80 TREATING BOAR SPERM WITH CHOLESTEROL IMPROVES CRYOSURVIVAL

2011 ◽  
Vol 23 (1) ◽  
pp. 145
Author(s):  
E. A. Moraes ◽  
C. A. A. Torres ◽  
J. K. Graham ◽  
P. L. Romualdo ◽  
P. S. Lopes
Keyword(s):  
Liquid Nitrogen ◽  
Zona Pellucida ◽  
Lipid Composition ◽  
Plasma Membranes ◽  
Egg Yolk ◽  
Glass Container ◽  
Epifluorescence Microscopy ◽  
Freezing And Thawing ◽  
Sperm Binding ◽  
Boar Sperm

Altering the lipid composition of plasma membranes not only affects the ability of sperm to capacitate and acrosome react, but also affects the way sperm respond to cryopreservation. When cyclodextrins are preloaded with cholesterol to form cholesterol-loaded-cyclodextrin (CLC) and then incubated with bull sperm before cryopreservation, higher percentages of motile and viable cells are recovered after freezing and thawing compared with control sperm. The amount of cholesterol in a membrane is important for maintaining its integrity during cryopreservation, and CLC alters the lipid composition of sperm, affecting their cryosurvival. This study evaluated the effect of adding cholesterol to boar sperm on cryosurvival rates and the ability of cryopreserved sperm to bind to the zona pellucida. Methyl-β-cyclodextrin was loaded with cholesterol as follows: 0.45 mL of cholesterol (200 mg mL–1 in chloroform) was added to 1 g of methyl-β-cyclodextrin dissolved in 2 mL of methanol, and the solution was stirred until clear. The mixture was poured into a glass dish and the solvents removed using a stream of nitrogen gas. The resulting crystals were allowed to dry for an additional 24 h, at which time they were removed from the dish and stored in a glass container at 22°C. A working solution of the cholesterol-loaded cyclodextrin was prepared by adding 50 mg of CLC to 1 mL TALP at 37°C and mixing the solution briefly using a vortex mixer. Ejaculates from each of 8 boars were collected, diluted 1:1 in BTS® (Minitub, Brazil), and maintained for 2 h at room temperature. The ejaculates were then cooled to 15°C over 60 min. The ejaculates were then centrifuged at 400 × g for 10 min (at 15°C), the supernatant was discarded, and the sperm were suspended to 120 × 106 cells in cooled diluent (80 mL of lactose solution 11%, 20 mL of egg yolk). The sperm were divided into 2 treatments (T): T1 = control and T2 = 1.5 mg of CLC mL–1. The samples incubated for 15 min at 15°C, after which they were cooled to 5°C over 90 min and diluted 1:1 (v:v) with Freeze diluent (2.5 mL of lactose solution 11%, 6 mL of glycerol, and 1.5 mL of Orvus-es-Paste). The sperm were then packaged into 0.5-mL French straws and frozen in static liquid nitrogen vapor (4.5 cm above the liquid nitrogen) for 20 min before being plunged into liquid nitrogen for storage. Straws were thawed and the efficiency of the sperm to bind to both the chicken egg perivitelline membrane (EPM) and porcine zona pellucida (PZP) were determined using epifluorescence microscopy. The post-thaw motility and binding efficiency of sperm to salt-stored EPM and PZP were analysed by analysis of variance. Boar sperm treated with CLC maintained higher post-thaw motility than control sperm (47 and 34%, respectively; P < 0.05) and had higher numbers of sperm binding to the PZP and EPM (101 sperm/EPM and 166 sperm/PZP) than control samples (77 sperm/EPM and 65 sperm/PZP; P < 0.05). In addition, sperm were easier to visualise on the EPM than the porcine zona pellucida. Adding CLC to boar sperm before cryopreservation increased the number of sperm surviving cryopreservation. Fapemig, CNPq, and CAPES from Brazil.

67 DELIVERING CHOLESTANOL OR DESMOSTEROL TO BULL SPERM MEMBRANES IMPROVES CRYOSURVIVAL

2008 ◽  
Vol 20 (1) ◽  
pp. 114
Author(s):  
E. A. M. Amorim ◽  
J. K. Graham ◽  
M. Meyers ◽  
B. Spizziri
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Plasma Membranes ◽  
Egg Yolk ◽  
Positive Control ◽  
Epifluorescence Microscopy ◽  
Osmotic Tolerance ◽  
Nitrogen Vapor ◽  
Bull Sperm ◽  
And Function

Altering the lipid composition of sperm plasma membranes affects sperm cryosurvival. Cryopreservation induces many stresses on the spermatozoa, including destabilization of the plasma membrane, which results in the loss of sperm motility and function. Treating bull spermatozoa with cholesterolloaded cyclodextrin (CLC) prior to cryopreservation increases sperm cryosurvival rates. This study compared the effect of adding other sterols, which should incorporate into the membrane and increase membrane fluidity at low temperatures, thereby increasing cryosurvival. Ejaculates from four bulls were divided into two experiments (E). In E1, ejaculates were extended with Tris, and then subdivided into four treatments: No additive (control), 1.5 mg CLC/120 million sperm (positive control), and 1.5 mg/120 million sperm in cyclodextrin pre-loaded with either cholestanol or desmosterol. Spermatozoa were incubated for 15 min at 22�C after which both the ability of fresh spermatozoa to bind to the zona pellucida (ZP) and chicken egg perivitelline membrane (EPM) and their osmotic tolerance were evaluated. In E2, sperm were diluted to 120 million cells mL–1 in a Tris diluent and treated as described for E1. Then, samples were diluted 1:1 (v:v) in Tris with 20% Egg Yolk (EY) and cooled to 5�C. After dilution 1:1 (v:v) with Tris containing 10% EY and 16% glycerol, samples were allowed to equilibrate for 15 min, and then were packaged into 0.5-mL straws, frozen in static liquid nitrogen vapor for 20 min, and plunged into liquid nitrogen for storage. Straws were thawed and the motility and zona-binding ability were determined using a Hamilton Thorne Motility Analyzer (Hamilton Thorne Biosciences, Beverly, MA, USA) and epifluorescence microscopy, respectively. Treatment differences for sperm motility, osmotic tolerance, and zona binding were determined using analysis of variance. Treating spermatozoa with CLC resulted in more fresh bull spermatozoa binding to the EPM and ZP compared to cholestanolor desmosterol-loaded cyclodextrin-treated spermatozoa or control cells (P < 0.05). No differences were observed between EPM and ZP binding (P > 0.05). The percentages of total and progressively motile spermatozoa were higher for fresh samples treated with cholesterol-, cholestanol-, or desmosterol-loaded cyclodextrin than for control cells (P < 0.05) when spermatozoa were exposed to anismotic conditions, and then returned to isosmolality. After cryopreservation, the percentages of motile spermatozoa and number of spermatozoa binding to ZP were similar for spermatozoa treated with CLC (56% and 115 sperm/ZP) and cholestanol (53% and 108 sperm/ZP) compared to spermatozoa treated with desmosterol (42% and 86 sperm/ZP; P < 0.05). All treatments provided higher motility and binding efficiency than control spermatozoa (32% and 62 sperm/ZP; P < 0.05). Therefore, adding cholesterol or cholestanol to bull sperm membranes improved cell cryosurvival. Studies to determine if cholestanol affects sperm capacitation need to be conducted.

103 CHOLESTEROL-LOADED CYCLODEXTRIN ADDED TO FRESH BOAR EJACULATES AND SPERM CRYOSURVIVAL

2010 ◽  
Vol 22 (1) ◽  
pp. 211
Author(s):  
E. A. Moraes ◽  
C. A. A. Torres ◽  
J. K. Graham ◽  
P. L. Romualdo ◽  
P. S. Lopes
Keyword(s):  
Liquid Nitrogen ◽  
Plasma Membranes ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Cumulus Cells ◽  
Polished Glass ◽  
Maximum Speed ◽  
Petroleum Jelly ◽  
Boar Sperm ◽  
Working Solution

Sperm cryosurvival is affected by altering the lipid composition of sperm plasma membranes and causes damage to spermatozoa during the cryopreservation process as loss of motile cells and functionality, compared with fresh sperm. Our objective was to compare the effect of adding cholesterol-loaded cyclodextrin (CLC) on sperm quality after freezing boar sperm. The CLC was prepared as described: 200 mg of cholesterol was dissolved in 1 mL of chloroform, and 1 g of methyl-β-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass dish and the solvents removed using a hot plate for 24 h. The crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL of BTS at 37°C. Thirty-five ejaculates from 5 boars were collected, diluted 1:1 in Beltsville thawing solution, and kept to 2 h at 22°C. The ejaculates were held at 15°C for 60 min and centrifuged at 15°C for 400g for 10 min; the pellet was suspended to 120 million cells in cooled diluent (80 mL of lactose solution 11%, 20 mL of egg yolk) and divided in 2 treatments: control and 1.5 mg of CLC/mL. The samples were incubated for 15 min at 15°C, cooled to 5°C over a 90-min period, and diluted 1:1 with freeze diluent (72.5 mL of lactose solution 11%, 6 mL of glycerol, 1.5 mL of Equex). Sperm were packaged into 0.5-mL straws, frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen. Straws were thawed in a water bath at 37°C for 30 sec, extended in Beltsville thawing solution, and analyzed by optic microscopy. Sperm were stained with 35 μg mL-1 of Hoechst 33342 and incubated for 15 min at 37°C, centrifuged at 400g for 5 min, and suspended in BTALP to a final concentration of 2 million spermatozoa/mL. A total of 10 000 spermatozoa (5 μL) from each sample were added to droplets containing 10 porcine oocytes. Porcine cumulus oocyte complexes were aspirated and placed in BTALP. The cumulus cells of the oocytes were removed by vortexing for 2 min at maximum speed. Denuded oocytes were washed 4 times in BTALP and incubated for 1 h at 38.5°C in an atmosphere of 5% CO2 in air, following which 10 oocytes per treatment were randomly placed into 45 μL droplets of BTALP, using a small bore fire polished glass pipette to remove loosely bound spermatozoa. Five oocytes were placed onto glass slides and covered with a cover slip supported by a mix of paraffin wax and petroleum jelly. Oocytes were viewed using an epifluorescence microscope, and the total number of spermatozoa bound to each zona pellucida (ZP) was determined at 400× magnification. Treatment differences for sperm motility and zona binding were determined using ANOVA. The addition of CLC to boar sperm before cryopreservation resulted in higher percentages of motile sperm and higher numbers bound to the ZP (35% and 67 sperm/ZP) compared with control cells (26% and 36 sperm/ZP; P < 0.01). In summary, adding CLC to boar sperm before cryopreservation improved cells. FAPEMIG, Piglandia, CNPq, FACEPE.

121 ACROSOME REACTION AS EFFECT OF ADDITION OF CHOLESTEROL TO BOAR SPERM MEMBRANES

2010 ◽  
Vol 22 (1) ◽  
pp. 219
Author(s):  
C. A. A. Torres ◽  
E. A. Moraes ◽  
J. K. Graham ◽  
P. L. Romualdo
Keyword(s):  
Liquid Nitrogen ◽  
Acrosome Reaction ◽  
Plasma Membranes ◽  
Petri Dish ◽  
Egg Yolk ◽  
Live Cells ◽  
Boar Sperm ◽  
Working Solution ◽  
C 30

Altering the lipid composition of sperm plasma membranes not only affects the ability of sperm capacitation and acrosome reaction, it also affects the way sperm respond to cryopreservation. The objective was to determine if increasing sperm membrane cholesterol levels, by adding cholesterolloaded cyclodextrin (CLC) to boar sperm, alter the cryopreservation sperm to undergo acrosome reaction in vitro. The CLC was prepared as described by Purdy and Graham (2004) with some modification: 200 mg of cholesterol was dissolved in 1 mL of chloroform, and 1 g of methyl-β-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass Petri dish and the solvents removed using a hot plate for 24 h. The resulting crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL of BTS at 37°C. Ejaculates (n = 5) from 5 boars were collected, diluted 1:1 in Beltsville thawing solution, and kept for 2 h at 22°C. Afterward, the ejaculates were put at 15°C/ for 60 min. Later, the ejaculates were centrifuged at 15°C at 400g/10 min, the pellet was suspended to 120 million cells in cooled diluent (80 mL of lactose solution 11%, 20 mL of egg yolk) and divided in 2 treatments: control and 1.5 mg of CLC/mL. These treatments were incubated for 15 min at 15°C. The samples were cooled to 5°C/90 min period and diluted 1:1 with freeze diluent (72.5-mL lactose solution 11%, 6 mL of glycerol, 1.5 mL of Equex). The sperm were packaged into 0.5-mL straws and frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen. Straws were thawed in a water bath 37°C/30 s. A 90% Percoll solution was prepared by diluting 1 mL of 10× PBS with 9 mL of Percoll. A 35% Percoll solution was then prepared by diluting 90% Percoll (0.67 mL) with Medium 199 (1.33 mL). Frozen/thawed spermatozoa (2 mL) were then layered onto 2 mL of 35% Percoll solution in a 15-mL conical tube and centrifuged at 400g/5.5 min. The resulting pellet was suspended with Medium 199 to 100 million cells/mL, and the cells were stained with 5 μL of PI (1 mg mL-1 in water) and 10 μL of FITC-PNA (1 mg mL-1 in 10× PBS). The cells were incubated for 5 min at room temperature to allow PI and FITC-PNA to become incorporated. The acrosomal status of viable cells for each treatment was then determined by epifluorescence microscope at 400× magnification, and the percentage of acrosome reacted cells was calculated as the proportion of FITC-PNA stained and PI negative cells (acrosome reacted, live)/total live cells (PI negative, FITC-PNA positive and negative). Treatment differences for acrosome reaction were determined using ANOVA. The addition of CLC to boar sperm before cryopreservation resulted in higher acrosome reaction (28%) compared with control cells (22%; P < 0.05). Several studies evaluated the ability of bull and stallion sperm treated with CLC to capacity and acrosome react. Adding cholesterol might alter the plasma membrane structure, improving the acrosome reaction in CLC-treated boar spermatozoa. FAPEMIG, Piglandia, CNPq, FACEPE.

75 GLUCOSE CONCENTRATION OF FREEZING EXTENDER MODULATES THE TYROSINE PHOSPHORYLATION PATTERN OF FROZEN-THAWED BOAR SPERMATOZOA

2009 ◽  
Vol 21 (1) ◽  
pp. 138
Author(s):  
J. E. Rodríguez-Gil ◽  
M. Hernández ◽  
M. M. Rivera ◽  
L. Ramió-Lluch ◽  
J. Ballester ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Tyrosine Phosphorylation ◽  
Glucose Concentration ◽  
Egg Yolk ◽  
Freezing And Thawing ◽  
Precise Control ◽  
Boar Sperm ◽  
Phosphorylation Pattern ◽  
Thawing Process ◽  
Boar Spermatozoa

The optimization of freezing extenders is an essential issue for enhancing boar sperm cryosurvival. The aim of the present study was to disclose the role of glucose concentration of freezing extender on the metabolic activity of frozen–thawed spermatozoa. To achieve it, pooled sperm-rich ejaculate fractions from 5 mature and fertile boars (3 ejaculates per boar) were collected using the gloved-hand method. After centrifugation (2400g for 3 min), the sperm pellet was split into 7 aliquots. The aliquots were diluted to a final concentration of 1 × 109 sperm mL–1, in a Tris-citric extender supplemented with 20% egg-yolk, 3% glycerol, and 0, 0.05, 2, 4, 10, 55, or 185 mm glucose. All the extenders were adjusted to a pH of 6.8 and 310 mOsm kg–1 to avoid osmolarity effects. Extended semen samples were dispensed into 0.5-mL straws, and frozen in a programmable cell freezer at 20°C min–1. Thawing was carried out in a water bath at 37°C for 20 s. Afterward, an analysis of protein phosphorylation in tyrosine residues was carried out through bi-dimensional electrophoresis followed by a Western blot analysis. This analysis indicated that sperm samples frozen in extenders without glucose showed specific changes in the tyrosine phosphorylation pattern compared with fresh sperm. Furthermore, the addition of glucose in increasing concentrations to the freezing extender was accompanied by a concentration-dependent decrease in the overall tyrosine phosphorylation pattern, especially in proteins with a molecular weight ranging from 150 to 200 kDa and an acidic isoelectric point (pI). The maximal decrease was observed in spermatozoa frozen in the extender containing 185 mm glucose, in which an additional decrease in the tyrosine phosphorylation of proteins ranging from 60 to 80 kDa, and a basic pI was also observed. These results suggest that glucose is a modulator in the resistance of boar sperm to support freezing and thawing process, because the precise protein phosphorylation pattern of spermatozoa is directly linked to their functional status. In this way, a precise control of the glucose concentration of the freezing extender would be required to improve boar sperm cryoresistance. Supported by CICYT (AGL2005-00760 and AGL2004-04756-C02-02/GAN), Madrid and GERM (04543/07), Murcia, Spain.

Egg yolk enhances the receptiveness of zona pellucida to sperm binding

1994 ◽  
Vol 11 (7) ◽  
pp. 332-334
Author(s):  
Deborah E. Johnson ◽  
Wendy J. Holmgren ◽  
Rajasingam S. Jeyendran
Keyword(s):  
Zona Pellucida ◽  
Egg Yolk ◽  
Sperm Binding

73 SUPPLEMENTATION OF FROZEN BOAR SEMEN WITH β-MERCAPTOETHANOL INCREASES THE INCIDENCE OF INTACT CELLS AFTER THAWING

2008 ◽  
Vol 20 (1) ◽  
pp. 117
Author(s):  
H. Funahashi ◽  
S. Yamaguchi ◽  
W. Fujii ◽  
T. Murakami
Keyword(s):  
Liquid Nitrogen ◽  
Dna Fragmentation ◽  
Egg Yolk ◽  
Molecular Probes ◽  
Sperm Cells ◽  
Freezing And Thawing ◽  
Intact Cells ◽  
Boar Semen ◽  
Post Hoc ◽  
Boar Spermatozoa

During the process of freezing and thawing of boar spermatozoa, a large number of the cells appear to be injured by some stresses such as osmotic forces and oxidation, causing reduced viability and penetrability. β-Mercaptoethanol (bME), a strong reducing agent, may ease oxidative stress and rescue sperm cells from those injuries. The aim of this study was to determine the effect of the presence of bME during freezing and thawing of boar spermatozoa on the viability and acrosome status of the sperm cells. Semen samples were collected from 3 boars; only samples with a high motility (more than 80%) were used for this experiment. Each sample was diluted 1:1 with modified Modena solution and kept overnight at 15�C. After centrifugation at 800g for 10 min, the diluent supernatant was removed; spermatozoa were re-suspended at 2 � 109 cells mL–1 in the first diluent (8.8% trehalose solution containing 20% egg yolk and antibiotic) supplemented with 0, 25, or 50 µm bME, and then cooled to 5�C over 2–3 h. At 5�C, semen samples were further diluted 1:1 with the second diluent (same as the first diluent + 5% glycerin + 1.48% Orvus ES Paste (Equex STM; Minitube, Verona, WI, USA)) supplemented with 0, 25, and 50 µm bME, respectively. After packaging the semen into 0.5-mL straws, it was frozen by keeping the straws 4 cm above the surface of liquid nitrogen for 15 min and then storing them in liquid nitrogen until use. After thawing at 37�C for 30 s, semen samples were re-suspended in 10 mL of BTS solution containing 1.15 mm caffeine and 4 mm Ca chloride, and incubated at 37�C under 5% CO2 in air for 90 min. Viability, DNA fragmentation, and acrosome status of spermatozoa were assessed by flow cytometry after staining with SYBR�14/PI (Molecular Probes, Inc., Eugene, OR, USA), acridine orange, and PNA/PI, respectively. Statistical analyses of data from at least 3 replicated trials were carried out by ANOVA and Fisher's protected least-squares difference (PLSD) post-hoc test. Just after thawing, no differences in viability (45.6–51.1%; P = 0.67), DNA fragmentation (0.7–0.9%; P = 0.76), and acrosome status (intact acrosome: 79.2–83.0%; P = 0.26) of the spermatozoa were observed when sperm cells were frozen and thawed in 0, 25, and 50 µm bME. After culture for 90 min, however, the incidence of spermatozoa with an intact acrosome was significantly higher (P < 0.05) when the semen was frozen and thawed in the presence of 50 µm bME (70.9%), compared with 0 (61.7%) and 25 µm bME (61.0%). Chlortetracycline (CTC) analyses were peformed to confirm that the incidence of intact spermatozoa was higher (P < 0.01) in 50 µm bME (67.6%) than that of non-supplementation controls (51.4%). These results demonstrate that supplementation of semen with 50 µm bME during freezing and thawing processes reduces acrosome damage of boar spermatozoa.

Inhibition of Boar Sperm Binding to Homologous Zona Pellucida by Antibodies against ZP3α and ZP3β Glycoproteins

1998 ◽  
Vol 33 (1) ◽  
pp. 21-25 ◽  
Author(s):  
N Blase ◽  
AR Fazeli ◽  
E Topper ◽  
MM Bevers ◽  
H Woelders ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Sperm Binding ◽  
Boar Sperm

12 FERTILITY OF FROZEN EQUINE SPERM IN SYSTEMS FOR CRYOPRESERVATION

2012 ◽  
Vol 24 (1) ◽  
pp. 117
Author(s):  
R. R. D. Maziero ◽  
P. N. Guasti ◽  
I. D. P. Blanco ◽  
I. Martin ◽  
G. A. Monteiro ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Membrane Integrity ◽  
Skim Milk ◽  
Egg Yolk ◽  
Automated System ◽  
Epifluorescence Microscopy ◽  
Computer Assisted ◽  
Plasma Membrane Integrity ◽  
Sperm Analysis

Optimizing cryopreservation of equine sperm will facilitate genetic banking and propagation of important horse strains through assisted reproduction. This study aimed to evaluate the motility pattern using computer-assisted sperm analysis (CASA) and plasma membrane integrity by epifluorescence microscopy of equine semen frozen in 0.5 mL straws at different freezing rates; also, a fertility trial was performed according to the freezing protocol. Three ejaculates from four stallions of various breeds (Mangalarga Marchador, Westfallen, Hanovarian and Arabian) and ages (5 to 20 years) were collected and processed for cryopreservation. The stallions were housed at the CERBEQ, Reproduction Centre of the Department of Animal Reproduction and Veterinary Radiology, UNESP. The ejaculates were filtered and submitted to analysis by CASA (HTM IVOS 12, Hamilton Thorne Research, USA). In addition, the plasma membrane integrity was determined by fluorescent probes. After evaluation, the ejaculates were diluted at 1:1 (extender:semen) with skim milk extender Botu-Semen™ and centrifuged at 600 × g for 10 min. The supernatant was removed and the pellet resuspended to a final concentration of 100 × 106 sperm mL–1 with milk-egg yolk freezing extender (Botu-Crio™). Semen was packaged in 0.5-mL straws (IMV, LAigle, France) and was placed in nitrogen for 20 min and then from room temperature to 5°C and then frozen in two different cooling systems: an isothermic box (42 cm × 28 cm × 12.5 cm) was placed upon racks suspended 6 cm above liquid nitrogen or other 20 min then immersed into nitrogen and automated system Mini Digitcool™ (IMV Technologies, France), cooling at a –40°C min–1 rate. All straws were stored in liquid nitrogen until thawing and analysis. The straws were thawed in a water bath at 46°C for 20 s and the samples were evaluated for progressive motility, angular progressive velocity, progressive velocity, track speed, percentage of rapid sperm and percentage of sperm with plasma membrane integrity. For the fertility trial, 65 clinically healthy mares had their oestrous cycle monitored by ultrasound and inseminated postovulation with sperm into the uterus. Ovulation was induced with 1 mL of deslorelin acetate (GnRH) injected IM when a 35-mm follicle was detected. Thirty-six hours later, mares were monitored every 6 h until ovulation was detected. When it was detected, mares were inseminated with 800 × 106 total sperm. Pregnancy was confirmed via ultrasound examination 15 days after ovulation. Pregnancy rate was 52.2% using the isothermic box and 60% using the automated machine. Statistical analysis from the frozen–thawed semen evaluated parameters was performed using the statistics software Proc. MIXED of SAS 9.1 and for the fertility trial, logistic regression using the Proc GENMOD from SAS 9.1. The conventional method using the isothermic box was similar to the automated machine with a fast freezing rate. Additionally, AI with 800 × 106 sperm frozen in the isothermic box or automated system resulted in similarly acceptable conception rates.

85 THE COMPARISON OF SPERM FREEZABILITY USING TWO EGG YOLK-FREE DILUENTS IN ZANDI RAM

2010 ◽  
Vol 22 (1) ◽  
pp. 201
Author(s):  
R. Ardebili ◽  
A. Towhidi ◽  
S. Zeinodini ◽  
M. Bahreini ◽  
E. Dirandeh
Keyword(s):  
Least Squares ◽  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Recovery Rate ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Freezing And Thawing ◽  
Automatic Equipment ◽  
Progressive Motility ◽  
Motility Of Sperm

The aim of this study was to investigate the effect of 2 different commercial egg yolk-free extenders, Bioxcell and AndroMed, on post-thaw ram sperm motility and recovery rate. A total of 10 ejaculates were collected from 5 adult Iranian Zandi rams using artificial vagina during 3 weeks, once per week. Preexamination was conducted on semen, and semen motility >70% were selected. Semen samples were pooled and diluted with Bioxcell or AndroMed. Pooled semen was split into 2 treatments and extended to a final concentration of 200 × 108 spermatozoa per mL with extenders. The extended semen was manually packed into 0.25-mL mini-straws and sealed with PVC powder. After 4 h of equilibration in a waterbath at 5°C, the straws were dried with paper tissue, placed horizontally on a rack, and transferred to an isothermal box to be frozen in vapor 5 cm above liquid nitrogen. After 10 min, the straws were plunged into liquid nitrogen. Freezing was conducted using IMV semi-automatic equipment. The percentage of motility and progressive motility of sperm were evaluated before freezing (at 37°C), after refrigeration (at 5°C) and thawing, and recovery rate was calculated. To evaluate indices of sperm motility including the percentage of motile sperm and the progressive forward motility, semen was diluted with 0.9% NaCl w/v (1:100). Ten μL of diluted semen was placed on the prewarmed (37°C) microscope slide (76.2 mm × 24.5 mm; Pearl, China) covered with a cover slip (24 × 24 mm; Menzel-Glaser, Germany) and examined 10 different fields under a phase contrast microscope (Nikon, Tokyo, Japan), at ×200. Dilution factor 1:5 was used. The microscope was equipped with warm plate set at 37°C. Data were analyzed using Proc GLM of SAS. The effect of extender on sperm qualitative characteristics after thawing was significant. Least squares mean percent motility and progressive motility of AndroMed (36.05% ± 0.75; 29.0% ± 0.84) was significantly higher (P < 0.01) than Bioxcell (8.02% ± 0.35; 4.0% ± 0.60); least squares mean recovery rate in AndroMed (34.23% ± 1.05) also was higher (P < 0.01) than Bioxcell (10.0% ± 0.92). Results suggested that AndroMed in comparison with Bioxcell had more ability to preserve sperm quality during the freezing and thawing process.

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