remain to be elucidated.

Combination of nanoparticle green tea extract in tris-egg yolk extender and 39 °c thawing temperatures improve the sperm quality of post-thawed Kacang goat semen.

Kacang goats are small ruminants produced by low-income households in smallholder and farm to reduce poverty and prevent undernutrition. Studies to find a cryopreservation protocol for Kacang goat semen are expected to multiplication of genetically superior animals selected by the paternal lineage. This study evaluated the effect of thawing temperature and supplementation of the green tea extract nanoparticle in skim milk-egg yolk (SM-EY) extender on post-thaw sperm quality of Kacang goat semen. Six ejaculates of Kacang goat were diluted in SM-EY supplemented or not (control group) with 0.001 mg/mL NPs GTE. The diluted semen was packaged with 0.25 mL straws (insemination dose: 60x106 sptz/mL) and cryopreserved. Then, six samples of the control group and NPs GTE groups were thawed at 37°C or 39°C sterile water for 30 s and submitted to sperm quality evaluations. The sperm viability, motility, and intact of the plasma membrane (IPM) were higher (p<0.05) in NPs GTE group than control group. In contrast, the NPs GTE group presented lower (p<0.05) malondialdehyde levels and sperm DNA fragmentation (SDF) compared with the control group. The catalase levels were not significantly different (p > 0.05) between the control and NPs GTE groups. Thawing at 39°C resulted in higher (p<0.05) sperm viability, motility, and IPM than thawing at 37°C. However, thawing at 39°C group presented lower (p<0.05) malondialdehyde levels compared with thawing at 37°C. SDF and catalase levels were similar (p>0.05) between thawing at 37°C and thawing at 37°C. In conclusion, supplementation of 0.001 mg/mL of NPs GTE in SM-EY extender and thawing temperature of 39°C resulted in a better quality of frozen-thawed Kacang goat semen.

A 150 kDa Protein Derived from Bull Seminal Plasma Extended the Survival Time of Kacang Goat Sperm Stored at 5°C.

Artificial insemination has proven to be an effective method for increasing population size and genetic quality of Kacang goats. However, innovation is required to maintain the quality of Kacang goat semen in storage. This study aimed to examine the effects of supplementing the 150 kDa protein assumed as IGF-I complex derived from bull seminal plasma in skim milk-egg yolk extender on the quality of Kacang goat sperm stored at 5°C. Twelve ejaculates collected from three Kacang goats were divided into three groups. In the control group (T0), the ejaculates were extended with skim milk-egg yolk only. In the treatment groups (T1 and T2), the ejaculates were extended with skim milk-egg yolk supplemented with the IGF-I complex protein at 12 μg and 24 μg/100 mL, respectively. The extended semen was stored at 5°C, and the viability, motility, intactness of the plasma membrane, malondialdehyde concentration, and apoptotic sperm percentage were evaluated daily for five days. The results showed that the T1 was the most effective treatment for maintaining Kacang goat semen at a quality acceptable for artificial insemination over five days of storage at 5°C. However, the T0 and T2 groups retained acceptable qualities for only three days at 5°C. It could be concluded that supplementation of 12 μg of the 150 kDa protein derived from bull seminal plasma per 100 mL extender successfully extended the life span of Kacang goat sperm for five days.

Maintaining the Quality of Kacang Buck Semen in Chilled Storage with the Addition of Green Tea Extract in Extender

Kacang goats can be bred through artificial insemination techniques using liquid semen from a superior buck. The study was aimed to determine the quality of Kacang buck semen in chilled storage when green tea extract (GTE) was added to the semen extender. Specifically, 12 ejaculates from three Kacang bucks were diluted in skim milk–egg yolk extender containing 0 mg, 0.05, 0.10, and 0.15 mg of GTE/100 mL. The extended semen was stored at 5°C, and its quality was evaluated daily for 5 d. Comparatively, semen with 0.10 mg of GTE/100 mL had the best quality (p<0.05): it had the highest sperm viability, sperm progressive motility, and sperm intact plasma membrane values, and the lowest malondialdehyde levels and DNA fragmentation percentage. However, as the storage period increased, there was a decrease in sperm viability, sperm motility, and sperm intact plasma membrane values with an increase in malondialdehyde levels and DNA fragmentation (p<0.05). The sperm motility of semen with 0.10 mg of GTE/100 mL was maintained during 5 d of chilled storage to meet the qualification for artificial insemination. In contrast, the sperm motility of semen with 0.05 mg and 0.15 mg of GTE/100 mL was maintained for 4 d, whereas that of the control semen was maintained only for 3 d. Thus, the addition 0.10 mg of GTE/100 mL of skim milk-egg yolk extender seems to help maintain the quality of Kacang goat sperm in chilled storage.

The The Efectiveness of Jamblang (Syzygium cumini) Leaves Extract Inclusion Skim Milk-Egg Yolk Extender on Motility and Viability of Aceh Cattle Spermatozoa during Pre-Freezing and Post-Thawing

The process of semen freezing causes an increase in free radicals concentration which can damage spermatozoa. The addition of natural ingredients in semen diluent is expected to solve this challenges. One of the natural ingredients that can be used is jamblang (Syzygium cumini) leaves. The objective of the current study was to investigate the quality of spermatozoa in Aceh cattle which was added with jamblang leaves extract in skim milk- egg yolk extender during pre-freezing and post-thawing. This study applied Completely Randomized Design (CRD) with 5 treatments and 5 replications. The treatments consisted of J0 = skim milk-egg yolk; J1 = skim milk-egg yolk + jamblang leaves extract 0.2%; J2 = skim milk-egg yolk + jamblang leaves extract 0.4%; J3 = skim milk-egg yolk + jamblang leaves extract 0.6%; and J4 = skim milk-egg yolk + jamblang leaves extract 0.8%. The parameters observed in this study were the percentage of motility and viability of frozen semen of Aceh cattle. The data obtained were analyzed using Analysis of Variance (ANOVA) and if differences were found, then it would be continued with Duncan's Multiple Distance test. The results showed that the addition of jamblang leaves extract in egg yolk skim milk significantly affected the percentage of motility during pre-freezing and post-thawing, significantly affected spermatozoa viability during pre-freezing and significantly affected the spermatozoa viability during post-thawing. J3 treatment (jamblang leaves extract 0.6 gram/100 ml) it should be higher than the other treatment, where the percentage of motility at pre-freezing and post-thawing were 55.48% and 52.71%, respectively, and the percentage of viability during pre-freezing and post-thawing were 56.59% and 53.94%, respectively. It was concluded that the addition of jamblang leaves extract in the skim milk-egg yolk extender affected the percentage of spermatozoa motility and viability of Aceh cattle during pre-freezing and post-thawing.

Kualitas post-thawing semen domba Merino dalam bahan pengencer berbasis susu skim-kuning telur yang ditambah isolat crude protein Tirosine Kinase

This study was conducted to determine the effect of crude protein tyrosine kinase (PTK) isolates supplementation into skim milk-egg yolk based diluent to maintain the quality of Merino ram spermatozoa. Four ejaculates of two Merino rams were divided into two groups for the control group (P0): Merino ram semen was diluted in skimmed milk-egg yolk based diluent, and the treatment group (P1): Merino ram semen was diluted in skim milk-egg yolk based diluent contained crude PTK 1,597 mg/ml diluent. All diluted semen was equilibrated for 2 hours at 5 °C and filled into 0.25 mL French straws. The filled straws were placed on steel racks (Cooltop, Minitube) held in liquid nitrogen vapour for 10 minutes at –140 °C, immersed immediately in liquid nitrogen at –196 °C, and stored for 48 hours for later assessment. Post-thawed semen samples were evaluated for spermatozoa motility, viability, and morphological abnormality. The results showed that the spermatozoa motility of fresh semen of Merino ram was 82.5 ± 2.89, which was qualified for freezing. The post-thawing spermatozoa motility, viability, and morphological abnormalities of Merino ram in the P1 group were 34.11 ± 3.26%, 38.00 ± 3.00%, and 12.89 ± 4.54%, respectively. It were higher (p &lt;0.05) than the control group of 24.44 ± 2.9%, 26.67 ± 3.32%, and 21.11 ± 3.02%. It was concluded that the addition of crude PTK isolates of 1.597 mg/ml skim milk-egg yolk diluent improved the quality of post-thawed spermatozoa of Merino ram.

Effect of insulin-like growth factor-1 complex of Simmental bull seminal plasma on post-thawed Kacang buck semen fertility.

Background and Aim:Kacang buck sperm is cryosensitive due to the seminal plasma of semen itself. Meanwhile, bull seminal plasma contains the insulin-like growth factor-1 (IGF-1) complex, which is cryoprotective. The addition of the crude protein of Simmental bull seminal plasma increased the quality of post-thawed semen of Kacang buck. The study was conducted to determine the effects of Simmental bull seminal plasma with IGF-1 on the fertility of post-thawed Kacang buck semen.Materials and Methods:Buck semen was diluted in the following skim milk-egg yolk extender preparations: Without the addition of Simmental bull seminal plasma IGF-1 complex protein (T0); with the addition of 12-μg Simmental bull seminal plasma IGF-1 complex protein (T1); and with the addition of 24-μg Simmental bull seminal plasma IGF-1 complex protein (T2). The extended semen was packed in 0.25-mL straws and frozen. Post-thawed semen fertility was evaluated based on the following variables: Sperm motility, viability, intact plasma membrane (IPM), malondialdehyde (MDA) levels, capacitation status, and acrosome reaction. The difference in each variable among the groups was evaluated using analysis of variance, followed by Tukey’s honestly significant difference test, at a 95% level of significance. Meanwhile, principal component analysis (PCA) was used to identify the principal component of semen fertility among the seven parameters.Results:The T1 group showed the highest sperm motility, viability, IPM, and percentage of incapacitated sperm and the lowest MDA levels, percentage of capacitated sperm, and acrosome reaction. PCA revealed that sperm motility had a moderate to very robust correlation with other variables and is the most crucial parameter, accounting for 80.79% of all variables.Conclusion:The IGF-1 complex in Simmental bull seminal plasma was useful for increasing the fertility of post-thawed Kacang buck semen, and sperm motility was the principal component of semen fertility.

Green tea extract increases the quality and reduced DNA mutation of post-thawed Kacang buck sperm

The study aimed to determine the addition of green tea extract (GTE) in extender on the quality and DNA mutation of post-thawed Kacang buck sperm. The sperm DNA mutation was observed on nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase 1 (ND1) of mitochondrial Deoxyribonucleic Acid (mtDNA). A pool of 12 Kacang buck ejaculates was diluted in skim milk-egg yolk extender contained 0, 0.05, 0.10, and 0.15 mg of GTE/100 mL for T0, T1, T2, and T3 group, respectively. Each of the aliquot groups was packaged in 0.25 mL French mini straw contained 60 million alive sperm and froze according to the protocol. The ND1 mtDNA amplification of samples was carried out Polymerase Chain Reaction machine, followed by DNA sequencing using the Sanger method. Meanwhile, the phylogenetic tree was constructed using the neighbor-joining (NJ) method with MEGA 7.0 software. The results showed that the T2 group maintained the highest quality for Kacang buck post-thawed semen. There was the highest percentages of sperms viability, motility, intact plasma membrane (IPM), the lowest of malondialdehyde (MDA) concentration, sperm DNA fragmentation (SDF), the total and types of ND1 mtDNA mutation frequency. The phylogenetic tree analysis revealed that the clade of the T2 group was most closely related to the sequence reference. However, there was no correlation between the semen quality parameters (sperm viability, motility, IPM, MDA concentration, and SDF) with ND1 mtDNA mutation of post-thawed Kacang buck semen. It could be concluded that GTE was useful as an antioxidant for Kacang buck semen extender for frozen sperm.

The Effect of Jamblang Leaves Extract (Syzygium cumini) Inclusion Skim Milk-Egg Yolk Extender on Motility, Abnormality and Viability of Aceh Cattle Spermatozoa Stored at 4ºC

This research aimed to know the effect of the addition of jamblang leaves extract (Syzygium cumini) in skim milk-egg yolk diluent material on motility, abnormality, and viability of Aceh cattle stored at 4ºC. The research design used was Complete Randomized Design consisting of 5 treatment and 5 repetitions. The treatments consisted of J0 (SSKT), J1 (SSKT+ EDJ 0. g/ 100 ml), J2 (SSKT+ EDJ 0.4 g/ 100 ml), J3 (SSKT+ EDJ 0.6 g/ 100 ml) and J4 (SSKT+ EDJ 0.8 g/ 100 ml). The data obtained were analyzed descriptively by determining the mean value and standard deviation using Microsoft Excel. The research result indicated that the addition of jamblang leaves extract in skim milk-egg yolk diluent material maintaining the motility, viability and abnormality of the spermatozoa of Aceh cattle stored at the temperature of 4ºC. Based on the research that has been carried out, it can be concluded that the addition of jamblang leaves extract in skim milk-egg yolk diluent material can maintain the quality of Aceh cattle spermatozoa, while the use of incorrect dose becomes toxic and disrupt the spermatozoa activities to cause death.

Penambahan alfa-tokoferol dalam pengencer susu skim - kuning telur terhadap kualitas spermatozoa domba Sapudi yang disimpan pada suhu 5°C

Spermatozoa in fresh semen of Sapudi ram has a limited life span. The storage of semen in cold temperatures (5 °C) is intended to prolong the spermatozoa's life. However, storage in cold temperatures can lead to increased production of reactive oxygen species (ROS). This condition reduces the quality of spermatozoa. The purpose of this study was to determine the effect of alphatocopherol supplementation in skim milk-egg yolk extender on viability, motility, and plasma membrane integrity of Sapudi ram spermatozoa. Fresh semen derived from Sapudi ram was divided into four treatment groups. Control treatment (P0): semen was added in the extender of skim milkegg yolk without alpha-tocopherol. Three other treatments: P1, P2, and P3 semen were added in skim milk-egg yolk extender with the supplementation of 0.25, 0.5, and 1 gram alpha-tocopherol/ 100 mL extender, respectively. The results showed that the viability, motility, and integrity of the spermatozoa plasma membrane decreased gradually according to the storage length. Supplementation of skim milk-egg yolk extender with 0.5 gram of alpha-tocopherol/100 mL (P2) was able to maintain spermatozoa quality longer (p &lt;0.05) than the control group. It can be concluded that alpha-tocopherol with a concentration of 0.5 g/100 mL of skim milk-egg yolk extender effectively maintains the quality of Sapudi ram spermatozoa in storage at 5 ° C.

Effect of α-tocopherol supplementation in diluents on the motility, viability and plasma membrane integrity of Simmental bull spermatozoa after cooling

Semen storage in cold temperatures might cause an increase in reactive oxygen species (ROS) production. This condition resulted in spermatozoa damage and quality decrease. This study was conducted to investigate the effects of α-tocopherol supplementation in diluents on the motility, viability, and plasma membrane integrity of Simmental bull spermatozoa after cooling. Semen samples were diluted in skim milk egg yolk supplemented with 0, 0.5, 1.0, and 1.5 mM α-tocopherol respectively for control, Tl, T2, and T3. Spermatozoa were evaluated for their motility, viability, and membrane integrity in cooling temperature (5°C). The daily evaluation showed that 1.5 mM α-tocopherol was the best in maintaining motility, viability, and plasma membrane integrity, while 1.0 mM α-tocopherol was only good for maintaining viability. Therefore, it can be concluded that α-tocopherol at the concentration of 1.5 mM was an efficient antioxidant supplement for Simmental cattle semen in skim milk egg yolk diluent.

Effect of green tea extract in extender of Simmental bull semen on pregnancy rate of recipients.

ObjectiveThe aim of this study was to ascertain the effects of adding green tea extract (GTE) to skim milk-egg yolk (SM-EY) extender on both the quality of post-thawed bull semen and the pregnancy rates of the recipient cows.MethodsTwelve ejaculates from four Simmental bulls, aged 3 to 5 years and weighing 900 to 950 kg, were diluted SM-EY extender, added with 0, 0.05, 0.1, and 0.15 mg GTE/100 mL extender and then frozen. After four weeks storage in liquid nitrogen, the sperm were thawed and evaluated for viability, motility, intact plasma membrane (IPM), and DNA fragmentation. Meanwhile, the estrus cycles of 48 recipient cows were synchronized by intramuscular administration of a single injection of 5 mg prostaglandin F2α. Estrus cows were divided into four equal groups and inseminated artificially 18 to 20 h after the onset of estrus by using semen from each extender group. Pregnancy was diagnosed by measuring serum progesterone levels at 21 days, followed by transrectal palpation 90 days after insemination.ResultsThe findings revealed that adding 0.1 mg of GTE/100 mL extender produced the highest percentages of sperm viability (70.67%±1.75%), motility (69.17%±1.47%), and IPM (69.23%±1.21%) and the lowest percentage of DNA fragmentation (3.00%±0.50%). The pregnancy diagnosis revealed that all cows (36/36) inseminated using frozen semen in GTE addition extender were pregnant (pregnancy rate 100%), whereas the pregnancy rate of the control group was 83.33% (10/12).ConclusionIt may be concluded that 0.1 mg GTE/100 mL extender yields the best quality of spermatozoa and that all variants doses of GTE in extender produce a higher pregnancy rate among recipient cows.

KUALITAS MOTILITAS DAN VIABILITAS SPERMATOZOA DARI SEMEN AFKIR SAPI LIMOUSIN PADA PENGENCER SUSU SKIM KUNING TELUR SITRAT DENGAN PENAMBAHAN BERBAGAI KADAR GLUKOSA

Quality of spermatozoa motility and viability from rejected limousin bull semen diluted with skim milk egg yolk sitrat added with various levels of glucose. Glucose level used were 0%, 0,5%, 1,5%, 2,5%, and 3,5%. Writer was using on four years old Limousine bull. Bull semen used in this research was bull rejected semen with bellow 70% motility. Semen observation was done at 0 hour, 24 hours and 48 hours. Research design used in this study was completely randomized design with faktorial pattern with 5 replicates. Highest result in motility this research was showed at 24 hours with 30% value in glucose 2,5% treatment and 48 hours with 10% value. The lowest result showed in glucose 0% treatment at 24 hours and 0% at 48 hours. Highest result in viability showed on glucose 2,5% treatment with 62,6% value at 24 hours and at 48 hours with 53,4% value. Lowest result in viability showed on glucose 0% treatment with 44,2% value at 24 hours and 31,4% value at 48 hours.

39 Nanowater enhances cryoprotective effects of glycerol during ram semen freezing

It was suggested that unique physicochemical properties of nanowater (NW, water declustered in the cold plasma generator), such as a low dielectric constant and high diffusivity, might ameliorate ram semen freezing by improving bioavailability of extender constituents. This study was undertaken to evaluate the effects of NW added to glycerol-containing semen extenders on the characteristics of frozen-thawed ram semen. A total of 18 ejaculates collected from 6 Olkuska breed rams during the breeding season were divided into six equal portions. The ejaculates were frozen in the modified fructose-skimmed milk-egg yolk Kareta extender containing 3 or 7% glycerol and diluted in deionized water (control extenders; Aqua Purificata, Prolab; C3% and C7%, respectively) or NW (declustered for 15min (NW15’) or 30min (NW30’); Nanotechnology Systems) to a final concentration of 800×106 spermatozoa/mL. They were placed in 0.25-mL plastic straws and frozen in liquid nitrogen. The two declusterization times were chosen on the basis of previous laboratory tests and fertility trials yielding the best results in terms of semen quality post-thawing and pregnancy rates after AI. All semen samples were evaluated for sperm concentration, progressive motility (Sperm Class Analyzer), and morphological defect rates (Nikon Eclipse 80i microscope, Nikon Corp.). In addition, the ex situ survival time at 37°C and extender content of alanine transferase, alkaline phosphatase, and aspartate aminotransferase were measured, along with the proportions of apoptotic, necrotic, and live spermatozoa determined post-thawing with flow cytometry (BD Accuri™ C6 Plus, Becton Dickinson) at 0 and 1h post-thawing. Data were analysed by one-way analysis of variance and Holm-Sidak method using SigmaPlot statistical software (Systat Software Inc.). The proportion of spermatozoa with mid-piece defects was significantly (P&amp;lt;0.05) reduced in NW15’-3% compared with C3% (1.3±0.3% vs. 4.0±06%; mean±s.e.m.). The proportion of necrotic spermatozoa 1h after thawing was greater (P&amp;lt;0.05) in C7% compared with NW30’-7% (20.7±0.4% vs. 17.6±1.0%), whereas the proportions of live cells detected immediately (0h) and 1h after thawing were greater (P&amp;lt;0.05) in NW30’-7% than in C30’ (0 h: 54.0±0.6% vs. 50.4±1.2%; 1 h: 54.4±1.2% vs. 59.2±0.4%, respectively). The mean survival time of spermatozoa was greater (P&amp;lt;0.05) in extenders dissolved in NW30’compared with their respective controls (NW30’-3% vs. C3%: 215±4min vs. 189±6 min; and NW30’-7% vs. C7%: 242±8min vs. 217±4min, respectively). Alkaline phosphatase concentrations in extenders prepared with NW30’ were lower (P&amp;lt;0.05) compared with the control groups (NW30’-3% vs. C3%: 3105±231IU vs. 4490±458 IU; and NW30’-7% vs. C7%: 2516±128IU vs. 3956±263IU, respectively). Our results indicate that NW significantly improves the effects of glycerol on ram sperm viability post-thawing, with the reduction in sperm necrosis/overall enhancement of sperm survivability being greater with 7% compared with 3% of glycerol in semen extender.

195 Sperm motion characteristics of ram semen liquid-stored in a milk egg yolk extender at four temperatures

Abstract Varying temperatures have been used for liquid storage of ram semen and that of other species. This study evaluated motility characteristics of ram semen stored at 5, 10, 15, and 20°C for up to 96 h. Two ejaculates were collected and pooled from each of 6 rams using an artificial vagina and evaluated for motility and concentration. Samples were extended in ultra-high temperature pasteurized skim milk and egg yolk (10% v/v) containing penicillin and streptomycin, diluted to 250 million sperm/mL, and packaged in 0.5 mL straws. Semen was held at 32°C during processing, and straws placed in 500 mL jars for storage at 5°C (refrigerator), 10 and 15°C (refrigerated water bath) and 20°C (air conditioned room temperature). Cooling rates were 0.04, 0.25, 0.44, and 0.02 °C/min, and final temperatures reached after 672, 83, 36, and 567 min in the four storage environments, respectively, with cooling rates faster in a liquid than air environment. Straws from individual rams were removed at 6, 24, 48, 72 and 96 h of storage and analyzed with a computer-assisted sperm analyzer after warming to 36°C. Data were analyzed for the effect of storage time, temperature, and their interaction on sperm motion characteristics. Sperm motion characteristics were not affected over time during storage at 5 and 10°C. At 15°C motility parameters decreased in a curvilinear (P &lt; 0.05) relationship with time (progressive motility: 52.6 to 29.7%; rapid motility: 36.5 to 16.5%), and at 20°C in linear (P &lt; 0.001) relationship (progressive motility: 51.9 to 13.1%; rapid motility: 36.1 to 6.3%). Circular motility decreased (P &lt; 0.05) with increasing temperatures after 72 and 96 h of storage, while local motility was not affected by storage temperature and time. Results suggest storage at 10°C may be a viable alternative to storage at 5°C as retention of motility was similar.

Effects of extender, cryoprotectants and thawing protocol on motility of frozen-thawed stallion sperm that were refrozen for intracytoplasmic sperm injection doses

We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose–EDTA–egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk–egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially thawed to 5 °C, diluted and refrozen in SMEY containing 2, 4, 6 or 8% GLY or GMF. In Experiment 1, sperm motility was reduced after each cryopreservation cycle (P < 0.05). Extender type did not affect motility after refreezing (P > 0.05), but sperm initially thawed to 5 °C exhibited higher motility than sperm thawed to 37 °C (P < 0.05). In addition, sperm refrozen in SMEY containing MF or GMF exhibited higher motility than sperm refrozen in GLY alone (P < 0.05). In experiment 2, there was an interaction between CPA and CPA concentration (P < 0.05). Sperm refrozen with GMF had higher motility than refrozen sperm with GLY (P < 0.05), and while GLY concentration did not affect post-thaw motility (P > 0.05). Sperm refrozen with 6 or 8% GMF exhibited the highest motility (P < 0.05). In conclusion, sperm motility is best maintained when thawing and refreezing stallion sperm in low sperm concentration ICSI doses by initially thawing the sperm to 5 °C and diluting the sperm in a freezing extender with 8% GMF.

Cryopreservation of Swamp Buffalo Semen in Skim Milk Yolk-based Diluent with Two Different Cryoprotectants

The successful of artificial insemination (AI) in buffalo is still low. The quality of frozen semen determines the successful of AI program. Glycerol is widely used for cryopreservation of buffalo semen with unsatisfactory results due to its toxicity and antifertility properties. This study aimed to investigate the effect of glycerol and dimethylformamide (DMF) concentrations in skim milk egg yolk (SMEY)–based diluent on the quality of frozen-thawed swamp buffalo bull semen. Fresh semen was divided into eight aliquots. Each aliquot was diluted in SMEY containing 4%, 5%, 6%, and 7% glycerol and 4%, 5%, 6%, and 7% DMF. Diluted semen was packed into mini straw (0.25 mL), equilibrated at 4 °C for 4 hours, frozen in liquid nitrogen vapor for 10 minutes, and stored at liquid nitrogen container. The straws were thawed after 24 hours at 37 °C for 30 seconds for evaluation. The results showed that the post thawed sperm motility and recovery rate in SMEY extender containing 7% glycerol and 5% DMF were higher than the other glycerol or DMF concentrations. Post thawed sperm viability and plasma membrane integrity in SMEY containing 4%-6% DMF did not differ significantly, however both values were higher than those in 7% DMF. Post thawed sperm viability and plasma membrane integrity did not differ between SMEY containing 6% and 7% glycerol. However, both values were higher than those in 4% and 5% glycerol. It is concluded that 7% glycerol or 5% DMF is considered to be the most suitable cryoprotective agent for swamp buffalo semen cryopreservation.The successful of artificial insemination (AI) in buffalo is still low. The quality of frozen semen determines the successful of AI program. Glycerol is widely used for cryopreservation of buffalo semen with unsatisfactory results due to its toxicity and antifertility properties. This study aimed to investigate the effect of glycerol and dimethylformamide (DMF) concentrations in skim milk egg yolk (SMEY)–based diluent on the quality of frozen-thawed swamp buffalo bull semen. Fresh semen was divided into eight aliquots. Each aliquot was diluted in SMEY containing 4%, 5%, 6%, and 7% glycerol and 4%, 5%, 6%, and 7% DMF. Diluted semen was packed into mini straw (0.25 mL), equilibrated at 4 °C for 4 hours, frozen in liquid nitrogen vapor for 10 minutes, and stored at liquid nitrogen container. The straws were thawed after 24 hours at 37 °C for 30 seconds for evaluation. The results showed that the post thawed sperm motility and recovery rate in SMEY extender containing 7% glycerol and 5% DMF were higher than the other glycerol or DMF concentrations. Post thawed sperm viability and plasma membrane integrity in SMEY containing 4%-6% DMF did not differ significantly, however both values were higher than those in 7% DMF. Post thawed sperm viability and plasma membrane integrity did not differ between SMEY containing 6% and 7% glycerol. However, both values were higher than those in 4% and 5% glycerol. It is concluded that 7% glycerol or 5% DMF is considered to be the most suitable cryoprotective agent for swamp buffalo semen cryopreservation.