Post-thaw viability of european bison (Bison bonasus) semen frozen with extenders containing egg yolk or lipids of plant origin and examined with a heterologous in vitro fertilization assay.

Basic characteristics of European bison (Bison bonasus) semen were described and the efficacies of two extenders-Triladyl, containing egg yolk, and a synthetic extender, containing soybean lipids-were tested for semen cryopreservation. Seven ejaculates were collected by electroejaculation from a 10-yr-old, European bison bull. Each ejaculate was diluted at 37 degrees C to a final concentration of 200 x 10(6) sperm/ml with Triladyl or the synthetic extender. Extended semen samples were frozen according to a standard bull semen freezing protocol. After 2 wk of storage, one straw from each extender and ejaculate was thawed, and postthaw quality was evaluated by individual sperm motility and movement rate, numbers of sperm morphologic abnormalities and intact acrosomes, functional integrity of the sperm membranes determined by hypoosmotic swelling test (HOST), viability (live-dead, eosin-nigrosin stain), and a heterologous in vitro sperm penetration assay (SPA). A total of 600 in vitro-matured bovine oocytes were inseminated with 1 X 10(6) spermatozoa of Holstein semen frozen-thawed in Triladyl (control) or of European bison semen frozen in Triladyl or the synthetic extender. Nuclear status of the oocytes was determined after 18 h of sperm-oocyte coincubation. Extender had no effect on any evaluated parameters of semen after dilution and cooling (4 hr at 5 degrees C) or in postthaw individual motility, quality of movement, and sperm morphology. However, significantly (P < 0.05) higher numbers of spermatozoa with intact acrosomes, intact membranes (HOST), and viable sperm (P < 0.01) were in semen frozen in Triladyl than in the synthetic extender. Mean values for heterologous SPA for bull (control) and for bison semen frozen in the synthetic extender were very much alike-63.3+/-10.6% and 63.1 +/- 15.9%, respectively; bison semen frozen in Triladyl was lower, 43.0+/-24.2% but not significantly different. Cumulative results from a variety of viability assays of diluted/cooled and frozen-thawed semen, including the heterologous SPA, suggest that European bison semen can be successfully frozen in both extenders tested in this study.     

Glycerol,Methyl‐Formamide and Dimethyl‐Formamide in Canine Semen Cryopreservation

The aim of the present work was to compare the efficiency of methyl‐formamide (MF), dimethyl‐formamide (DF) and glycerol (GL) as cryoprotectants in canine semen cryopreservation. For the experiment, pooled semen was submitted to one of the three cryoprotectants, with a final concentration of 3% in egg yolk–TRIS extender. Semen was subjectively evaluated for total and progressive motility, vigour and morphology. Sperm membrane functional integrity was assessed by hypo‐osmotic swelling test (HOST), and longevity was assessed using the thermoresistance test (TRT). Fresh semen showed normal physical and morphological characteristics. After thawing, differences were observed between semen frozen using GL and DF, regarding total and progressive motility and vigour (p < 0.05), but not between MF and GL or MF and DF. Means for total motility, progressive motility, vigour and morphologically normal spermatozoa were, respectively, 69.0 ± 5.4%, 61.0 ± 7.4%, 2.9 ± 0.5 and 57.1 ± 5.0% for GL; 59.0 ± 8.9%, 50.0 ± 10.0%, 2.5 ± 0.7 and 66.9 ± 7.7% for MF; and 44.0 ± 21.0%, 37.0 ± 19.8%, 2.1 ± 0.6 and 61.1 ± 5.5% for DF. On HOST, GL was superior (p < 0.05) to MF and DF (57.8 ± 12.4%, 35.8 ± 18.4% and 34.4 ± 9.4%, respectively). During the TRT, both GL and MF were superior to DF, with no differences between GL and MF. In conclusion, the use of MF as cryoprotectant showed results similar to GL, and can be considered as an alternative in canine semen cryopreservation. Further studies testing different concentrations of MF may improve its effects on cryopreservation of canine semen.     

Comparative study of different methods for dog semen cryopreservation and testing under clinical conditions

The extenders and freezing rates from three different freezing protocols were combined and compared to each other in order to study the post-thawing acrosome integrity and fertility of frozen dog sperm. A commercial bovine TRIS-base extender (TRILADYL) and two self-made canine semen extenders (Norwegian and Dutch) were combined with a conventional bovine and two canine freezing regimes, and acrosome integrity of frozen/thawed spermatozoa was assessed by fluorescein isothiocyanate conjugated peanut agglutinin staining (FITC-PNA). Differences between freezing/thawing protocols were reflected in the proportion of cells with acrosomal damage and not based on motility results. It was concluded that during dog semen cryopreservation extenders had less influence on the post-thawing sperm quality than did the freezing rates. The optimal extender/freezing rate combination (TRILADYL/Norwegian) was used in the clinical practice to evaluate the fertility of frozen sperm administered by intrauterine insemination using a surgical approach. The pregnancy rate was 57% (4/7), but the average litter size was low (2.8). This may have been due to the insufficient sperm numbers contained in an insemination dose and/or to the incorrect timing of artificial insemination (AI). The final conclusion is that the commercial bovine extender is useful for freezing dog semen, and the TRILADYL/Norwegian freezing protocol is recommended as the most advantageous combination for the freezing of canine semen in the clinical practice.     

Effect of Oviductal Proteins on Structural and Functional Characteristics of Cryopreserved Sperm in Murrah Buffaloes*

The present study was undertaken to elucidate the effect of non‐luteal oviductal proteins on sperm characteristics in Murrah buffaloes. Oviducts from healthy buffaloes were collected immediately after slaughter and the oestrous cycle phase was determined as either luteal or non‐luteal based on ovarian morphology. Non‐luteal oviducts (n = 80) were flushed from the isthmic end of the oviduct with PBS, fluid was centrifuged at 10 000 g at 4°C for 20 min and then dialysed and clarified. The supernatant obtained was lyophilized to concentrate the protein and stored at ?20°C till use. Sixteen good quality ejaculates from four Murrah buffalo bulls were collected using an artificial vagina. After fresh semen analysis, each ejaculate was split into two parts and extended in Tris–citrate–egg yolk glycerol dilutor. Part I of the split ejaculate was treated with non‐luteal oviductal proteins at the dose rate of 1 mg/ml of diluted semen, while part II remained as control. The extended semen was equilibrated for 4 h at 5°C, filled in 0.5 ml French straws, exposed to LN₂ vapour, plunged into LN₂ and then stored at ?196°C. The equilibrated and frozen–thawed semen was evaluated for sperm motility, viability, acrosomal integrity, cervical mucus penetration test and hypo‐osmotic sperm swelling test (HOST). In frozen–thawed semen, the percentage of sperm motility, viability and acrosomal integrity was significantly (p < 0.05) higher in the treatment group compared to the control group. The incorporation of non‐luteal oviductal proteins in the extender increased the ability of sperm to penetrate cervical mucus both after equilibration and the freeze‐thaw process. Similarly, the proportion of sperm with intact plasma membrane, as revealed by HOST values, was also significantly (p < 0.05) higher in the treatment group (32.6%) than the control group (27%) in frozen–thawed semen. It was inferred that incorporation of non‐luteal whole oviductal fluid proteins improved the sperm quality in frozen–thawed semen in Murrah buffaloes.     

Effects of Rapid Cooling Prior to Freezing on the Quality of Canine Cryopreserved Spermatozoa

The aim of this study was to evaluate the effects of rapid cooling prior to freezing on frozen-thawed canine sperm quality. In experiment 1, centrifuged ejaculates from 6 dogs were pooled, split into 4 aliquots and cryopreserved by the Uppsala procedure using different cooling rates (control, cooling speed 18 C/90 min and average cooling rate 0.2 C/min; rapid, cooling speed 18 C/8 min and average cooling rate 2.25 C/min) in combination with 2 glycerol addition protocols (fractionated or unfractionated). In experiment 2, centrifuged ejaculates from 4 dogs were processed individually using the same cooling rates described in experiment 1 in combination with an unfractionated glycerol addition protocol. Each of the experiments was replicated 5 times. Sperm quality was evaluated after 30 and 150 min of post-thawing incubation at 38 C. Total motility (TM), progressive motility (PM) and quality of movement parameters were assessed using a computerized system, and sperm viability (spermatozoa with intact plasma and acrosome membranes) was assessed using flow cytometry (H-42/PI/FITC-PNA). Values for TM, PM, viable spermatozoa and the quality of movement parameters after thawing were not significantly affected by the cooling rate. The interaction between the cooling rate and the added glycerol protocol was not significant. There were significant differences among the males (P<0.01) in the sperm quality parameters evaluated after thawing. The interaction between the males and the cooling rate was not significant. In conclusion, canine spermatozoa can be cryopreserved using the Uppsala method at an average cooling rate of 2.25 C/min prior to freezing together with addition of fractionated or unfractionated glycerol.     

Tris-Lecithin Extender Supplemented With Antioxidant Catalase for Chilling of Canine Semen

The aims were to evaluate the suitability of a non-commercial TRIS-lecithin (LC) extender and the effect of different concentrations of catalase (CAT) on motility, capacitation status (Chlortetracycline-assay) and zona pellucida (ZP) binding capacity of canine spermatozoa stored at +5°C for 4 days. The sperm-rich fractions of the ejaculates of four stud dogs were divided into four aliquots. After centrifugation, sperm pellets were diluted (200 × 10⁶ sperm/ml) in TRIS buffer, citric acid, fructose, antibiotics, supplemented with 20% egg yolk (TRIS-EY) or 0.04% soybean lecithin (TRIS-LC) with CAT (150 or 450 UI/ml) or without CAT, and then preserved at 5°C for 4 days. The results showed that LC is a valid alternative to EY for chilling canine semen, as similar rates of motility, number of uncapacitated spermatozoa and of spermatozoa binding the oocyte ZP were obtained in semen chilled in TRIS-LC or TRIS-EY. Different concentrations of CAT in a TRIS-LC based extender did not improve the quality of semen after chilling. However, a concentration of 150 UI/ml CAT resulted in an increased number of spermatozoa bound to the oocyte ZP, after 4 days of chilling when compared to semen chilled with TRIS-EY and TRIS-LC. In conclusion, an animal protein-free extender with soybean LC, as a replacement of EY, is suitable for 4 days chilling of canine spermatozoa, but the addition of CAT does not improve general semen quality except for a slight effect on sperm-ZP binding.     

Effect of linoleic acid albumin in a dilution solution and long-term equilibration for freezing of bovine spermatozoa with poor freezability

Despite normal eucrasia, mating desire and semen quality, sire bulls sometimes have spermatozoa with poor freezing tolerance. This study assessed effects of the addition of linoleic acid albumin (LAA) and long-term (LT) equilibrium to frozen semen on their sperm freezing tolerance. Immediately after collection using an artificial vagina and a breeding mount, semen was diluted with yolk citrate buffer; then, it was cooled slowly to 4°C during more than 5 h. Equilibrium treatment at 4°C was applied using the same extender supplemented with glycerol. Semen of bull A, with low sperm freezing tolerance, was treated with 1 mg/ml of LAA added to the first extender. The equilibrium treatment at 4°C was prolonged to 30 h. Significantly higher motility rates were obtained for the LT + LAA-treated sperm before and after freezing-thawing. However, for semen of bulls B and C with normal sperm freezing tolerance, the LT + LAA treatment barely exhibited a small effect on the motility rate. Almost no difference was found among bulls A, B and C in the motility rates of LT + LAA-treated sperm after freezing-thawing. No difference of fertility was apparent on LT + LAA-treated frozen sperm in comparison with normal sperm in embryonic collection and in vitro fertilization. It was not an aberration of fertility in vivo or in vitro. In addition, the conception rate of artificial insemination did not have a difference, and a normal calf was obtained. Results show that addition of LAA to an extender for frozen bovine spermatozoa and 30 h of low-temperature equilibrium might improve the motility of freezing-thawing spermatozoa with poor freezability. Sperm exhibited normal fertilization capability and ontogenic capability.     

Effects of final dilution rate, sperm concentration and times for cooling and glycerol equilibration on post-thaw characteristics of canine spermatozoa

This study re-evaluated a protocol for cryopreservation of canine semen. Semen from 4 beagle dogs was pooled, concentrated by centrifugation and adjusted to increasing sperm concentrations by adding back seminal plasma. The prepared or original semen was diluted with an extender (Egg yolk-Tris-citrate-glucose) and cooled to 4 degrees C (cooling), followed by a second dilution with the same extender including glycerol, equilibrated at 4 degrees C (equilibration), then stored in liquid nitrogen. The semen was diluted for frozen samples having a fixed sperm concentration with increasing dilution rates or for those having the reverse combinations. Various dilution rates of 2.5-10 folds or sperm concentrations of 0.25-2.5 x 10(8)/ml had no significant effect on post-thaw sperm characteristics. When cooling was done for different times (0-26 hr) with glycerol equilibration for 1 hr, post-thaw characteristics were better at 2 and 3 hr of cooling, while various times for equilibration (0-4 hr) with cooling for 3 hr had no effect. These results suggest that different dilution rates and sperm concentrations within the ranges tested may not affect the post-thaw sperm characterisitics and that sufficient time for cooling may be essential but a specific equilibration time may not necessarily be required.     

Rotation of Boar Semen Doses During Storage Affects Sperm Quality

It is common practice to rotate boar semen doses during storage for prevention of sperm sedimentation. In this study, the effect of rotation of boar semen doses during storage on sperm quality was investigated. Manual turning twice daily and automatic rotation five times per hour resulted in the following effects: alkalinization of the BTS‐extender, loss of membrane integrity at day 3, and loss of motility and changes in sperm kinematics during a thermoresistance test at day 5. Using a pH‐stabilized variant of BTS extender, sperm motility and velocity decreased in continuously rotated samples, whereas membrane integrity and mitochondrial activity remain unaffected. It is concluded that rotation of semen samples adversely affects sperm quality and, therefore, should no longer be recommended for AI practice.     

Enhanced freezability of goat spermatozoa collected into tubes containing extender supplemented with bovine serum albumin (BSA)

The purpose of this study was to investigate the effects of semen collection into tubes containing extender supplemented with BSA on the cryosurvival of goat spermatozoa. Semen was collected from two goats into empty tubes or tubes containing 10 ml extender supplemented with 0, 0.1, 1, or 5% BSA, and the washed spermatozoa were frozen as pellets in egg yolk-trehalose extender with the addition of 0.04% SDS and 4% glycerol. Sperm motion parameters were evaluated after post-thawing and during a thermal resistance test. The acrosome status of frozen-thawed spermatozoa was also observed using FITC-PNA staining. In frozen semen that was collected into tubes containing extender supplemented with 5% BSA, the post-thawed spermatozoa exhibited a significant improvement in motion parameters and maintained high motility throughout incubation and acrosome integrity, as compared with semen collected into tubes containing extender supplemented with lower concentrations of BSA. In conclusion, semen collection into tubes with a large volume of extender containing high concentrations of BSA dramatically improves the motility and acrosome integrity of frozen-thawed spermatozoa. This suggests that the in vitro functional freezability of spermatozoa is abruptly modified by reducing contact with seminal plasma and by flash contact with BSA at ejaculation.     

Influence of Ascorbic Acid and Glutathione Antioxidants on Frozen-Thawed Canine Semen

Poor sperm viability post-thaw has resulted in constant research into methods of cryopreservation of canine semen. One factor that may be involved in poor viability is sperm oxidative stress caused by excessive formation of reactive oxygen species. The present study was performed in order to evaluate the effect of different concentrations of ascorbic acid (AA) and glutathione (Glu) added to an extender for the freeze-thawing of dog sperm. Semen from five mature dogs was collected and frozen in two studies. Prior to and after freezing, sperm motility, morphology and membrane status were examined. In addition, sperm motility was examined up to 120 min after thawing to evaluate thermo-resistance. In study I, semen was collected twice from each dog. On both occasions, semen was divided into three aliquots: control, Glu 1 m m and Glu 5 m m . In study II, semen was collected twice and divided into three aliquots; control, AA 50 μ m and AA 250 μ m . Initial sperm motility was significantly higher in sperm diluted with AA 50 μ m ; sperm longevity, however, measured by a thermal-resistance test (TRT), was higher for Glu treatments. Higher concentration of Glu produced significant improvement in TRT and membrane status, whereas higher concentration of AA had a negative impact in sperm longevity. Antioxidant supplementation to semen freezing extenders improved semen quality post-thaw. Moreover, Glu had the most beneficial effect when supplemented at 5 m m .     

Preservation capability of horse semen by the use of two diluents and preservation temperatures

The effect of a skim milk extender and a glycine-containing extender on sperm motility and acrosome morphology of stallion semen was examined. There was no difference concerning acrosome morphology. After 24 hours of preservation motility of the ejaculates diluted with glycine extender was significantly superior to those handled with skim milk extender. Storage at 5 degrees C in all cases gave better results than storage at room temperature. Skim milk extender is an appropriate diluent when the semen is used for al on the day of its collection, whereas the glycine-containing extender offers the possibility to maintain sperm viability beyond 24 hours up to 72 hours.     

Effect of N‐acetyl‐L‐cysteine Supplementation in Semen Extenders on Semen Quality and Reactive Oxygen Species of Chilled Canine Spermatozoa

The objective of this study was to evaluate the quality of chilled dog semen processed with extenders containing various concentrations of N‐acetyl‐L‐cysteine (NAC). Ejaculates from five dogs were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF‐movement), viability, acrosomal integrity and by the hypo‐osmotic swelling test (HOST). In addition, superoxide anion (O₂^‐?) production, hydroxyl radicals (OH^?) and total reactive oxygen species (tROS) were determined. The pool was divided into five aliquots, which were diluted to a final concentration of 66.66 × 10⁶ spermatozoa/ml with Tris‐glucose‐egg yolk extender containing one of the following concentrations of NAC (0, 0.5, 1, 2.5 or 5 mm ). The semen aliquots were chilled and preserved at 4°C. Semen quality was evaluated after rewarming at 72 h. Sperm motility was significantly higher with the 0.5 mm concentration compared with the control group (p = 0.001). Rapid steady forward movement was higher with the 0.5 and 1 mm concentrations compared with the control and 5 mm group (p < 0.001). Viability and HOST percentages were not significantly altered. Compared with the control, the 5 mm concentration showed significantly reduced percentages of spermatozoa with normal acrosomes (p = 0.049). None of the ROS values at 72 h were significantly affected by the presence of NAC in semen extenders, although all NAC concentrations showed lower O₂^‐? and OH^? values compared with the control. Only the concentrations of 1 and 5 mm inhibited the significant increase of tROS values after 72 h, compared with the fresh semen value. In conclusion, NAC supplementation of semen extenders is beneficial to semen motility of canine spermatozoa during chilling with the 0.5 mm concentration being the most effective, although no significant ROS inhibition was observed at 72 h.     

Effect of chilling temperature on the long-term survival of rabbit spermatozoa held either in a tris-based or a jellified extender

As the preservation of the fertilizing capacity of rabbit spermatozoa for several days after semen collection remains a major target for the artificial insemination programs of rabbit breeding, a study was conducted to compare the efficacy of 5 or 15°C as holding temperature in lengthening the preservability of rabbit semen quality during 192 h of storage both in a solid (Cunigel) and a liquid (Tris-Citric acid-Glucose; TCG) extender. Six pooled semen samples (two ejaculates/male; two-three males/pool) were taken and made four aliquots: two aliquots were tenfold diluted with the TCG extender, whereas the other two were tenfold diluted with the Cunigel extender. One aliquot per diluent was stored at 5°C and the second one at 15°C. Sperm motility (light microscope), viability (SyBr-PI staining), plasma membrane functional integrity (Hypo-osmotic swelling test) and acrosome integrity (PSA-FITC staining) were recorded at 0, 48, 120 and 192 h of storage. In liquid-stored spermatozoa, mass motility and viability were significantly higher (p ≤ 0.05) in samples stored at 5°C than at 15°C at all the storage times; at 5°C resulted also higher (p ≤ 0.05) the percentages of both forward motility at 48 h and sperm functional integrity at 120 and 192 h of storage, whereas chilling temperature did not affect acrosome integrity. With the Cunigel extender, all the semen qualitative parameters were significantly higher in sample stored at 5 than 15°C over storage time (p ≤ 0.05); only acrosome integrity at 192 h was not different according to the chilling temperatures. In conclusion, 5°C were better than 15°C for the long-term storage of rabbit semen both in the TCG and Cunigel extender.     

红腹锦鸡精液冷冻稀释液配方的筛选

为加强濒危珍稀动物种质资源的保护,开展了濒危珍稀禽类—红腹锦鸡的人工授精研究。2005年5月26日~6月8日,对8只红腹锦鸡用手按摩其背、尾部采精12次。初测其精液品质,结果表明,采精量平均(0.114±0.016)mL(0.01~0.2 mL),每毫升精液中精子平均3.2亿个(3.0~3.3亿),活力9级以上,pH值6.5,偏酸性,淡乳白色(半透明似冲熟的藕粉),微腥。鲜精分别加11%蔗糖—卵黄稀释液(3号液);11%蔗糖—0.1%柠檬酸三钠—卵黄稀释液(5号液);5%葡萄糖—卵黄稀释液(8号液)。精子活力达8~9级。用3%柠檬酸三钠—卵黄稀释液(2号液)稀释,精子活力2级。冷冻时,用11%蔗糖溶液100 mL中加16 mL鲜卵黄,加5 mL甘油,配制的3号冷冻稀释液稀释,解冻后,精子活力达4级。优于试验中选拟的其它配方(加入5 mL甘油的5号冷冻液,解冻后精子活力为3级;加入5 mL甘油的8号冷冻液,解冻后未见活的精子)。如果冷冻稀释液中甘油改为6 mL,则解冻后精子全部死亡。     

Effects of Egg Yolk and Cooling Rate on the Survival of Refrigerated Red Deer (Cervus elaphus hispanicus) Epididymal Spermatozoa

Egg yolk is a common component to sperm refrigeration for most of the deer species, the role of which is to protect sperm membranes against cold shock. In addition, there have been many studies of conservation of ejaculated semen from stags, but few have been reported for epididymal spermatozoa. This work was designed to investigate the combined effects of cooling rates (slow: 0.23 degrees C/min vs rapid: 4.2 degrees C/min) from room temperature to 5 degrees C, and egg-yolk concentration (0, 5 or 20%) in the extender on the survival of Iberian red deer epididymal spermatozoa refrigerated at 5 degrees C. Heterospermic sperm samples were diluted to a final sperm concentration approximately 400x10(6) sperm/ml with a Tris-citrate-fructose (TCF)-egg-yolk diluent. Sperm quality was in vitro judged by microscopic assessments of individual sperm motility [sperm motility index (SMI)], and of plasma membrane (hypo-osmotic swelling test) and acrosome (NAR) integrities. Our results first showed that the presence of egg yolk in the extender significantly improves (p=0.01) the viability and sperm motility after sperm dilution. In addition, acrosome and plasma membrane integrities post-refrigeration did not differ significantly between cooling procedures; however, the SMI differed significantly between cooling procedures (slow: 46.6% vs rapid: 50.0%; p=0.01). Our results also showed that sperm quality was significantly (p<0.01) affected by the combined effects of egg-yolk concentration and cooling procedure, being rapid cooling with 20% of egg yolk the most suitable combination for epididymal sperm refrigeration. In conclusion, egg-yolk improved red deer epididymal spermatozoa characteristics after dilution. Rapid cooling protocol using TCF with 20% egg-yolk significantly improved sperm motility of red deer epididymal spermatozoa after cooling.     

Antioxidant effects of supplementation of 3,4-dihydroxyphenyl glycol on sperm parameters and oxidative markers following cryopreservation in canine semen

Supplementing the extender with antioxidants with low molecular weight can enhance the quality of the post-thaw sperm during the freezing process. This study was aimed at determining the impacts of 3,4-dihydroxyphenyl glycol (DHPG) on the spermatozoa of the canine undergoing freeze-thawing process. In this study, 24 ejaculates were obtained from three mixed-breed dogs and were diluted in a Tris-based extender. The diluted semen was divided into aliquots for supplementation of 10, 30, 50 and 70 µg/ml of DHPG, control (without antioxidant) and control sham (DMSO). After being extended, the semen was equilibrated at a temperature of 4°C and then transferred to the straws and kept 4 cm above the liquid nitrogen for 20 min and was finally immersed in the liquid nitrogen. They were cryopreserved for seven days; then, sperm parameters including sperm motility evaluation, motility characteristics, viability, DNA and plasma membrane integrity, total antioxidant capacity (TAC), reduced glutathione content (GSH), antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT] and glutathione peroxidase [GPx]) activity malondialdehyde (MDA) levels were evaluated. This study showed that spermatozoa cryopreservation with 50, 30 and 70 µg/ml of DHPG concentrations had better progressive motility, Curvilinear Velocity, Linearity, viability, intact plasma membrane and the levels of TAC, GPx and GSH were higher than the control group. The 50, 30 and 70 µg/ml of DHPG concentrations led to the significant decrease of DNA damage compared to the control group. Total motility, average path velocity, straight-line velocity and CAT activity were significantly improved in 30 and 50 µg/ml of DHPG concentrations, compared to the control group. Also, the 50 and 30 µg/ml of DHPG concentrations, decreased MDA levels compared to the other groups, significantly. In conclusion, our study showed that the addition 50 µg/ml of DHPG to the canine semen extender improved the semen characteristics and oxidative markers in the cryopreservation process.     

Influence of Cool Storage before Freezing on the Quality of Frozen–Thawed Semen Samples in Dogs

The aim of this study was to determinate the semen quality of frozen–thawed samples that were chilled for up to 2 days before freezing. The ejaculates (n = 18) from six dogs were collected, pooled and divided into six aliquots. The first aliquot (C, control) was frozen in liquid nitrogen using a conventional protocol to reach a final concentration of 100 × 10⁶ spermatozoa/ml, 20% egg yolk and 5% glycerol. The remaining five aliquots were diluted with a chilled extender (Tris‐glucose and 20% egg yolk) and cooled at 4°C as follows: R1, the semen was cooled for 1 h; R6, the semen was cooled for 6 h; R12, the semen was cooled for 12 h; R24, the semen was cooled for 24 h and R48, the semen was cooled for 48 h. After the chilling period, a second extender was added (Tris‐glucose, 20% egg yolk, 10% glycerol and Equex at 1%) to reach a final composition similar to aliquot C, and then, the semen samples (R1, R6, R12, R24 and R48) were frozen in liquid nitrogen. The post‐thaw sperm quality was assessed in 30 straws from each experimental group. After freezing–thawing, the total sperm motility (approximately 60–70%) in the semen chilled for up to 48 h did not show any differences from the samples frozen by the conventional cryopreservation method (63.2%). No significant differences were detected in the percentages of abnormal sperm cells among the fresh semen, the control group and the frozen samples after the different cooling times. Finally, the post‐thaw percentages of damaged acrosomes showed a very uniform distribution, with mean values ranging between 7% and 10.5%. The results clearly demonstrated that cooling the semen up to 48 h before freezing did not produce a decrease in the semen quality when was compared with semen frozen by a traditional procedure.     

Effect of washing on motility and acrosome morphology of frozen-thawed goat spermatozoa

Semen from 4 bucks was collected using an artificial vagina and was pooled and divided into 6 aliquots. Three aliquots were washed twice, 15 minutes each time, with Ringer's solution, and the fluid was removed by centrifugation at 950 X g between washes. All 6 aliquots (3 washed and 3 unwashed) were extended with skim milk-glycerol, lactose-egg yolk-glycerol, or tris (hydroxymethyl) aminomethane-citric acid-egg yolk-glycerol and were frozen in straws to -196 C. The semen was then thawed and kept at 37 C for 8 hours. Percentage of sperm motility was estimated, and the percentage of normal acrosomes (NA) was determined at 0, 2, 4, 6, and 8 hours after thawing. The experiment was repeated 7 times. The data indicated a significant positive effect (P = 0.0009) of washing on motility, but no effect (P = 0.5347) of extender. There was also a significantly higher percentage of NA in washed semen (P less than 0.0001). Sperm extended in tris aminomethane-citric acid-egg yolk-glycerol had more NA than those extended in lactose-egg yolk-glycerol. Sperm motility and acrosome morphology were depressed also in the presence of seminal plasma for the milk extender, which did not contain egg yolk. Removal of seminal plasma from goat semen was beneficial in preserving the integrity of the spermatozoa after freezing, regardless of the extender used.     

Successful Cryopreservation of Domestic Cat (Felis catus) Epididymal Sperm after Slow Equilibration to 15 or 10°C

To support conservation strategies in wild species, simple but highly reproducible procedures of sperm cryopreservation are required for an application under field conditions. We used epididymal sperm of the domestic cat to optimize a sperm freezing procedure for felid species, particularly questioning the demand for sperm cooling to 4°C. We equilibrated sperm during slow cooling to only 15 or 10°C in a Tes–Tris–fructose extender with final concentrations of 4.7% (v/v) glycerol and 10% (v/v) of the water‐soluble fraction of hen's egg yolk (low‐density lipoproteins). Subsequently, sperm were frozen over liquid nitrogen. Total and progressive motility (mean ± SD) after thawing was 60.7 ± 8.6% and 53.9 ± 9.6% in samples cooled to 15°C or 61.6 ± 9.5% and 55.3 ± 9.9% in samples cooled to 10°C. Therefore, a one‐step addition of glycerol to sperm at room temperature together with the freezing extender, the use of cryovials (loaded with diluted sperm aliquots of 300 μl), an equilibration period of 40 min comprising slow cooling to 15°C at a rate of approximately ?0.14 K/min before rapid freezing over liquid nitrogen, yielded satisfying results. Cooling, freezing and thawing rates were exactly characterized as a prerequisite for further optimization and to provide a repeatable protocol to other practitioners.