62 EFFECTS OF VARYING GLUTATHIONE CONCENTRATIONS IN SEMEN EXTENDER ON THE QUALITY OF FROZEN–THAWED CANINE SPERM

2014 ◽  
Vol 26 (1) ◽  
pp. 145
Author(s):  
K. Ogata ◽  
B. Sarentonglaga ◽  
M. Yamaguchi ◽  
A. Sasaki ◽  
Y. Kato ◽  
...  

Trans-cervical insemination (TCI) with cryopreserved semen offers a potentially effective approach for breeding canids with specific genetic traits, such as guide dogs for the blind. However, there are technical difficulties in canine sperm cryopreservation, such as the composition of semen extender. The aim of this study was to evaluate the effects of glutathione (GSH) as an antioxidant in the semen extender to improve the quality of frozen-thawed dog sperm. A Tris-egg yolk-citrate extender containing 15.7 mg mL–1 of TRIS, 8.8 mg mL–1 of citric acid, 14.1 mg mL–1 of lactose, 25.4 mg mL–1 of raffinose, 1% (vol/vol) antibiotics, and 20% (vol/vol) egg yolk in ultra-pure water was used as the base medium. Twelve ejaculates were collected from 7 dogs. Each ejaculate was divided into 2 to 5 aliquots and extended with base extender supplemented with 0, 2.5, 5, 7.5, and 10 mM GSH as first dilution. The extended semen was equilibrated for 3 h at 4°C. An equal volume of second extender was added to obtain a final concentration of 6.5% glycerol and sperm per milliliter. The sperm samples were loaded in straws and frozen at 6 cm above the surface of LN2 for 15 min in a styrene foam box and plunged into the LN2. The frozen semen was thawed for evaluation. The motility of sperm was estimated with a phase-contrast microscope and the motile patterns were classified into the following grades: progressively motile at a high speed (+++), progressively motile at a moderate and low speed (++), motile without progression (+), and immotile (–). Then, the sperm motility index (SMI) was determined from the following formula as described previously (Iritani et al., 1975), with some modifications: the percentage of (+++) sperm + the percentage of (++) sperm × 0.75 + the percentage of (+) sperm × 0.5. Sperm motility and the SMI were determined at 0, 1, 2, 3, 4, 12, and 24 h after thawing. Acrosome status was evaluated at 4 h after thawing. Lipid peroxidation (LP) levels at 0 and 12 h after thawing were used to examine the antioxidant ability of GSH. Trans-cervical insemination was carried out on 5 bitches to evaluate the fertility of GSH-treated sperm. The TCI were performed nonsurgically with a laparoscope and deposited 2 mL of semen through a catheter. Each bitch was inseminated 1 to 2 times during oestrus. Data were analysed using ANOVA with the Tukey-Kramer method. We found that the rate of (+++) sperm in the 5 mM GSH group was higher than that in the 0 mM group from 1 to 24 h after thawing (P < 0.05). The SMI was higher in the 5 and 7.5 mM GSH groups than in the 0 mM group (P < 0.05). There were no significant differences in the control and 2.5 and 10 mM GSH groups. Long-term survival was increased in the 5 mM GSH group. Acrosome integrity was higher in the GSH-treated group. The level of LP was lower in the GSH-treated groups at 0 h after thawing (P < 0.05). Trans-cervical insemination with the 5 mM GSH-treated semen resulted in the delivery of 5 pups from 2 bitches. These results indicate that the cryopreservation with 5 mM GSH can improve the motility, viability, and fertility of frozen-thawed canine sperm by its antioxidant effects on the sperm membrane.

2016 ◽  
Vol 47 (2) ◽  
pp. 60-67
Author(s):  
P. Folková ◽  
J. Šichtař ◽  
O. Šimoník ◽  
A. Dokoupilová ◽  
R. Rajmon

Abstract The aim of the study was to evaluate the effect of repeated semen collection and the substitution of normal egg yolk with clarified egg yolk to commercially produced semen extender on qualitative parameters of frozen-thawed canine semen. Two semen collections were scheduled in a 24-hour interval and in each of six dogs, three 1st and three 2nd collections were performed. The frozen-thawed sperm samples were prepared either with clarified or normal egg yolk and motility and viability were evaluated. The effect of the sequence of semen collection was demonstrated by significant differences in motility and also in viability of sperms both in native and frozen-thawed ejaculate. The percentage of viable sperms was significantly higher in samples from the 2nd compared to the 1st collection. This trend was the same also in motility except in native ejaculate. The addition of clarified egg yolk was beneficial for higher survival of sperms immediately after thawing and also after 30 min of incubation, compared to samples with normal egg yolk. Sperm motility evaluated after thawing was higher in samples with clarified egg yolk, without an apparent connection with semen collection sequence. The decrease of values of the qualitative parameters of sperms observed in the period of 30 min of incubation was significantly slowed down when clarified egg yolk was used. This was especially obvious in samples from the 2nd collection.


2004 ◽  
Vol 16 (2) ◽  
pp. 127 ◽  
Author(s):  
G. Brogliatti ◽  
G. Barreiro ◽  
G. Larraburu ◽  
A. Laborde

Flow citometry cell sorting has been proven successfully to separate X and Y sperm; however, the technology is still too stressfull for the viability of the sorted semen. The objective of this study was to evaluate nonsexed and sexed frozen sperm motility characteristics using a CASA technology. Ejaculates from 4 different bulls (3 Holstein and 1 Angus) were collected, and processed as split non-sexed and sexed semen samples using Tris egg yolk extenders. X and Y sperm were separated using a high-speed sorter (SX Moflo). Cryopreservation was done at the same time under appropiate conditions using a programmed cryochamber. Thawing procedure was done at 37°C for 30s and a sample of each straw was placed in the evaluation chamber. The experiment was repeated twice and two chambers with 30 observations each were analyzed each time. Mean and standard deviation of each characteristic were calculated, compared and analyzed statistically. The sperm concentration was determined by means of a burker counting chamber. Sperm quality was determined at 0h after thawing, and later at 1h, 2h and 3h after incubation in a glass tube at 30°C. The following sperm motility parameters were determined with the Hamilton Thorne (HTM-ceros 12.1) on at least 1000 spermatozoa: velocity average path (VAP), velocity straight line (VSL), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and percentage of progressively motile spermatozoa (PMS). Linearity of nonsexed spermatozoa was 53±3.5, 47±0.8, 43±7.8 and 42±4.5 for the 0h and the 3 test incubation times and 49.5±3.7, 51.2±3.7, 43.3±7.8 and 44.5±7.6, respectively, for sexed semen. There were no significant differences (P&gt;0.05) in the progressive velocity, track speed and linearity between sexed and nonsexed semen. The percentage of static cells was 33%, 30%, 47% and 50% for the 0h and the 3 test incubation periods; however, the percentage of static cells for the sexed semen was 53%, 71%, 77% and 82%, respectively. Results from the analysis indicate a significant increase (P&lt;0.01) in the number and the percentage of static cells with time. The lateral amplitude of sperm motility for nonsexed semen was 5.9±0.5, 6.8±0.8, 6.0±0.4 and 5.1±0.7, and for sexed semen 6.6±0.7, 6.8±0.4, 6.4±0.4 and 5.5±1.7, respectively. The percentage of progressively motile sperm was significantly different at 0 time 49.7±4.9 and 23.1±4.9 for nonsexed and sexed semen respectively. After 3 hours of incubation the percentage of progressively motile sperm was 38.7±10.2 and 3.7±3.2 for nonsexed and sexed semen, respectively. In conclusion, sexed frozen semen seems to have characteristics similar to those of normal nonsexed semen. However, a significant decrease in the percentage of progressively motile cells could affect pregnancy rates. More research needs to be done to detect differences between bulls and cryoprotectans.Research supported by Centro Genetico Bovino de EOLIA sa Argentina.


2021 ◽  
Vol 44 (2) ◽  
pp. 146-151
Author(s):  
Nurcholis Nurcholis ◽  
A. Furqon ◽  
R. I. Arifiantini ◽  
S. M. Salamony

Antioxidants such as tocopherol, ß-carotene, and polyunsaturated fatty acids (PUFA) from red fruit oil of Papua may be used to protect frozen semen. The study is aimed to test the effect of red fruit oil supplementation on motility, viability, and recovery rate of frozen sperm of Ongole-grade bulls. Semen was collected twice a month from eight 4-5-year-old male Ongole grade using an artificial vagina, followed by macro- and microscopical evaluations. Collected semen was divided into four tubes and diluted with tris egg yolk diluents (TEY) as a control, TEY supplemented with 0.5% red fruit oil (RFO) (TEY RFO0.5), TEY supplemented with 1% RFO (TEY RFO1.0), and TEY supplemented with 1.5% RFO (TEY RFO1.5). The diluted semen was then packed into the straw and equilibrated for 2, 4, and 6 h prior to frozen on liquid nitrogen vapor for 10 minutes. The observed variables in this study were sperm motility, sperm viability, and morphology after equilibration, after thawing, and recovery rate. The experimental design is a completely randomized factorial design. The data were analyzed using ANOVA and were further tested using Duncan multiple range test. The results showed that the sperm motility of fresh semen was 81.10±1.42%. The percentage of sperm motility in TEY RFO1.5 treatment at 6 h equilibration was 60.00±1.06%, significantly higher compared to TEY RFO1.0 and TEY RFO0.5. The percentage of post-thawing sperm motility in TEY RFO1.5 treatment was 62.40±1.09%. The best post-thawing sperm viability in TEY RFO1.5 was 80.70±1.20%, significantly increase from the treatment of TEY RFO1.0 and TEY RFO0.5. The recovery rate (RR) for TEY RFO1.5 treatment had the best percentage at 76.94%. In conclusion, RFO supplementation in semen diluents for 2 h of equilibration resulted in the best motility and viability at 0 h of post thawing observation.


2020 ◽  
Vol 13 (11) ◽  
pp. 2422-2428
Author(s):  
Ani Atul Arif ◽  
Tulus Maulana ◽  
Ekayanti Mulyawati Kaiin ◽  
Bambang Purwantara ◽  
Raden Iis Arifiantini ◽  
...  

Background and Aim: Indonesia has two National Artificial Insemination centers and 17 Regional Artificial Insemination Centers. The frozen semen production techniques differed between the centers, including the type of diluent and semen dilution technique. The aim of the research was to compare the quality of frozen Limousin bull semen diluted using different techniques. Materials and Methods: Semen was collected from three sexually mature Limousin bulls using an artificial vagina. Immediately after collection, the semen was evaluated macroscopically and microscopically. Semen that had >70% motile sperm and <20% sperm abnormality was divided into three tubes and diluted with skim milk-egg yolk (SMEY) using three different dilution techniques: One-step dilution (100% SMEY with 8% glycerol) at room temperature ([RT] 20°C until 25°C) two-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol after 1 h stored at 5°C); and three-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol added twice at 1 h and 1.5 h after being stored at 5°C). The diluted semen was loaded into 0.25 mL mini straws, equilibrated, and frozen using a freezing machine. Sperm motility, viability, membranes, DNA integrity, and concentrations of malondialdehyde (MDA) and aspartate aminotransferase (AST) enzymes were evaluated after thawing. Results: The results showed that there were no significant differences in sperm motility and DNA integrity between dilutions (p>0.05). However, sperm viability and membrane intactness of one-step dilutions were higher than those of three-step dilutions. The concentrations of MDA and AST enzymes of sperm in one-step dilutions were lower than those of three-step dilutions (p<0.05). Conclusion: It was concluded that the one-step-dilution technique was better than three-step dilution for cryopreservation of Limousin bull semen.


2017 ◽  
Vol 16 (1) ◽  
pp. 31-38 ◽  
Author(s):  
Herdis Suharman

The objective of this research was to examine the effect of sucrose in improving the quality of the plasma membrane intact and sperm motility of frozen semen of Garut ram. Semen was collected using artificial vagina weekly from six mature garut rams.   Immediately after initial evaluation, fresh semen was divided into four parts and diluted with Tr s extender without sucrose (T0), Tris extender + sucrose 0.2g/100 ml (T1), Tris extender + sucrose 0.4g/100 ml (T2) and Tris extender + sucrose 0.6g/100 ml (T3), respectively.  Results of this research showed that the percentage of sperm motility after thawing in T2 (49.00 ± 5.48%)  was significantly (P<0.05) higher than T0 (42.00 ± 2.74%) but was not significantly difference (P>0.05) than T1 (46.00 ± 4.18%)and T3 (48.00 ± 4.47%).   Evaluation of plasma membrane intact showed that T1 (62.33 ± 6.51%) was s gnificantly different (P <0.05) w h T0 (49.40 ± 2.19%) but was not significantly different (P> 0.05) than T2 (58.50 ± 4.97%) and T3 (56.40 ± 5.90%).  In conclusion, he addition of sucrose in semen extender  improved the quality of frozen semen of Garut ram.  Concentration of 0.2g / 100 ml is the op ma dose to improve the quality of the plasma membrane intact and motility of spermatozoa during the freezing process.


2020 ◽  
Vol 30 (3) ◽  
pp. 246-253
Author(s):  
Sri Wahjuningsih ◽  
Muhammad Nur Ihsan ◽  
Aulia Puspita Anugra Yekti ◽  
Muhammad Agus Tahar

This research aims to evaluate the effect of soybean extract (Glycine max (L.) Merr.) supplementation on tris aminomethane egg yolk extender to the post thawing quality of Simmental bull frozen semen. The fresh semen was collected from 3 Simmental bulls aged at 3 to 3.5 years for twice a week by using artificial vagina. The semen was then selected for sperm motility and abnormality, and the semen that had >70% motility and <15% abnormality was used for the research. The research was conducted in a completely randomized with 4 treatments and 30 replications. The research treatments include 0% (T0), 7.5% (T1), 10% (T2), and 12.5% (T3) soybean extract supplementation on tris aminomethane egg yolk extender. The observed variables include spermatozoa motility, viability, abnormality, and plasm membrane integrity. The collected data were analysed with ANOVA and followed with Duncan’s Multiple Range Test to determine significant differences. The results showed that soybean extract supplementation gave significant effect (P<0.05) to the post thawed quality of Simmental semen. The research concludes that 10% (v/v) soybean supplementation on tris aminomethane egg-yolk extender was effective to maintain sperm motility, viability, and plasma membrane integrity of post thawed Simmental semen.


2019 ◽  
Vol 6 (1) ◽  
pp. 78
Author(s):  
Muhammad Ade Salim ◽  
Muhammad Nur Ihsan ◽  
Nur Isnaini ◽  
Trinil Susilawati

ABSTRAKAir kelapa muda varietas viridisdapat dijadikan pengencer aletrnatif semen cair bagi program IB di daerah minim sarana semen beku. Tujuan penelitian ini untuk menguji pengaruh penggunaan air kelapa muda viridissebagai bahan pengencer terhadap kualitas semen cair kambing Boer setelah didinginkan. Dilaksanakanselama 3 bulan di Laboratorium Fakultas Peternakan UBUnit SumberSekar,Malang. Metodenya yaitu eksperimen. Semen dari  3 pejantan Boer umur 3-5 tahun, dikoleksi seminggu sekali dengan VB. Air kelapa mudaviridis umur 5-7 bulan serta tris aminomethane sebagai kontrol. Didesain menggunakan Rancangan Acak Kelompok (RAK) dengan 2 perlakuan yaitu P0 (tris aminomethane + 10% KT) dan  P1 (air kelapa muda viridis + 10% KT) masing-masing diulang 10 kali. Data dianalisis dengan analisis Ragam (Anova) dengan software Genstat 18. Variabelnya yaitu motilitas individu, viabilitas dan abnormalitas. Hasil penelitian yaitu motilitas individu pada P1bertahan sampai 4 hari (40,5± 24,3%), viabilitas terbaik sampai hari ke-5 (42±24,6%), abnormalitas terendah di hari ke-7(1,31± 0,6). Kesimpulannya, Pengencer air kelapa muda viridis dapat mempertahankan kualitas semen cair kambing Boer selama 4 hari untuk motilitas dan 5 hari untuk viabilitas.Kata Kunci:pengencer, air kelapa, varietas viridisABSTRACTYoung viridis coconut water could be used as an alternative to liquid semen diluent for artificial insemination program in the area with limited facility for frozen semen production. This study evaluated the use of young coconut water as a diluent on liquid semen quality of Boer goat after cold storage. This study was carried out for 3 months at Sumber Sekar Laboratory, Faculty of Animal Husbandry, University of Brawijaya, Malang. The semen was collected from 3 Boer bucks aged at 3 to 5 years old. The semen collection was done once a week with the aid of artificial vagina. The diluents used were young Viridis coconut (5 to 7 months old) and tris aminomethane. The method used was an experiment in a randomized block design with 2 treatments and 10 replicates. The treatments used were T0: tris aminomethane + 10% egg yolk (control) and T1:  young Viridis coconut water + 10% egg yolk. Data were analyzed by analysis of variance using Genstat 18 software. The variables measured were sperm individual motility, viability, and abnormality. The results showed that the sperm individual motility in T1 survived up to 4 days (40.5± 24.3%), the best viability at 5 days (42.0±24.6%),  while the lowest abnormality at 7 days (1.31±0.6). It could be concluded that: 1. Tris aminomethane diluent has higher quality with the storage length up to 9 days, 2. Young Viridis coconut water diluent could preserve liquid semen quality of Boer goat up to 4 days for sperm motility and 5 days for sperm viability.Keywords: diluents, coconut water, viridis variety


2020 ◽  
Vol 25 (2) ◽  
pp. 68
Author(s):  
Nurul Afzan Hilda Zakiya ◽  
A H Yanti ◽  
T R Setyawati

The use of liquid semen for artificial insemination program of Etawah crossbreed goat (PE) is an alternative to replace frozen semen which is constrained by limited and expensive facilities. Production of liquid semen is faster than frozen semen, but the viability of liquid semen which preserved with a standard extender such as tris egg yolk is very short. The purpose of this study was to determine the viability of PE goat semen in egg yolk tris substituted with energy sources such as glucose, galactose, and mannose and to determine the most efficient energy source for semen preservation. This research was conducted from August to September 2018 at the Artificial Insemination Center in Lembang, West Java. This study was designed in a randomized block design (RBD) consist of three experimental groups divided into five groups. Fresh semen of PE goats were preserved using extender which energy source has been modified. Results showed that using glucose in PE goat semen extender produced the best motility among other groups (64.29 ± 9.2%). The highest viability was found in extender with fructose substitution (86.76 ± 2.3%). The longest viability of liquid semen was found in the extender with glucose substitution. It lasted for six days.


2013 ◽  
Vol 25 (1) ◽  
pp. 185
Author(s):  
M. M. Seshoka ◽  
M. L. Mphaphathi ◽  
F. V. Ramukhithi ◽  
T. R. Netshirovha ◽  
C. Hlungwani ◽  
...  

There are bull shortages in South African poor rural areas. Artificial-insemination technology could play a significant role on breeding emerging farmer’s cattle. The objective of this study was to compare glycerol concentrations (0, 4, 8, or 12%) during freezing of Nguni bull semen to conduct AI in different villages. Semen was collected by electro ejaculator from 2 Nguni bulls of known and proven fertility. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories within 10 min after collections. Semen samples were pooled and evaluated by Sperm Class Analyser® and allocated randomly per treatment. Semen was then diluted (1 : 2 v:v) with egg-yolk citrate extender supplemented with either 0% (negative control), 4, 8, and 12% of glycerol concentration or AndroMed® (positive control). Semen samples were equilibrated for 4 h at 5°C. After equilibration period, samples were loaded into 0.5-mL straws and placed horizontally into the controlled rate (–5, –8, –10, –12, –15, –25, –35°C min–1) from 5°C until target temperature of –80°C is reached. The frozen semen straws were stored in a liquid nitrogen tank (–196°C) until thawing. Treatment means were separated using Fisher’s protected t-test least, and data are presented as mean ± SD. There was a significant differences (P < 0.05) between raw total sperm motility (83.3 ± 19.3) and frozen–thawed sperm with either 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), or 12% (61.5 ± 4.7) of glycerol and on AndroMed® (27.7 ± 17.8) group. Regardless of the glycerol concentrations used, the freezing-thawing process reduced (P < 0.05) the Nguni total sperm motility rate compared to uncryopreserved sperm (83.3 ± 19.3). In conclusion, egg-yolk citrate extender supplemented with 12% glycerol yielded a better (P < 0.05) total sperm motility rate (61.5 ± 4.7) as compared with the 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), and AndroMed® (27.7 ± 17.8) group. Further studies are required to test other levels of glycerol concentrations (>12%) on freezing Nguni semen and conducting AI.


2018 ◽  
Vol 41 (1) ◽  
pp. 21-31
Author(s):  
Mohamed Ali ◽  
Musa M. Musa ◽  
Sulaiman Alfadul ◽  
K. Al-Sobayel

Abstract This study is aimed at investigating effects of supplementation of stallion’ semen extender with various concentrations of Gum Arabic (GA) versus egg yolk (EY) on viscosity, sperm motility and survival during cooling and freezing. Physical sperm characteristics; i.e. curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN) and straightness index (STR) were evaluated. Based on the sperm velocity (velocity of the average path), individual spermatozoons were classified into two major groups; i.e., progressively motile (>45 μm/sec) and immotile (0-45 μm/sec) spermatozoa. Addition of 3, 9 or 15% of GA to HF-20 extender resulted in linear decreases in VCL, VSL and VAP and a decrease in the percentage of progressively motile spermatozoa. Dilution of horse semen samples with high viscosityextenders (i.e., high percentage of GA) decreased the VCL, VSL and VAP in fresh and chilled semen. Freezing semen in high viscosity-extenders reduced percentage of progressively motile spermatozoa compared with those of low viscosity-extenders. In refrigerated and frozen semen samples, the extender containing 15% GA had detrimental effects on the percentage of progressively motile sperm cells and velocity of progressive motile sperm. Moreover, cooling sperm in extenders containing 9 or 15% of GA for 72 hours resulted in complete motility cessation. In conclusion, GA could replace EY in stallion semen extenders at a level of 3% to maintain the physical and biological characteristics of cold and frozen semen.


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