scholarly journals Ability of the ISAS3Fun Method to Detect Sperm Acrosome Integrity and Its Potential to Discriminate between High and Low Field Fertility Bulls

Biology ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1135
Author(s):  
Jesús L. Yániz ◽  
Inmaculada Palacín ◽  
Miguel A. Silvestre ◽  
Carlos Olegario Hidalgo ◽  
Carolina Tamargo ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Low Fertility ◽  
Head Width ◽  
Low Field ◽  
Frozen Semen ◽  
Acrosome Integrity ◽  
Sperm Acrosome ◽  
Acrosomal Integrity

The objective of the present study was to investigate whether fertility differences in bulls are reflected in variations of sperm quality when analysing only one ejaculate per male. Two experiments were performed. In the first experiment, frozen semen samples from 20 adult bulls were tested; 10 bulls had high field fertility and 10 bulls had low field fertility. Analyses of sperm motility, membrane integrity, and membrane–acrosome integrity with the ISAS3Fun method were performed. Sperm morphometry of the fluorescence sperm subpopulations obtained with the ISAS3Fun method was also analysed. Significant differences between high- and low-fertility groups were only found with the ISAS3Fun technique, specifically in sperm acrosome integrity, the proportion of spermatozoa with an intact acrosome and damaged membrane, and in sperm head width of spermatozoa with intact structures. Discriminant analyses allowed us to correctly classify 90% of sperm samples in their fertility group using sperm quality parameters. Given that only the results obtained with the ISAS3Fun technique were related to bull fertility, we performed a second experiment aimed to validate the efficacy of this technique to detect the acrosomal integrity of bull spermatozoa, comparing them with the conventional FITC-PNA/propidium iodide (PNA/PI) combination under capacitating conditions. The results indicated that the ISAS3Fun combination provided an accurate assessment of both viability and acrosomal integrity for ejaculated spermatozoa, while the PNA/PI combination underestimated the extension of acrosomal damage due to false negatives. It was concluded that the simultaneous assessment of sperm plasma membranes and acrosome integrity with the ISAS3Fun method is precise and seems to have a greater potential to discriminate between high- and low-fertility bulls than more conventional in vitro sperm quality tests.

Cryoprotective Effect of Glycerol Concentrations on Indian Red Jungle Fowl (Gallus Gallus Murghi) Spermatozoa

2018 ◽  
Vol 11 (2) ◽  
pp. 80-88 ◽  
Author(s):  
Bushra Allah Rakha ◽  
Muhammad Sajjad Ansari ◽  
Shamim Akhter ◽  
Elisabeth Blesbois
Keyword(s):  
Plasma Membrane ◽  
Semen Quality ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Gallus Gallus ◽  
Recovery Rates ◽  
Plasma Membrane Integrity ◽  
Red Jungle Fowl ◽  
Frozen Semen ◽  
Acrosome Integrity

Semen cryopreservation protocols for wild avian species need to be optimised in order to achieve optimum post-thaw sperm quality and fertility. The present study was designed to evaluate the cryoprotective effect of different glycerol concentrations (11%, 15% and 20%) on post-thaw quality, recovery rates, absolute livability index and fertility of Indian Red Jungle Fowl (Gallus gallus murghi) semen. Semen was collected from eight mature cocks and cryopreserved for storage at −196 °C. Frozen semen was thawed at 37 °C for 30 s and assessed for motility, plasma membrane integrity, viability and acrosome integrity at 0, 2 and 4 h incubation at 37 °C. Percentages of motility, plasma membrane integrity, viability and acrosome integrity were recorded higher (P<0.05) post-thaw at 0, 2 and 4 h at 37 °C with 20% glycerol compared to 15% and 11% glycerol. Likewise, recovery rates (%) of aforementioned parameters after cryopreservation and absolute livability index were observed highest (P<0.05) with 20% glycerol. By comparing values of R2 after multivariate regression analysis, least negative effects of hours of incubation were observed on semen quality in extenders with 20% glycerol followed by 15% and 11% glycerol. The fertility outcomes (number of fertile eggs, fertility [%], number of hatched chicks, percent hatch and hatchability of fertilised eggs) were recorded higher (P<0.05) with 20% glycerol followed by 15% and 11% glycerol. It is concluded that the concentration of 20% glycerol gives the best cryoprotection for quality and fertility of Indian Red Jungle Fowl semen.

scholarly journals A comparative study of two methods to determine acrosome integrity of frozen-thawed boar sperm

2021 ◽  
Vol 52 (6) ◽  
Author(s):  
Janyaporn Rungruangsak ◽  
Junpen Suwimonteerabutr ◽  
Kakanang Buranaamnuay ◽  
Arun Chankrachang ◽  
Padet Tummaruk
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Blue Staining ◽  
Computer Assisted ◽  
Sperm Viability ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Boar Sperm ◽  
Acrosome Integrity ◽  
Sperm Acrosome

Coomassie blue staining has been reported as an effective and inexpensive method for evaluating the acrosome integrity of spermatozoa, though to date its use to evaluate cryopreserved boar sperm has not been reported. Moreover, there is no information concerning the agreement between Coomassie blue staining and fluorescein isothiocyanate conjugated peanut agglutinin and ethidium homodimer (FITC-PNA/EthD-1) methods for assessing sperm acrosome integrity for any species. The current study was performed to determine the efficacy and agreement between Coomassie blue and FITC-PNA/EthD-1 staining methods for evaluating the acrosome integrity of frozen-thawed boar sperm. A total of 25 semen samples were cryopreserved using lactose-egg yolk-based extender and loaded into 0.5 PVC-French straws. Sperm motility and motion characteristics were determined using a computer-assisted sperm analysis system. Sperm viability and plasma membrane integrity were evaluated using the SYBR-14/EthD-1 and hypo-osmotic swelling test, respectively. Acrosome integrity of frozen-thawed boar sperm was evaluated using both FITC-PNA/EthD-1 and Coomassie blue staining to assess the association between sperm acrosome integrity and agreement between these two methods. The average percent acrosome integrity of frozen-thawed boar sperm as determined by FITC-PNA/ EthD-1 and Coomassie blue staining was 48.8 ± 12.6% and 52.6 ± 13.6%, respectively (P&gt;0.05). Interestingly, Coomassie blue staining found a correlation between sperm viability and acrosome integrity (r=0.609, P=0.002), while FITC-PNA/EthD-1 staining did not (P&gt;0.05). However, the acrosome integrity of frozen-thawed boar sperm evaluated by FITC-PNA/ EthD-1 and Coomassie blue staining was significantly correlated (r=0.448, P=0.025, n=25). The Bland-Altman plot determined that this agreement was acceptable. In conclusion, the acrosome integrity of the frozen-thawed boar sperm assessed via Coomassie blue staining was significantly correlated with that obtained via the FITC-PNA/EthD-1 staining method, and the two methods showed good agreement. Moreover, the significant association between the acrosome integrity of frozen-thawed boar sperm determined by Coomassie blue staining with other sperm quality parameters indicates that this is an effective method for assessing the acrosome integrity of frozen-thawed sperm in pigs.

scholarly journals COMBINED EFFECT OF STREPTOMYCIN AND SWEET ORANGE ESSENTIAL OIL TO MEMBRANE AND ACROSOME INTEGRITY BOER GOAT FROZEN SEMEN

2019 ◽  
Vol 3 (2) ◽  
pp. 89
Author(s):  
Sukma Aditya Sitepu ◽  
Julia Marisa
Keyword(s):  
Essential Oil ◽  
Sweet Orange ◽  
Membrane Integrity ◽  
Boer Goat ◽  
Randomized Design ◽  
Frozen Semen ◽  
Acrosome Integrity ◽  
Completely Randomized Design ◽  
Orange Essential Oil

One of factors that cause a bad quality of Boer Goat frozen semen is the growth of bacterial. This can be overcome by adding antibiotics such as streptomycin. To further suppress the growth of bacteria can be added other ingredients that contain antibacterials such as sweet orange essential oil. The purpose of this research is to know the percentage value of Membrane Integrity and Acrosome Integrity on Boer Goat frozen semen with addition sweet orange essential oil and streptomycin. The method used was experimental using Completely Randomized Design with 5 treatments and 5 replications. The treatment in this research is addition 0%, 0,25%; 0.5%; 0.75% and 1% sweet orange essential oil on tris yolk and streptomycin extender. The results showed the best treatment addition combination streptomycin and sweet orange essential oil to percentage Membrane Integrity and Acrosome Integrity is increase 1% sweet orange essential oil.Keywords: Boer Goat, essential oil, frozen semen, streptomycin, sweet orange.

8 THE USE OF ANNEXIN V MAGNETIC-ACTIVATED CELL SORTING TO SEPARATE APOPTOTIC SPERM FROM THE EJACULATE OF STALLIONS

2011 ◽  
Vol 23 (1) ◽  
pp. 110
Author(s):  
M. A. Coutinho da Silva ◽  
C. R. F. Pinto ◽  
J. M. Young ◽  
K. Cole
Keyword(s):  
Cell Sorting ◽  
Semen Quality ◽  
Sperm Quality ◽  
Cleveland Clinic ◽  
Membrane Integrity ◽  
Annexin V ◽  
Sperm Parameters ◽  
Control Group ◽  
Frozen Semen ◽  
Magnetic Activated Cell Sorting

Magnetic-activated cell sorting (MACS) has been used successfully in humans to remove apoptotic sperm from the ejaculate. Annexin V-conjugated microbeads recognise sperm with externalized phosphatidylserine, which is considered one of the features of apoptosis, and the labelled sperm is separated by MACS. The goals of the study were to determine if MACS can be used to separate apoptotic sperm from the ejaculate of stallions; and to determine if removal of apoptotic sperm improves the quality of stallion sperm. Our hypothesis was that MACS would improve semen quality by removing apoptotic sperm, resulting in samples with higher motility and viability. Two ejaculates from three different stallions of good fertility were used. Sperm were diluted with Tyrode’s albumin lactate pyruvate (TALP) and incubated with annexin V-conjugated microbeads for 15 min at 37°C. Control samples were incubated in the absence of annexin V microbeads. The suspension was then loaded into the separation column containing iron globes, which were fitted in a magnet (MiniMACS; Miltenyi Biotec Inc., Auburn, CA, USA). The effluent sample containing annexin-negative sperm was collected and then, the column was removed from the magnetic field and rinsed with TALP to collect the annexin-positive cells. Sperm viability, motility, morphology and caspase activation were determined in all three samples: control, annexin-negative, and annexin-positive. Data were evaluated by ANOVA and individual comparisons were performed by Tukey’s hsd test. Significance was set at P < 0.05 and data is presented as means ± SEM (Table 1). The main effect of stallion was significant only for sperm motility parameters. Sperm recovery rate following MACS was 46 ± 3%. In conclusion, the use of MACS was effective in removing apoptotic sperm from the ejaculate. The annexin-positive population displayed a higher proportion of sperm with activated caspases and lower membrane integrity and motility. However, removal of apoptotic sperm from the ejaculate did not improve sperm parameters in the annexin-negative group compared to control group. In addition, sperm morphology was not affected by MACS. Further studies are necessary to determine if MACS could be used successfully to improve sperm quality from subfertile stallions and frozen semen. Table 1.Sperm parameters following annexin V MACS (mean ± SEM) The authors are thankful to Mark Williams at Miltenyi Biotec Inc. for providing supplies; and Dr Ashok Agarwal at The Center for Reproductive Medicine, Cleveland Clinic, for scientific input.

scholarly journals Evaluation of motility, membrane status and DNA integrity of frozen–thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation

Reproduction ◽  
2012 ◽  
Vol 143 (6) ◽  
pp. 799-813 ◽  
Author(s):  
G A Montano ◽  
D C Kraemer ◽  
C C Love ◽  
T R Robeck ◽  
J K O'Brien
Keyword(s):  
Bottlenose Dolphin ◽  
Sperm Quality ◽  
Critical Evaluation ◽  
Membrane Integrity ◽  
Ex Situ ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Freezing Method ◽  
Frozen Semen ◽  
Motility Parameters

Artificial insemination (AI) with sex-sorted frozen–thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen–thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen–thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze–thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

scholarly journals Effects of Taurine on Sperm Quality during Room Temperature Storage in Hu Sheep

Animals ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 2725
Author(s):  
Liuming Zhang ◽  
Yanhu Wang ◽  
Tariq Sohail ◽  
Yan Kang ◽  
Haoyuan Niu ◽  
...  
Keyword(s):  
Room Temperature ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Quality Parameters ◽  
Control Group ◽  
Negative Effects ◽  
Positive Effects ◽  
Antioxidant Parameters ◽  
Acrosome Integrity ◽  
Hu Sheep

The present study aimed to investigate whether the presence of Tau protected Hu sheep sperm from ROS stress during storage at room temperature. The semen was diluted with extender (Tris-based) at room temperature, supplemented with different concentrations of Tau (0, 10, 20, 40, 80, or 100 mM), and stored at 15 °C. Sperm quality parameters (sperm progressive motility, kinetic parameters, plasma membrane integrity rate, acrosome integrity rate, and MMP) and antioxidant parameters (ROS, MDA, SOD, CAT, and T-AOC) were evaluated during the preservation of semen. The addition of Tau, especially at a concentration of 20 mM, exerted positive effects on sperm quality parameters and antioxidant parameters compared to the sperm without Tau treatment (control group). The addition of Tau, especially at a concentration of 100 mM, exerted negative effects on sperm quality parameters and antioxidant parameters compared to the control group. Interestingly, the results indicated that the sperm acrosome integrity rate did not change during storage time. In conclusion, the addition of Tau to sperm preserved at room temperature can enhance the antioxidant ability of sperm, reduce the LPO on the 5th day, and improve the quality of semen preserved at room temperature. These results implied that Tau had potential to enhance Hu sheep sperm reproductive performance.

scholarly journals Cryopreservation of semen from goats fed a diet supplemented with flaxseed

2019 ◽  
Vol 20 ◽  
Author(s):  
Rosileia Silva SOUZA ◽  
Larissa Pires BARBOSA ◽  
Cristiane Silva AGUIAR ◽  
Renan Luiz Albuquerque VIEIRA ◽  
Márcio Oliveira RIBEIRO ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Analysis Of Variance ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Maximum Level ◽  
Integrity Test ◽  
Plasma Membrane Integrity ◽  
Integrity Tests ◽  
Acrosomal Integrity

ABSTRACT The objective of this study was to evaluate the effects of the inclusion of flaxseed in the diet of male goats on the resistance of semen to the cryopreservation process. Sixteen males were distributed in four groups and fed a diet supplemented with 0, 4, 8, and 12% of flaxseed for a period of 60 days. Only the ejaculates that presented motility and vigor above 70% and 3, respectively, were sent through the cryopreservation process. After thawing, the semen was evaluated through thermo resistance, hypoosmotic, and acrossomal integrity tests. The data were submitted to analysis of variance and regression at 5% of significance. There was positive quadratic behavior for motility after 60, 120, and 180 min in the thermoresistance test (TTR), and positive quadratic behavior for sperm vigor after thawing after 120 and 180 min in the TTR (P < 0.05). However, negative quadratic behavior was obtained for plasma membrane integrity according to the hypoosmotic test (P < 0.05) and there was a difference in the acrosomal integrity test, presenting an optimum maximum level of 3.25% of flaxseed for acrosomal integrity of 65.83% (P < 0.05). The results obtained demonstrated that the addition of as much as 12% of flaxseed to the diet of goat breeders improved post-thawing sperm quality.

68 DIFFERENCE IN THAWING CURVE OF STALLION FROZEN SEMEN DILUTED IN 2 EXTENDERS

2015 ◽  
Vol 27 (1) ◽  
pp. 127
Author(s):  
C. P. Freitas-Dell'aqua ◽  
C. Ramires Neto ◽  
Y. F. R. Sancler-Silva ◽  
P. M. Papa ◽  
J. A. Dell'aqua ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Freezing Process ◽  
Freezing And Thawing ◽  
Becton Dickinson ◽  
Plasma Membrane Integrity ◽  
Frozen Semen ◽  
Group 2 ◽  
Artificial Vagina

Commercial freeze extenders have different composition and ratio of cryoprotectors; freezing and thawing protocols are different for each extender. The aim of this experiment was to observe the effect of thawing curve in stallion frozen semen with 2 commercial extenders. Two ejaculates from each of 9 stallions of different breeds (Quarter Horses and Mangalarga Marchador) were used. Semen was collected using an artificial vagina, and the ejaculate was divided into 2 groups following the manufacture's protocol: group 1 (INRA), in which the semen was diluted 1 : 1 with the extender INRA 96TM (IMV, Paillette Crista, France) and group 2 (BC), in which the semen was diluted (1 : 1) with the extender Botu-SemenTM (Botupharma, Brazil). The samples of the 2 groups were centrifuged at 600 × g for 10 min, the supernatant was discarded, and the pellet was resuspended with INRA FreezeTM (group INRA, IMV) and with BotucrioTM (group BC, Botupharma) at the concentration of sperm 100 × 106 sperm mL–1. After this, the semen was packaged in 0.5-mL straws. For each group the freezing process was carried out according to the manufacturer's instructions. The straws were thawed in a water bath with 3 different thawing curves: 37°C for 30 s (37/30), 46°C for 20 s (46/20), and 75°C for 7 s (75/7) before analysis. The aim of these rates is to keep the semen in 37°C post-thaw. The sperm kinetic analysis was performed by computerized method (CASA, HTM-IVOS, IMV, USA) and the analysis of plasma membrane integrity by flow cytometer (BD LSR Fortessa, Becton Dickinson, Mountain View, CA, USA). Data of sperm kinetic and of plasma membrane integrity were compared among the 3 thawing curves for one extender using analysis of variance. Differences were considered significant at a probability level of 5%. No differences were observed in total motility (%, BC 37/30 = 72.8 ± 14.4; BC 46/20 = 70.0 ± 14.2; BC 75/7 = 70.3 ± 12.0 v. INRA 37/30 = 57.2 ± 19.1; INRA 46/20 = 50.0 ± 21.9; BC 75/7 = 58.8 ± 20.8), progressive motility (%, BC 37/30 = 36.9 ± 8.2; BC 46/20 = 34.4 ± 10.5; BC 75/7 = 33.6 ± 7.8 v. INRA 37/30 = 25.3 ± 12.7; INRA 46/20 = 21.9 ± 13.9; BC 75/7 = 28.9 ± 14.8), rapid sperm (%, BC 37/30 = 59.7 ± 16.4; BC 46/20 = 56.8 ± 17.1; BC 75/7 = 58.1 ± 14.9 v. INRA 37/30 = 38.3 ± 20.9; INRA 46/20 = 35.3 ± 22.9; BC 75/7 = 44.4 ± 23.8), and plasma membrane integrity (%, BC 37/30 = 49.1 ± 14.8; BC 46/20 = 43.1 ± 13.1; BC 75/7 = 46.7 ± 11.8 v. INRA 37/30 = 32.2 ± 10.7; INRA 46/20 = 29.6 ± 10.1; BC 75/7 = 37.4 ± 9.1) among the 3 thawing curves for INRA and BC groups. In this study, we can conclude there is no influence of the 3 tested thawing curves in sperm quality for stallion frozen semen with INRA Freeze and Botucrio extenders.

Semen quality and fertility of liquid stored and frozen-thawed semen in crossbred pigs of North-Eastern India

2018 ◽  
Author(s):  
G Kadirvel ◽  
M K Kalita ◽  
Raju Kr Dewry ◽  
Ashok Kumar ◽  
Nripendra Mahanta ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Semen Quality ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Eastern Region ◽  
Plasma Membrane Integrity ◽  
Frozen Semen ◽  
North Eastern ◽  
Farrowing Rate

Study was conducted to compare the semen quality and fertility of liquid stored semen for three days and frozen-thawed semen in the north-eastern region of India. For liquid semen, the semen ejaculates were extended in Beltsville Thawing Solution (BTS) extender and preserved at 17°C for three days. For cryopreservation, semen was diluted Lactose-egg yolk-glycerol extender and frozen in straw using programmable freezer with freezing rate of 40°C/min from -6 to -140°C. The preserved evaluated for sperm motility, viability, plasma membrane integrity and fertility. The results revealed that the liquid stored semen has maintained the sperm motility and viability up to day 3 without significant reduction. Similarly the plasma membrane integrity did not differ significantly up to day 2, but it was significantly (P less than 0.05) reduced on days 3 in liquid stored semen. After freezing and thawing, the mean sperm motility, viability and plasma membrane integrity were 58.25 ± 2.96%, 64.75 ± 2.47% and 47.06 ± 2.02%, respectively. These parameters were significantly (PP less than 0.01) lower as compared to the liquid stored semen from day 0 to day 3. After insemination with liquid semen, the farrowing rate was 77.7%, 80.76%, 73.07% and 69.8%, respectively from day 0, day1, day 2 and day 3. The pregnancy rate, farrowing rate and litter size did not differ significantly among different days of liquid storage. These parameters were significantly (PP less than 0.01) lower in frozen semen as compare to that of liquid stored semen. The study concluded that the liquid semen stored up to three days is more efficient than frozen-thawed semen in terms of preserving sperm quality and fertility.

scholarly journals Efek Lama Penyimpanan Semen Beku Sapi Bali pada Pos Inseminasi Buatan terhadap Membran Plasma, Tudung Akrosom Utuh, dan DNA Spermatozoa

2020 ◽  
Vol 3 (2) ◽  
pp. 58-66
Author(s):  
Fikri Ardhani ◽  
Hayatul Mufidah ◽  
Rahmah Samsuriati ◽  
Hilman Pratama Putra
Keyword(s):  
Dna Damage ◽  
Plasma Membrane ◽  
Artificial Insemination ◽  
Storage Time ◽  
Membrane Integrity ◽  
Frozen Storage ◽  
Plasma Membrane Integrity ◽  
East Kalimantan ◽  
Frozen Semen ◽  
Acrosome Integrity

The purpose of this study was to determine the effect of frozen storage time for Bali Bull in artificial insemination station in Samarinda City, East Kalimantan on the quality of motility, viability, velocity, abnormality, plasma membrane integrity (MIn), acrosome integrity (AIn), and DNA damage of spermatozoa. The study design used a completely randomized design (CRD) with 5 treatments (storage time) and 5 replications. Frozen semen of Bali Bull used in 2009 (10 years of storage), 2011 (7 years of storage), 2013 (5 years of storage), 2015 (3 years of storage), and 2017 (1 year of storage). The storage time of frozen semen stored for one to ten years in liquid nitrogen at the artificial insemination station in Kota Samarinda, East Kalimantan was still suitable for use in artificial insemination based on motility quality (44.99±2.40%), viability (55.33±2,60%), velocity (0.050±0.002 mm/sec), abnormality (12.87±1.09%), plasma membrane integrity (58.83 ± 1.86%), acrosome integrity (75.48 ± 1 , 61%), and DNA damage of spermatozoa (1.60 ± 0.21%).

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