Effect of osmolality of skim-milk diluents and thawing rate on cryosurvival of ram spermatozoa

The effect of osmolality of skim-milk diluents (200, 320, 450, 600, and 750 mOsm/kg water) on the survival of ram spermatozoa frozen in straws were investigated after thawing in 39 °C water or in 20 °C air.Spermatozoa motility improved with increasing osmolality of the freezing diluent, irrespective of thawing rate. Diluents of 600 and 750 mOsm resulted in highest motility immediately after thawing and after 60 min incubation at 39 °C. A significant decrease in spermatozoa motility was observed when straws were thawed at 20 °C air with the magnitude of decrease inversely related to osmolality of the freezing diluent. Fertility of progestagen synchronized ewes inseminated with semen frozen in the 600 mOsm hypertonic skim-milk diluent was comparable to that obtained with fresh semen.     

Cryopreservation of Bangladeshi ram semen using different diluents and manual freezing techniques

A study was conducted to establish a sustainable and effective manual freezing technique for cryopreservation of Bangladeshi ram semen. Three diluents and freezing techniques were tested, both as treatment combinations (diluent × freezing technique) and fixed effects (diluent or freezing technique) on post-thaw sperm motility (SM), viability (SV), plasma membrane integrity (SPMI) and acrosome integrity (SAI). Ten rams were selected, based on semen evaluation. Eight ejaculates were used for each treatment combination. Semen samples were diluted using a two-step protocol for home-made Tris-based egg yolk (20%, v/v) diluents: D1 (7% glycerol, v/v) and D2 (5% glycerol, v/v), and one-step for commercial diluent: D3 (Triladyl^®, consists of bi-distilled water, glycerol, tris, citric acid, fructose, spectinomycin, lincomycin, tylosin and gentamycin) at 35 °C. Fraction-A (without glycerol) was added at 35 °C, and following cooling of sample to 5 °C (−0.30 °C/min), Fraction-B (with glycerol) was added. The diluted semen samples were aspirated into 0.25 ml French straws, sealed, and equilibrated at 5 °C for 2 h. The straws were frozen in liquid nitrogen (LN) vapour, in a Styrofoam box. The freezing techniques were; One-step (F1): at −15.26 °C/min from +5 °C to −140 °C; Two-step (F2): at −11.33 °C/min from +5 °C to −80 °C, and −30 °C/min from −80 °C-140 °C; and Three-step (F3): at −11.33 °C/min from +5 °C to −80 °C, at −26.66 °C/min from to −80 °C to −120 °C, and at −13.33 °C/min from −120 °C to −140 °C. Two semen straws from each batch were evaluated before and after freezing. The group F3D3 exhibited significantly higher (p < 0.05) post-thaw SM 63.1 ± 2.5%, SV 79.0 ± 2.1% and SPMI 72.9 ± 1.7%, whereas SAI 72.9 ± 1.7% was significantly higher (p < 0.05) in group F3D2. The freezing technique F2 and F3 had significantly higher (p < 0.05) post-thaw sperm values compared to F1. The post-thaw SM and SV were above 50% and 65% with the freezing technique F2 and F3 but differed non-significant. The SPMI 67.6 ± 2.0% and SAI 76.1 ± 1.4% were significantly higher (p < 0.05) with F3. Likewise, the diluent D2 and D3 had significantly higher (p < 0.05) post-thaw sperm values compared to D1. The post-thaw SM, SV and SPMI were above 50%, 65% and 55% with the diluents D2 and D3 but differed non-significant. The SAI 76.1 ± 1.1% was significantly higher (p < 0.05) with D3. We concluded that the use of a simple home-made Tris-based diluent containing 20% (v/v) egg yolk and 5% glycerol (v/v), two-step dilution and a three-step freezing technique is a sustainable and effective method for freezing ram semen. For further validation, the fertility of ewes artificially inseminated with the frozen semen will be observed.     

Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws

The interaction of glycerol concentrations of 0-10% and cooling rates from 1 to 1,500 degrees C/min with boar spermatozoa motility and acrosomal integrity (proportion of spermatozoa with normal apical ridge) was studied after thawing 0.5 ml straws at a constant rate. While increasing the glycerol concentration from 0 to 4% progressively improved motility, the percentage of spermatozoa with a normal apical ridge gradually decreased. The magnitudes of the respective changes depended on cooling rate. A peak value of 48.1% and rating 3.8 were obtained in semen protected with 4% glycerol, frozen at 30 degrees C/min. Increasing the glycerol levels above 6% resulted in a gradual decrease in motility. The proportion of spermatozoa with normal apical ridge was highest in semen protected with 0-1% glycerol after cooling at 30 degrees C/min (64.4% and 66.1%, respectively), but at these glycerol concentrations the percentage of motile spermatozoa was low. At the 30 degrees C/min cooling rate, the decline in the proportion of cells with normal apical ridge due to increasing the glycerol levels to 3 and 4% was relatively slow (57.3% and 49.4%, respectively). Cooling at 1 degrees C/min was detrimental to acrosomal integrity, which decreased with increasing glycerol concentration, in contrast to increasing motility, which even at its maximum, remained low. The direct plunging of straws into liquid nitrogen (1,500 degrees C/min) resulted in damaged acrosomes in all spermatozoa with the total loss of motility. Balancing motility and acrosomal integrity, freezing boar semen protected with 3% glycerol by cooling at 30 degrees C/min resulted in optimal survival for boar semen frozen in 0.5 ml French straws.     

Successful pregnancies in cows following double freezing of a large volume of semen

The objective of the following paper is to describe a new technology for large volume and double freezing of semen in 12 mL test tubes. Semen from two different bulls was frozen with a new technique using 12 mL test tubes and was refrozen after thawing in mini straws. All freezing was done in a "Multi thermal gradient" (MTG) freezing apparatus, which moves the container at a constant velocity (V) through a thermal gradient (G) producing a controlled cooling rate B = (G) x (V). Each of the two bulls ejaculated were evaluated for post thaw motility in the lab and then in a field trial which was carried out in a split sample mode. We inseminated 105 cows after a double freezing/thawing cycle, and another 123 cows were inseminated with semen frozen in mini-straws and a conventional method. The results showed a 75 +/- 5% post thaw motility after freezing a 12 mL test tube and 50 +/- 5% after a second freezing/thawing in mini-straws, respectively. Controlled vapour freezing showed a 60 +/- 10% post thaw motility. The results of the field trial showed a pregnancy rate of 44% (47/105) for the double freezing group in comparison to 45.5% (56/123) for the controlled group. These results can be beneficial for large volume freezing, and therefore for bull semen cryobanking in a large volume which will be followed by second freezing in a regular insemination volume.     

Ultrastructural and cytochemical characterization of follicular cell types in bovine (Bos taurus) cumulus-oocyte complexes aspirated from small and medium antral follicles during the estrus cycle

In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.     

Evaluation of extenders and cryopreservatives for cooling and cryopreservation of spermatozoa from the western spotted skunk (Spilogale gracilis)

Experiments were conducted to develop a suitable protocol for cryopreservation of spotted skunk semen. Semen was collected by electroejaculation of captive male skunks (n = 16) from late January through late November. In the first experiment, fresh semen was diluted in either TEST (n = 10), TRIS (n = 9), or BF5F (n = 7) extenders and maintained at 4°C for 16 hr. Sperm motility in these extenders was not significantly different before cooling (P = 0.71), but samples diluted with BF5F exhibited significantly lower sperm motility than the other extenders at all time points after cooling (P < 0.05). In the second experiment, fresh semen was diluted in TEST containing either 3, 5, or 10% DMSO or 3, 5, or 10% glycerol as a cryopreservative. These samples were cooled to 4°C and frozen in 0.25 ml French straws on dry ice. Some samples containing 5% DMSO or 5% glycerol (n = 4), were also frozen on dry ice as pellets. Frozen samples were maintained in liquid nitrogen. Fresh samples had significantly greater sperm motility in dimethyl sulfoxide (DMSO) than in glycerol (P < 0.05), while frozen and thawed samples had the highest motility in 5 or 10% DMSO or 10% glycerol. Samples frozen in French straws had significantly greater sperm motility after freezing and thawing than those frozen by the pellet method (P < 0.05). Optimum cryoprotection was achieved with the TEST extender containing 5 or 10% DMSO, when used in conjunction with French straws. © 1992 Wiley-Liss, Inc.     

Two-step accelerating freezing protocol yields a better motility,membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols

The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to −35 °C (40 °C/min), from −35 °C to −65 °C (17 °C/min), and then from −65 °C to −85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to −5 °C (4 °C/min), from −5 °C to −110 °C (25 °C/min), and then from −110 °C to −140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to −10 °C (5 °C/min), and then from −10 °C to −130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates.     

Cryopreservation of seabream (Sparus aurata) spermatozoa

The aim of this research was to optimize protocols for freezing spermatozoa of seabream (Sparus aurata). All the phases of the cryopreservation procedure (sampling, choosing the cryoprotective extender, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa under examination, so as to be able to restore on thawing the morphological and physiological characteristics of fresh semen. Seabream spermatozoa were collected by stripping and transported to the laboratory chilled (0-2 degrees C). Five cryoprotectants, dimethyl sulfoxide (Me(2)SO), ethylene glycol (EG), 1,2-propylene glycol (PG), glycerol, and methanol, were tested at concentrations between 5 and 15% by volume to evaluate their effect on the motility of semen exposed for up to 30 min at 26 degrees C. The less toxic cryoprotectants, 10% EG, 10% PG, and 5% Me(2)SO, respectively, were added to 1% NaCl to formulate the extenders for freezing. The semen was diluted 1:6 with the extender, inserted into 0.25-ml plastic straws by Pasteur pipette, and frozen using a cooling rate of either 10 or 15 degrees C/min to -150 degrees C followed by transfer and storage in liquid nitrogen (-196 degrees C). The straws were thawed at 15 degrees C/s. On thawing, the best motility was obtained with 5% Me(2)SO, although both 10% PG and EG showed good results; no differences were found between the two freezing gradients, although semen frozen with the 10 degrees C/min gradient showed a slightly higher and more prolonged motility.     

Effects of supplementation of Tris-egg yolk extender with royal jelly on chilled and frozen-thawed ram semen characteristics

This study was conducted to examine the effect of supplementation of Tris-egg yolk extender with lyophilized royal jelly (RJ) on chilled and frozen-thawed ram semen parameters. Ejaculates were collected by artificial vagina from 4 mature rams, twice a week for 4 weeks. Only samples with motility of ≥70% were included, pooled and divided into four equal parts and then diluted in extenders with various concentrations of RJ (0, 1, 3 and 5%, vol/vol) to a final concentration of 200 × 10⁶ sperm/mL and was incubated at 37 °C for 30 min and were subsequently evaluated. After equilibration of extended semen for 2 h at 4 °C, some semen samples were packed in 0.25 mL plastic straws. Then, the straws were frozen in the liquid nitrogen vapor phase for 15 min and stored at −196 °C in liquid nitrogen. The frozen straws were thawed in warm water (37 °C) for 30 s and evaluated; whereas, other semen samples were stored in the refrigerator (4 °C) up to 7 days. The chilled samples were kept in water bath (37 °C) for 5 min and then were evaluated. After dilution, the lowest and highest sperm total abnormality was recorded in 3 and 5% RJ supplemented groups, respectively (P < 0.05). The chilled sperm total motility and membrane integrity were significantly (P < 0.05) higher in 3% than those in 0% and 5% RJ supplemented groups. The chilled sperm progressive motility and viability was significantly (P < 0.05) higher in 1 and 3% than those in 0 and 5% RJ supplemented groups. The frozen-thawed sperm total motility, progressive motility, membrane integrity and viability were significantly higher in 3% RJ supplemented group (P < 0.05). In conclusion, supplementation of Tris-egg yolk extender with 3% lyophilized RJ had a protective effect on chilled and cryopreserved ram spermatozoa.     

Effects of centrifugation, glycerol level, cooling to 5 degrees C, freezing rate and thawing rate on the post-thaw motility of equine sperm

Five experiments evaluated the effects of processing, freezing and thawing techniques on post-thaw motility of equine sperm. Post-thaw motility was similar for sperm frozen using two cooling rates. Inclusion of 4% glycerol extender was superior to 2 or 6%. Thawing in 75 degrees C water for 7 sec was superior to thawing in 37 degrees C water for 30 sec. The best procedure for concentrating sperm, based on sperm motility, was diluting semen to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium at 20 degrees C and centrifuging at 400 x g for 15 min. There was no difference in sperm motility between semen cooled slowly in extender with or without glycerol to 5 degrees C prior to freezing to -120 degrees C and semen cooled continuously from 20 degrees C to -120 degrees C. From these experiments, a new procedure for processing, freezing and thawing semen evolved. The new procedure involved dilution of semen to 50 x 10(6) sperm/ml in centrifugation medium and centrifugation at 400 x g for 15 min, resuspension of sperm in lactose-EDTA-egg yolk extender containing 4% glycerol, packaging in 0.5-ml polyvinyl chloride straws, freezing at 10 degrees C/min from 20 degrees C to -15 degrees C and 25 degrees C/min from -15 degrees C to -120 degrees C, storage at -196 degrees C, and thawing at 75 degrees C for 7 sec. Post-thaw motility of sperm averaged 34% for the new method as compared to 22% for the old method (P<0.01).     

Effect of initial freezing temperature on the semen characteristics of frozen-thawed ram spermatozoa in a semi-arid tropical environment

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including initial freezing temperature. The present study was conducted to observe the effect of initial freezing temperature on post-thawing motility of ram spermatozoa of native and crossbred rams maintained in a semi-arid tropical environment. Good quality semen obtained from native Malpura and crossbred Bharat Merino rams were pooled within breed and diluted at a rate of 1000 million spermatozoa per milliliter in TEST—yolk–glycerol extender. Diluted semen samples were loaded in 0.25 ml straws and cooled to −25, −75 or −125 °C freezing temperature at the rate of −25 °C/min under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at 50 °C in a water bath for 10 s and motility characteristics of the frozen-thawed spermatozoa were assessed by a computer-assisted spermatozoa analysis technique. Initial freezing temperature significantly affected the post-thawing motility of sperm in both the breeds. The post-thawing % motility and rapid motile spermatozoa were significantly higher at initial freezing temperature of −125 °C and lower at −25 or −75 °C. The percentage medium motile sperm were similar at all three initial freezing temperatures. The percentage of slow motile and linearity of sperm varied (P<0.01) between the different freezing temperatures. The curvilinear velocity, average path velocity and straight line velocity of spermatozoa were higher (P<0.01) at −125 °C than −25 or −75 °C. Although the lateral head displacement of spermatozoa did not vary significantly between the different initial freezing temperatures, the stroke frequency was significantly lower at −25 °C than −75 or −125 °C. Except for % linearity, the average path velocity and straight line velocity, other spermatozoa characteristics were not significantly different between breeds. The interaction between freezing temperature and breed was significant only for the % motility and linearity of the spermatozoa. The study indicates that initial freezing temperature has a significant effect on spermatozoa motility and velocity following post-thawing. The best motile spermatozoa following thawing were achieved at −125 °C freezing temperature.     

The effect of warming velocity on motility and acrosomal integrity of boar sperm as influenced by the rate of freezing and glycerol level

The effect of thawing velocities ranging from 10°C/min to 1.800°C/min on the motility and acrosomal integrity of boar spermatozoa frozen at 1°C/min (suboptimal), 5°C/min, and 30°C/min (optimal) rate was studied with the sperm suspended for freezing in diluent containing 2, 4, or 6% of glycerol (v/v). The influence of thawing on sperm survival depends on the rate at which the sperm had been frozen. In semen frozen at a suboptimal rate of 1°C/min, the percentage of motile sperm (FMP) initially fell to 3.5–4.0% when the thawing rose to 200°C/ min, but, with further increases in thawing rate, increased and reached peak values (10.3–11.0% FMP) after thawing at 1,800°C/min. The percentage of sperm with normal apical ridge (NAR) also increased moderately with thawing rate, but the degree of improvement decreased as the glycerol level was increased. In semen frozen at 1°C/min, acrosomal integrity (NAR) was best maintained in 2% glycerol, reaching 22.9% NAR after thawing at 1,800°C/min. In semen frozen at the optimal rate of 30°C/min, the increases in thawing rates above 200°C/min substantially improved motility. Motility was generally higher in semen protected by 4 or 6% glycerol, with the peak values of 44 or 46% FMP, respectively, after thawing at 1,200°C/min. The proportion of sperm with NAR also increased with thawing rate, but as in the case of suboptimally frozen sperm it was influenced negatively by the glycerol concentration. The peak value 53% NAR was recorded in semen protected by 2% glycerol, frozen at 30°C/min, and thawed at 1,200°C/min. In view of the inverse relationship between FMP and NAR, selection of optimal conditions from among the interacting variables, freezing rate, glycerol concentration, and thawing rate requires compromising between maximal FMP and maximal NAR. Accordingly, we have adopted as optimal a protocol with a thawing rate of 1,200°C/min, a freezing rate of 30°C/min and concentrations of 3% glycerol. © 1993 Wiley-Liss, Inc.     

The effect of glycerol concentration and cooling velocity on cryosurvival of ram spermatozoa frozen in straws

The effect of varying the concentration of glycerol from 0 to 16% on the survival of ram spermatozoa frozen at increasing rates of cooling (1–100 °C/min) or by direct plunging of spermatozoa in 0.5-ml straws in liquid nitrogen was studied after thawing at a constant rate (in water at 39 °C for 30 sec). For each glycerol concentration, the ram spermatozoa tolerated a range of cooling velocities and the best survival rates (percentage motility and rating) were obtained when the glycerol concentration was 4 or 6% and when the rate of freezing ranged from 10 to 100 °C/min. No spermatozoa survived in any glycerol concentration following freezing in straws plunged into liquid nitrogen. In general, the range of cooling rates shifts to lower values as the glycerol concentration increases for optimum cryosurvival. However, the toxic effect of increasing the concentration of glycerol over 8% contributes greatly to the gradual decrease in cryosurvival of spermatozoa at these particular concentrations.     

Influence of extender, cryoperservative and seminal processing procedures on postthaw motility of canine spermatozoa frozen in straws

Experiments were conducted to evaluate two extenders (egg-yolk Tris and egg-yolk lactose), varying concentrations of two cryopreservatives (glycerol and dimethyl sulfoxide), and rates for cooling to 5 degrees C, cooling from 5 to -100 degrees C, and warming for canine spermatozoa packaged in 0.5-ml French straws. At optimal concentrations of glycerol, egg-yolk Tris extender was superior to egg-yolk lactose in preserving spermatozoal motility. Addition of dimethyl sulfoxide, alone or in combination with glycerol in either extender, was not beneficial to spermatozoal survival after thawing. Canine spermatozoa withstood a range of cooling and equilibration times with no detrimental effect on spermatozoal motility prior to freezing. However, there were differences in spermatozoal motility immediately after thawing; these differences were variable, resulting in a cooling time by equilibration time interaction. Spermatozoal motility after thawing was best preserved by freezing in egg-yolk Tris extender containing 2-4% glycerol, using a moderate rate of cooling from 5 to -100 degrees C (-5 degrees C/min from 5 to -15 degrees C, then -20 degrees C/min from -15 to -100 degrees C). Three of 12 bitches inseminated intravaginally with semen frozen using this protocol became pregnant.     

Sperm survival during short-term storage and after cryopreservation of semen from striped trumpeter (Latris lineata)

Methods of short-term storage and cryopreservation were examined for semen from striped trumpeter (Latris lineata). For fresh semen at 18 degrees C, the percentage of motile sperm declined rapidly from over 80% immediately after activation with sea water to less than 2% within 9 min after activation. The motility after activation of undiluted fresh sperm stored at 5 degrees C was maintained for two days and then declined markedly so that by the eighth day, sperm were mostly immotile after activation. The post-thawing motility was higher for sperm frozen with a non-activating diluent containing 2.84 M DMSO in saline (117 mM NaCl) than in an activating glycerol (2 M) medium in dilute sea water (300 mOsm). Post-thawing motility was higher for a dilution rate of 1:5 (semen:diluent) than 1:2 or 1:11 but was similar when frozen semen was thawed at 10 degrees, 20 degrees or 30 degrees C. For semen stored at a range of volumes as pellets frozen on dry ice (0.2 to 2.0 mL) or straws frozen in liquid nitrogen vapor (0.25 to 0.5 mL) and thawed in a waterbath at 20 degrees C, the post-thawing motilities were similar even though the patterns of cooling and thawing differed markedly between methods of freezing and sizes of pellets and straws.     

Survival and fertility of ram spermatozoa frozen in pellets, straws and minitubes

The post-thaw survival and fertility of ram spermatozoa frozen in pellets, 0.25- and 0.5-ml PVC straws, and 0.25-ml minitubes were examined. In 5 experiments, a freezing height of 6 cm above the level of liquid nitrogen was optimal for 0.25- and 0.5-ml straws, whereas 4 cm was best for the 0.25-ml minitubes. Post-thaw motility of spermatozoa was lower for semen frozen in straws and minitubes than in pellets (Experiment 1: 43.7 vs 53.4%, P < 0.001), but after freezing was better in 0.5-ml straws and 0.25-ml minitubes than in 0.25-ml straws (Experiment 1: 44.9 vs 41.3%, P < 0.05; Experiment 2: 49.6 vs 46.8%, P < 0.01). Sperm motility was also better for 1:8 (semen:diluent) pre-freezing dilution rate (50.5%) than for 1:4 (45.6%, P < 0.01) and 1:2 (39.8%, P < 0.001) but not the 1:16 (49.5%) dilution rate. Dry ice was a better freezing medium than liquid nitrogen vapor (49.2 vs 46.9% motile spermatozoa, P < 0.001). The post-thaw motility of spermatozoa was similar for the three freezing packages if the semen was loaded at 5 degrees C, but motility was poorer for semen loaded into 0.25-ml straws than 0.25-ml minitubes at 30 degrees C (P < 0.05). In a fertility test, pregnancy rates were influenced by rams (3 rams, P < 0.05) and freezing package (pellets vs 0.25-ml minitube vs 0.25-ml straw vs 0.5-ml straw, P < 0.05) but not freezing medium (liquid nitrogen vapor vs dry ice). More ewes were pregnant after insemination with pellet-frozen semen (106/150, 71%) than with semen frozen in 0.25-ml straws (85/150, 57%; P < 0.05) and in 0.5-ml straws (83/150, 55%; P < 0.01) but not minitubes (98/150, 65%). It was concluded that minitubes provide a useful alternative to pellets as a storage package for ram spermatozoa, allowing for individual dose identification and easier storage while maintaining a fertility rate indistinguishable from that obtained with pellet-frozen semen.     

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5–ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 μM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 μM zinc sulphate.     

First pregnancies in jennies with vitrified donkey semen using a new warming method

Sperm vitrification has been recently developed, but fertility trials have not been performed yet in equine species. In this study, a new warming technique for vitrified donkey semen was developed and the uterine inflammatory response and fertility were compared to conventional freezing. In Experiment 1, sperm was vitrified in straws and warmed in 3 ml of extender or in a water bath at: 37 °C/30 s; 43 °C/10 s; and 60 °C/5 s. Sperm motility, plasma and acrosome membranes and DNA integrity were compared between treatments. In Experiment 2, jennies were inseminated twice (500 × 10⁶ sperm) in the uterine body either with vitrified or frozen semen (2 cycles/jenny). Pregnancy rates and the uterine inflammatory response (polymorphonuclear neutrophil concentration; PMN) were evaluated after artificial insemination (AI). No differences between warming in extender/water bath were found and 43 °C/10 s was better than lower temperatures in terms of total (53.8 ± 13.2%) and progressive sperm motility (41.4 ± 11.4%). No differences in PMN concentration (× 10³ PMN/ml) were found between vitrified (276.8 ± 171.6) or frozen (309.7 ± 250.7) semen after AI. However, PMN decreased faster (P < 0.05) using vitrified semen. Pregnancy rates were greater for vitrified (22%) than frozen semen (10%) but not statistically different. In conclusion, donkey sperm vitrified in straws could be directly warmed in a water bath at 43 °C/10 s, reducing the uterine inflammatory response obtained after AI and promoting positive pregnancy outcomes. These findings confirm the possibility to use vitrified semen as an alternative for AI in jennies.     

Post-thaw functional status of boar spermatozoa cryopreserved using three controlled rate freezers: a comparison

This study compared variation in the quality of cryopreserved boar spermatozoa and the control and accuracy of cooling rates between three semen freezers (CryoLogic Freeze Control CL3000, Planer Products Kryo Save Compact KS1.7/Kryo 10 Control module and a controlled rate 'Watson' freezing machine developed within our laboratory). Five ejaculates were collected from each of 15 boars (five boars from each of three breeds). Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% v/v glycerol) and placed into 0.5 ml straws. Three straws per treatment, from each ejaculate were cooled to -5 degrees C at 6 degrees C/min, held at -5 degrees C for 30s while ice crystal formation was induced, then further cooled from -5 to 80 degrees C at either 40 degrees C/min (Kryo Save Compact KS1.7 and Watson) or 6 degrees C/min (Freeze Control CL3000). Precise measurements of temperature fluctuations during the programmed cooling curves were made by inserting thermocouples into the semen filled straws. Semen was assessed for %motile cells, motility characteristics using computer-assisted semen analysis (CASA), plasma membrane integrity (%SYBR-14 positive stained spermatozoa) and acrosome integrity (%FITC-PNA positive stained spermatozoa). Spermatozoa cryopreserved using the Freeze Control CL3000 system (maximum rate of 6 degrees C/min) exhibited reduced post-thaw viability (14.2+/-2.8% mean plasma membrane intact spermatozoa) when compared to both the KS1.7 and Watson freezers (optimal rate of 40 degrees C/min) (18.4+/-3.2 and 25.7+/-3.7% mean plasma membrane intact spermatozoa, respectively). Differences in motility characteristics were observed between spermatozoa cryopreserved at 40 degrees C/min with the Watson apparatus preserving a larger proportion of sperm with progressive motility. Cooling curves in the CL3000 and KS1.7 were interrupted by a pronounced increase in temperature at -5 degrees C that corresponded with the latent heat of fusion released with ice crystal formation. This temperature change was significantly reduced in the cooling curves produced by the Watson freezer. These findings suggest that preserving spermatozoa using the Watson freezer improved post-thaw semen quality, with regard to sperm motility characteristics. Furthermore, that post-thaw semen viability was enhanced by minimising temperature fluctuations resulting from the release of the latent heat of fusion at ice crystal formation.     

The effect of straw size, freezing rate and thawing rate upon post-thaw quality of dog semen

Optimal freeze-thaw processes for dog semen will yield a maximal number of insemination doses from an ejaculate. The objectives of this study were to compare the effects of two straw sizes (0.25- and 0.5-mL French), two freezing rates (straws suspended 3.5 and 8 cm above liquid nitrogen) and two thawing rates (in water at 37 and 70 degrees C) upon post-thaw quality of dog semen, and to determine the best treatment combination. Quality was expressed in terms of the percentage progressively motile sperm 5 and 60 min after thawing and the percentage of abnormal acrosomes 5 min after thawing. One ejaculate from each of eight dogs was frozen. Two straws from each ejaculate were exposed to each of the eight treatment combinations. Data were analyzed by means of a repeated measures factorial analysis of variance and means compared using Bonferroni's test. Dog affected each response variable (P < 0.01). Neither straw size, nor freezing rate, nor thawing rate affected motility 5 min after thawing (P > 0.05). Half-milliliter straws resulted in 5.7% more progressively motile sperm 60 min after thawing and 6.5% fewer abnormal acrosomes than 0.25-mL straws (P < 0.05, n = 64). The percentage progressively motile sperm 60 min after thawing tended to be higher for semen thawed at 70 degrees C compared to 37 degrees C (P < 0.06, n = 64). Semen thawed in water at 70 degrees C had 6.6% fewer abnormal acrosomes than semen thawed in water at 37 degrees C (P < 0.05, n = 64). Freezing rate interacted with thawing rate (P < 0.05) in their effects upon acrosomal morphology and freezing 8 cm above liquid nitrogen and thawing in water at 70 degrees C was best. Dog semen should be frozen in 0.5-mL straws, 8 cm above liquid nitrogen and thawed in water at 70 degrees C.