86 EFFECTS OF THREE CRYOPRESERVATION SYSTEMS ON LONGEVITY OF STALLION SPERM AFTER THAWING

2008 ◽  
Vol 20 (1) ◽  
pp. 123 ◽  
Author(s):  
D. F. Pasquini ◽  
H. N. Ferreira ◽  
F. O. Papa ◽  
J. A. Dell Aqua Jr ◽  
M. A. Alvarenga
Keyword(s):  
Large Scale ◽  
Egg Yolk ◽  
Final Concentration ◽  
Sperm Cells ◽  
Progressive Motility ◽  
Hamilton System ◽  
Nitrogen Vapor ◽  
Frozen Semen ◽  
Horse Industry ◽  
Artificial Vagina

Use of frozen semen by the horse industry is popular. Several extenders have been used to freeze stallion semen; the most used are EDTA-lactose egg yolk and INRA 82. A new extender named Botu-Crio� (BioTech, Botucatu, Sao Paulo, Brazil) has been used on a large scale in Brazil. No comparisons of these extenders have been published. The present experiment was designed to compare the ability of these three extenders to preserve sperm longevity after incubation. One ejaculate from each of 13 stallions was used. After collection using an artificial vagina, semen was filtered and diluted 1:1 with a milk-base extender (Botu-Semen�), split into 3 parts, and then centrifuged (600g/10 min). After removal of the supernatants, each semen pellet was resuspended with an aliquot of one of the extenders (INRA 82, EDTA-Lactose, or Botu-Crio) to a final concentration of 100 � 106 sperm cells mL–1. Semen was packaged in 0.5-mL French straws, and cooled for stabilization for 20 min at 5�C for Botu-Crio (BC), for 2 h at 5�C for INRA 82 (IR), and without stabilization for EDTA-Lactose (EL) extender. After stabilization, the straws were placed in nitrogen vapor for 15 min and then plunged into liquid nitrogen. Before motility evaluation by CASA using the Hamilton System Analyzer (Hamilton Thorne Biosciences, Beverly, MA, USA), straws were thawed (46�C/20 s) and then incubated at 37�C in a dry block. The patterns of total motility (TM) and progressive motility (PM) were evaluated immediately after thawing (T0), and at 10 (T10), 30 (T30), and 60 (T60) min after incubation at 37�C. Statistical analysis was performed using ANOVA and Tukey's test. Total motility, respectively, for IR, EL, and BC was TM0: 10%, 35%, and 57%; TM10: 25%, 37%, and 68%; TM30: 20%, 21%, and 56%; and TM60: 13%, 10%, and 41%. The respective progressive motility was PM0: 3%, 16%, and 28%; PM10: 11%, 16%, and 32%; PM30: 10%, 8%, and 26%; and PM60: 5%, 2%, and 16%. Motilities were always superior (P < 0.05) at all incubation times with the utilization of Botu-Crio extender. A fertility trial comparing the three extenders is in progress.

Kualitas Semen Cair Kambing Boer Berbahan Pengencer Air Kelapa Muda Varietas Viridis Setelah Simpan Dingin

2019 ◽  
Vol 6 (1) ◽  
pp. 78
Author(s):  
Muhammad Ade Salim ◽  
Muhammad Nur Ihsan ◽  
Nur Isnaini ◽  
Trinil Susilawati
Keyword(s):  
Semen Quality ◽  
Block Design ◽  
Animal Husbandry ◽  
Egg Yolk ◽  
Coconut Water ◽  
Boer Goat ◽  
Randomized Block Design ◽  
Frozen Semen ◽  
Artificial Vagina

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

scholarly journals Comparison of Extenders With the Addition of Egg Yolk for Cooling Alpaca Sperm Obtained From Deferent Ducts

2020 ◽  
Vol 7 ◽  
Author(s):  
Mariana Lucía Bertuzzi ◽  
Edita Yola Torres ◽  
Teodosio Huanca ◽  
Deborah Neild ◽  
María Ignacia Carretero
Keyword(s):  
Sperm Motility ◽  
Chromatin Condensation ◽  
Egg Yolk ◽  
Final Concentration ◽  
Sperm Parameters ◽  
Membrane Function ◽  
Progressive Motility ◽  
Evaluation Time ◽  
Viable Sperm ◽  
Acrosome Integrity

The use of non-commercial and commercial extenders for cooling alpaca sperm has already been reported, the latter showing certain advantages over the first. The Andromed® (AM) extender was created for use in ruminants and has also been tested in ejaculated and epididymal alpaca sperm. According to the manufacturer, this extender does not need the addition of egg yolk (EY); however, it is known that the addition of EY to some extenders improves the preservation of cooled sperm. The objective of this study therefore was to compare a non-commercial extender (Tris) with the addition of EY vs. the commercial extender AM with and without the addition of EY, for cooling alpaca sperm obtained from diverted deferent ducts. Fifteen pools of deferent duct sperm were formed using samples from two or three different males for each. Each sperm pool was evaluated and then divided into three aliquots that were diluted to a final concentration of 30 × 106 sperm ml-1 (0 h) with either: (1) Tris with 20% EY (T-EY), (2) AM, or (3) AM with 20% EY (AM-EY). Samples were cooled to 5°C and the following sperm parameters were evaluated after 24 and 48 h of storage: motility, viability, membrane function, acrosome integrity, morphology, and chromatin condensation. Motility was also evaluated after 72 h of storage. The samples that best preserved progressive and total sperm motility at the 24 and 48 h evaluation periods were the ones diluted with AM-EY, observing that with this extender these motility patterns decreased significantly after 72 h of storage compared to time 0 h (p &lt; 0.05). A significant decrease (p &lt; 0.05) in total and progressive motility was observed at 48 h for the T-EY and AM extender compared to 0 h. AM was the only extender in which the percentages of viable sperm decreased significantly (p &lt; 0.05) after 48 h of conservation. For the rest of sperm parameters evaluated, no significant differences were observed between any of the extenders at any evaluation time. The Andromed® extender with the addition of 20% EY could be an alternative option for cooling alpaca sperm obtained from deferent ducts.

37 USE OF THERMO-RESISTANCE TEST (TRT) TO EVALUATE EQUINE COOLED SEMEN STORED AT 5°C

2010 ◽  
Vol 22 (1) ◽  
pp. 176
Author(s):  
G. Pugliesi ◽  
J. M. Silva Filho ◽  
C. A. A. Torres ◽  
D. M. Rates ◽  
P. G. Ker ◽  
...  
Keyword(s):  
Reproductive Potential ◽  
Egg Yolk ◽  
Resistance Test ◽  
Progressive Motility ◽  
Effective Use ◽  
Short Time ◽  
Bovine Species ◽  
Artificial Vagina ◽  
Sperm Movement ◽  
Made In

Evaluation of seminal characteristics is an important step to predict the reproductive potential of equine semen in natural or AI programs. Thermo-resistance test (TRT) has wide acceptance among tests in the bovine species, mainly because of its high correlation with fertility field. However, the TRT for stallion semen has not been widely studied. The objective of this study was to evaluate the effective use of TRT for equine cooled semen diluted with different extenders. Three stallions of Mangalarga Marchador breed aged between 8 and 14 years were used. Five semen samples per stallion were obtained, collected 3 times a week, with the aid of an artificial vagina (adapted Hannover model) using mares in natural estrus as dummy. The semen was diluted in 2 extenders: skim dried milk-glucose (E1) and glycine-egg yolk (E2), packaged in samples containing 12 mL of diluted semen to reach a final concentration of 30 million viable spermatozoa mL-1 and then stored at 5°C in an Equitainer® for 24 h. The cooled semen was warmed at 37°C in a water-bath. Spermatozoal progressive motility and vigor of semen were evaluated at 0 (TRT0), 30 (TRT30), 60 (TRT60), and 90 (TRT90) min after the start of warming. Treatment differences for sperm parameters were determined using ANOVA. The average values of sperm motility during TRT0, TRT30, TRT60, and TRT90 in E1 and E2 were, respectively, (E1) 37.0, 31.3, 23.7, and 19.7 and (E2) 30.3, 23.7, 18.3, and 15.7. The average values of vigor during TRT0, TRT30, TRT60, and TRT90 in E1 and E2 were, respectively, (E1) 2.4, 2.03, 1.53, and 1.43 and (E2) 1.97, 1.53, 1.33, and 1.17. During the test, the progressive motility obtained with E1 was higher (P < 0.05) than that with E2, and is within the patterns of motility considered acceptable only at 0 and 30 min of TRT. The E2 extender gave the worst result of the test, which was below the standards recommended for cooled semen. The seminal characteristics decreased in a very short time of TRT (30 min). This test is for use in insemination program. Thus, this demonstrates that changes in interpretation of the test need to be made in equine semen evaluation. A marked reduction of progressive motility at 30 min of test can be caused by loss of intracellular components or lesions in sperm movement structures. Possibly, availability of cyclic nucleotides involved in oxidative phosphorylation and motility are insufficient, although the mitochondria have the ability to produce energy. The TTR time of 90 min is long for equine cooled semen, and a duration for TTR of 30 min may be more appropriate in this species. Supported by grants from CNPq and CAPES.

scholarly journals An endeavor to improve longevity of cryopreserved equine sperm

2017 ◽  
Vol 57 (3) ◽  
pp. 195 ◽  
Author(s):  
TAA KCHALIFA ◽  
M. M. WAHEED ◽  
A. G. LYMBEROPOULOS (Α.Γ. ΛΥΜΠΕΡΟΠΟΥΛΟΣ)
Keyword(s):  
Oxidative Stress ◽  
Bovine Serum Albumin ◽  
Zinc Chloride ◽  
Bovine Serum ◽  
Free Fraction ◽  
Egg Yolk ◽  
Sperm Cells ◽  
Progressive Motility ◽  
In Vitro Effects

Exposure of sperm cells to the oxidative stress pending hypothermic storage of semen has been suggested to be responsible, in part, for the decline of their motility and fertility. This study was conducted to evaluate the in-vitro effects of antioxidants (AOs) and / or caffeine on longevity of cryopreserved stallion spermatozoa. Aliquots from the gel-free fraction of semen ejaculates (n=12), collected from 5 Arabian stallions (9-18 years old) of unknown sperm freezability, were mixed 1:1 with a Tris-egg yolk extender (TEYE), centrifuged at 500 χ g for 5 min and sperm cells were frozen in the form of 0.25-ml concentrated pellets after 2-step addition of TEYE supplemented with or without AOs (0.50 mg /ml Na pyruvate, 1 mg / ml Na thiosulfate, 5 mg / ml bovine serum albumin, 0.15 mg / ml zinc chloride and 0.50 mg / ml ferulic acid). The final pre-freeze concentrations of glycerol and sperm cells were 5% and 562-924 χ IO6 / ml, respectively. Frozen pellets from non-AOs and AOs-treated sperm were thawed in a Tris-citric acidglucose solution (40°C) containing 0, 0.49, 0.97 or 1.94 mg / ml caffeine and incubated (140-230 χ IO6 sperm / ml) at 30°C for 3 h. Sperm progressive motility (%) was assessed after centrifugation, before freezing and after 0, 1, 2 and 3 h of thawing. The results revealed significant (P<0.05) effects of sperm treatments only on post-thaw motility. Neither AOs alone nor caffeine alone could significantly ameliorate the maintenance of sperm motility. AOs plus 0.97 or 1.94 mg / ml caffeine were the superior supplements in improving the longevity of stallion spermatozoa.

scholarly journals Ram semen preserved at 0°C with soybean lecithin Tris-based extender substituted for egg yolk

2021 ◽  
Vol 34 (2) ◽  
pp. 192-197
Author(s):  
Jian-qing Zhao ◽  
Guo-liang Xiao ◽  
Wen-liang Zhu ◽  
Di Fang ◽  
Na Li ◽  
...  
Keyword(s):  
Soybean Lecithin ◽  
Egg Yolk ◽  
Control Group ◽  
Progressive Motility ◽  
Significant Difference ◽  
Acrosome Integrity ◽  
Normal Offspring ◽  
And Control ◽  
Ram Sperm ◽  
Artificial Vagina

Objective: The present study evaluated the preservation of ram semen at 0°C using soybean lecithin with a Tris-fructose extender.Methods: Semen was collected by artificial vagina ejaculation from six rams with proven fertility. High quality ejaculates were diluted by soybean lecithin (0.25%, 0.5%, 0.75%, 1.0%, 1.25%) using Tris-fructose extender and control (Tris-fructose egg yolk extender), respectively. The ejaculates were diluted to a concentration of 5×10<sup>8</sup> sperm/mL, followed by cooling to 0°C in 90 min and maintaining the temperature for 12 days. The diluted semen samples were examined and recorded for sperm progressive motility, acrosome integrity at 0, 24, 72, 144, 216, 288 h, respectively. Two hundred and twenty-three ewes were inseminated for 216 h with optimal soybean lecithin concentrated semen or control via trans-cervical insemination.Results: The results showed that there were no differences in sperm progressive motility at 0, 24, 72, and 144 h (p>0.05). After 216 h, the sperm progressive motility in the control group and 0.5% concentration groups was significantly higher when compared to 0.25% concentration (p<0.05). The 0.5% concentration group demonstrated the highest survival rate and had no difference with the control group (p>0.05). At 216 h, the sperm progressive motility of all groups was still above 50%. The acrosome integrity of all groups was decreased with prolongation of storage time, but there was no difference at each time point (p>0.05). There was no significant difference in the lambing rate and pregnancy rate between the 0.5% concentration group and the control group (p>0.05).Conclusion: These results suggest that ram sperm is capable of fertilization after preservation at 0°C with 0.5% of soybean lecithin in Tris-based extender substituted for egg yolk and produce normal offspring after insemination.

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

2019 ◽  
Vol 97 (Supplement_1) ◽  
pp. 78-78
Author(s):  
Stephan Wildeus ◽  
Dahlia O’Brien
Keyword(s):  
Storage Temperature ◽  
Skim Milk ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Cooling Rates ◽  
Progressive Motility ◽  
Liquid Storage ◽  
Motion Characteristics ◽  
Sperm Motion ◽  
Artificial Vagina

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 < 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 < 0.001) relationship (progressive motility: 51.9 to 13.1%; rapid motility: 36.1 to 6.3%). Circular motility decreased (P < 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.

scholarly journals The effect of heterologous seminal plasma from ram, buck or camel on the freezability of ram semen

2018 ◽  
Vol 63 (No. 11) ◽  
pp. 500-512 ◽  
Author(s):  
A.A. Swelum ◽  
I.M. Saadeldin ◽  
M. Bahadi ◽  
M. Afifi ◽  
M. Al-Mutary ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Reduced Glutathione ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Dromedary Camel ◽  
Glutathione Concentration ◽  
Nitrogen Vapor ◽  
Frozen Semen ◽  
Before And After ◽  
Primary Evaluation

Here, we compared the effects of ram, buck and dromedary camel seminal plasma mixed with TRIS-egg yolk glycerol extender on the freezing preservation of ram semen. Awassi ram semen samples underwent primary evaluation and were then pooled and diluted with the following diluents: TRIS-egg yolk glycerol mixed with (1) whole ram semen as a control (T); (2) ram sperm after seminal plasma removal (W); or (3) ram, (4) buck or (5) camel seminal plasma (R, B and C, respectively). The diluted semen was frozen using liquid nitrogen vapor. Various sperm parameters were evaluated in the frozen semen. Total motility before and after freezing was significantly higher in R, B and C diluents than in T and W diluents. Progressive motility after freezing was significantly higher in R, B, C and T diluents than in W diluent. Vitality after freezing was significantly higher in B than in W diluent. DNA fragmentation before and after freezing was significantly lower in R, B, C and T diluents than in W diluent. Plasma membrane integrity before and after freezing was significantly higher in R, B and C diluents than in W diluent. Sperm abnormalities before freezing were significantly lower in R, B and C diluents than in W diluent. Malondialdehyde concentration was significantly higher in T and W diluents than other diluents. Reduced glutathione concentration was significantly higher in B diluent than other diluents. Moreover, reduced glutathione concentration was significantly higher in C, R and W diluents than in T diluent. Thus, the addition of ram, buck or camel seminal plasma to TRIS-egg yolk glycerol extender improved the quality of frozen ram semen, while seminal plasma removal adversely affected it. Ram, buck and camel seminal plasma had similar effects, with no significant differences between them on the evaluated parameters of frozen ram semen.

scholarly journals In vitro evaluation of canine spermatozoa cryopreserved in different extenders

2006 ◽  
Vol 58 (6) ◽  
pp. 1116-1122 ◽  
Author(s):  
E.C.S. Oliveira ◽  
G.C. Juliani ◽  
A.P. Marques Jr. ◽  
M. Henry
Keyword(s):  
Plasma Membrane ◽  
Ethylene Glycol ◽  
Egg Yolk ◽  
Dimethyl Formamide ◽  
Sperm Cells ◽  
Fluorescent Staining ◽  
Progressive Motility ◽  
Functional Integrity ◽  
Hypoosmotic Swelling Test

The efficacy of three extenders, tris-egg yolk-5% ethylene glycol (T1), lactose-egg yolk-5% ethylene glycol (T2) and lactose-egg yolk-5% dimethyl formamide (T3) on preserving the viability of post-thawing canine spermatozoa was evaluated. Three ejaculates per dog were obtained of five animals. The semen was packaged in 0.5ml straws and cooled to 4°C for 120min. The straws were frozen 4cm above the nitrogen level for 15min and thawed in water-bath at 37°C for 60sec and at 75°C for 7sec. Progressive motility and vigour were evaluated immediately after thawing (time 0) and at 30, 60, 90 and 120min. Structural and functional integrity of plasma membrane of the spermatozoa were evaluated, respectively, by fluorescent staining probes and hypoosmotic swelling test. Lactose-egg yolk based extenders showed better cryoprotectant capability and dimethyl formamide was an alternative cryoprotectant agent for dog sperm cells.

scholarly journals Efficiency of Tris-Based Extender Steridyl for Semen Cryopreservation in Stallions

Animals ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1801
Author(s):  
Elena Nikitkina ◽  
Artem Musidray ◽  
Anna Krutikova ◽  
Polina Anipchenko ◽  
Kirill Plemyashov ◽  
...  
Keyword(s):  
Artificial Insemination ◽  
Egg Yolk ◽  
Dna Integrity ◽  
Normal Morphology ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Fertilizing Ability ◽  
Motility Of Sperm ◽  
In Pregnancy ◽  
Stimulation Of

The fertilizing ability of stallion sperm after freezing is lower than in other species. The search for the optimal extender, combination of extenders, and the freezing protocol is relevant. The aim of this study was to compare lactose-chelate-citrate-yolk (LCCY) extender, usually used in Russia, and Steridyl® (Minitube) for freezing sperm of stallions. Steridyl is a concentrated extender medium for freezing ruminant semen. It already contains sterilized egg yolk. Semen was collected from nine stallions, aged from 7 to 12 years old. The total and progressive motility of sperm frozen in Steridyl was significantly higher than in semen frozen in LCCY. The number of spermatozoa with normal morphology in samples frozen in LCCY was 60.4 ± 1.72%, and with Steridyl, 72.4 ± 2.10% (p < 0.01). Semen frozen in Steridyl showed good stimulation of respiration by 2.4-DNP, which indicates that oxidative phosphorylation was retained after freezing–thawing. No differences among the extenders were seen with the DNA integrity of spermatozoa. Six out of ten (60%) mares were pregnant after artificial insemination (AI) by LCCY frozen semen, and 9/12 (75%) by Steridyl frozen semen. No differences among extenders were seen in pregnancy rate. In conclusion, Steridyl was proven to be a good diluent for freezing stallion semen, even though it was developed for ruminants.

118 CRYOPRESERVATION OF CONVENTIONAL AND SEX-SORTED BULL SPERM USING A DIRECTIONAL FREEZING METHOD

2007 ◽  
Vol 19 (1) ◽  
pp. 176 ◽  
Author(s):  
H. Hayakawa ◽  
T. Yamazaki ◽  
M. Oshi ◽  
M. Hoshino ◽  
O. Dochi ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Interface Velocity ◽  
Egg Yolk ◽  
Sperm Viability ◽  
Freezing Method ◽  
Progressive Motility ◽  
Nitrogen Vapor ◽  
Bull Sperm ◽  
Directional Freezing ◽  
Acrosomal Integrity

The objective of this study was to find an optimal parameter (liquid-ice interface velocity; velocity) of the directional freezing method (Arav et al. 2002 Reprod. Nutr. Dev. 42, 583–586) for conventionally processed bull sperm stored in 0.5-mL straws. We also evaluated sex-sorted bull sperm frozen using this method. Experiment 1: Each single ejaculate from two Holstein bulls was split and processed in egg yolk citrate extender (2 steps, 6% glycerol final; EYC) or egg yolk Tris extender (1 step, 7% glycerol final; EYT1). After cooling and loading in 0.5-mL plastic straws, each batch of semen was frozen using a directional 0.5-mL straw freezer (MTG 550; IMT, Ltd., Ness-Ziona, Israel) in 6 different velocities (v2.0, v2.2, v2.4, v2.6, v2.8, v3.0; mm s-1). Ice seeding time was 45 s. Static freezing in liquid nitrogen vapor was used as the control. Sperm parameters were measured using computer-assisted semen analysis (CASA) at 0, 1, and 2 h after thawing. The thawed samples were also evaluated for sperm viability and acrosomal integrity by triple staining. The experiment was repeated 3 times. Data were analyzed by ANOVA. No significant differences in general and progressive motility were observed in each extender regardless of the freezing method. However, sperm viability and acrosomal integrity of sperm frozen in EYC were highest at v3.0 (73.2% and 84.7%, respectively) and were generally higher after MTG freezing (63.4 to 73.2% and 80.6 to 84.7%, respectively) than in the control (52.6% and 73.1%, respectively; P &lt; 0.05 or 0.01) except for v2.4 (54.5% and 70.8%). Sperm viability (74.6 to 77.9% vs. 63.2%) and acrosomal integrity (84.8 to 88.9% vs. 79.3%) in EYT after MTG freezing were also higher than in the control (P &lt; 0.01). Experiment 2: Two single ejaculates from a Holstein bull were flow cytometrically sex-sorted (Schenk et al. 1999 Theriogenology 52, 1375–1391) for X sperm. Each ejaculate was assigned to processing in EYC or egg yolk Tris extender (2 steps, 6% glycerol final; EYT2). Processed sperm was frozen using MTG 550 (velocity set at v3.0) or in liquid nitrogen vapor as control and analyzed as in Experiment 1. In the control, general (40.2% vs. 21.1%; P &lt; 0.01) and progressive (16.3% vs. 4.3%; P &lt; 0.05) motility in EYC were higher than in EYT2, respectively, at 0 h. In MTG freezing, motility (at 0 h, 42.0% vs. 28.3%; P &lt; 0.05), viability (60.5% vs. 40.8%; P &lt; 0.01), and acrosomal integrity (86.9% vs. 71.0%; P &lt; 0.05) in EYC were higher than in EYT2, respectively. But in experiment 2, there were no differences in motility, progressive motility, viability, and acrosomal integrity between MTG freezing and control. The above results suggest that MTG freezing improves membrane and acrosomal quality of bull sperm frozen in 0.5-mL straws. Faster interface velocity (3.0 mm s-1) seems optimal for the given condition. Egg yolk citrate extender seems to be beneficial for MTG freezing of sex-sorted bull sperm, but further studies using ejaculates from different bulls are required to confirm the apparent benefit.

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