Electroejaculation and semen cryopreservation of free-ranging Japanese black bears (Ursus thibetanus japonicus)

The Japanese black bear (Ursus thibetanus japonicus) is endangered for extinction in some areas of Japan, and semen collection and cryopreservation are an important means to preserve genetic resources. The aim of this study was to characterize and cryopreserve semen of free-ranging Japanese black bears. Semen was collected by electroejaculation procedure from 4 free-ranging Japanese black bears at the capture point in the field. Ejaculates containing motile sperm were recovered from all of the animals and ejaculate volume, total sperm count, % motility (percentage of motile spermatozoa), % viability (percentage of spermatozoa that excluded eosin) and % abnormal morphology (range (mean)) were 0.65-2.20 (1.51) ml, 99-1082 (490) x 10(6), 5-100 (31), 42-97 (66) and 20-87 (53), respectively. Three of the 4 ejaculates were diluted with an egg yolk-TRIS-citrate-glucose extender and cryopreserved in liquid nitrogen. Motile spermatozoa were observed after freezing and thawing in all cases. This study showed that electroejaculation would be a useful method for collecting semen from free-ranging Japanese black bears and that at least motile spermatozoa would be obtained by freezing the thus collected electroejaculates.     

Laparoscopic intra-uterine insemination of fallow deer with frozen-thawed or fresh semen after synchronisation with CIDR devices

This study investigated the efficacy of fixed-time laparoscopic intra-uterine insemination of farmed fallow deer (Dama dama) with frozen-thawed or fresh semen. In the trials with frozen-thawed semen, a total of 547 mature non-lactating does across five New Zealand farms were used. For oestrous synchronisation and artificial insemination, a standard control regimen was applied to at least 30% of the does on each farm, involving the insertion of single CIDR type-G devices intravaginally for 14 days, deposition of 50 x 10(6) frozen-thawed spermatozoa at 65 hours after withdrawal of the CIDR device and the continuous presence of vasectomised bucks from the insertion of the CIDR device until 10 days after insemination. Various aspects of this protocol were changed for the remaining does on each farm, including inseminations at 60 or 70 hours, the absence of vasectomised bucks, insemination with 25 x 10(6) or 10 x 10(6) spermatozoa, synchronisation with CIDR type-S devices and synchronisation with prostaglandin. The conception rate, based on rectal ultrasonography at 45 days after insemination, was 67% across all treatments (n=547). Corrected conception rates (+/-s.e.), calculated following between-farm adjustments, were 67+/- 3% for the control regimen, 67+/- 9% and 73 +/- 8% for inseminations at 60 and 70 hours respectively, 61 +/- 9% for absence of bucks, 80 +/- 8% and 74 +/- 9% for inseminations with 25 x 10(6) and 10 x 10(6) spermatozoa respectively, 62 +/- 10% for CIDR type-S device synchronisation, and 49 +/- 10% for prostaglandin synchronisation. Despite apparent differences, none of the treatments resulted in adjusted conception rates that were significantly different from the control regimen (P>0.01). In the trials with fresh semen, 216 does in the USA were inseminated at 69-71 hours after withdrawal of the CIDR device using either cryopreserved semen from New Zealand (n=158; 25 x 10(6) spermatozoa per inseminate) or fresh semen (n=58; 7.5 x10(6) to 20 x 10(6) spermatozoa per inseminate) collected less than 10 hours earlier. The overall conception rates were 77% and 81% respectively, with no significant differences between semen type (frozen v. fresh) or fresh spermatozoa number per inseminate (P>0.01). A further 102 does in New Zealand similarly received fresh semen from 3/4 Mesopotamian buck. Doses of 10 x 10(6) (n=35), 5 x 10(6) (n=32) or 2.5 x 10(6) (n=35) spermatozoa per inseminate were delivered at 69-71 hours after withdrawal of the CIDR device. The conception rates were 77%, 66% and 51% respectively, reflecting a dose effect (P<0.05). However, 1/4 Mesopotamian does in the group (n=19) exhibited higher conception rates (95% overall) irrespective of semen dose, possibly indicating a semen/recipient genotype interaction. It is concluded that laparoscopic intra-uterine insemination of fallow deer with frozen-thawed or fresh semen at fixed intervals after removal of a CIDR device can give acceptable conception rates under a range of on-farm management options and semen doses.     

Microencapsulation of bovine spermatozoa

Two experiments were conducted to examine the efficacy of microencapsulation of bovine spermatozoa for use in artificial insemination. In Exp. 1, sperm were encapsulated at three different concentrations (45, 90 and 180 X 10(6) sperm/ml) in either .75- or 1.5-mm (diameter) microcapsules and incubated in vitro for 24 h at 37 C. Unencapsulated samples of each concentration served as controls. Capsule contents were evaluated for percentage of sperm motility and intact acrosomes at 2, 12 and 24 h of incubation. Capsule fragility was evaluated after 24 h incubation. Viability of spermatozoa was not influenced by sperm concentration or capsule size, and compared with controls, cellular injury after encapsulation was not apparent. Fragility of capsules was unaffected by capsule size; however, as the sperm concentration increased, integrity of the capsules decreased (P less than .05). In Exp. 2, using frozen-thawed semen, the effect of egg yolk content, presence of glycerol and viability of spermatozoa on the success of microencapsulation was measured. The extender was 2.9% sodium citrate with glycerol (7% v/v) and either 0, 5, 10 or 15% egg yolk (v/v). Uniformity of capsules in size and shape was evaluated subjectively. Capsule integrity and uniformity were unaffected by glycerol, sperm viability or egg yolk level up to 10% v/v; however, encapsulation of spermatozoa in 15%-yolk buffer increased the heterogeneity in capsule size and shape. Viability of encapsulated spermatozoa was maximal for extenders containing 10 or 15% yolk v/v. Reduced viability for the 5% yolk extender was due to pre-encapsulation injury associated with freezing. Microencapsulation procedures are compatible with sperm viability and can be adapted to an acceptable extender system used in artificial insemination.     

Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid

The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 10⁶/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 10⁶ ± 21, 59 × 10⁶ ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.     

Trehalose in glycerol-free freezing extender enhances post-thaw survival of boar spermatozoa

Cryopreservation of boar semen is still considered suboptimal due to lower fertility as compared with fresh samples when glycerol, a permeating cryoprotectant, is used. Trehalose is a non-permeable cryoprotectant and nonreducing disaccharide known to stabilize proteins and biologic membranes. The aim of this study was to evaluate the cryosurvival and in vitro penetrability of boar spermatozoa when glycerol was replaced with trehalose in a freezing extender. Ejaculated Berkshire semen samples were diluted in egg yolk-based freezing extender containing glycerol (100 mM) or trehalose (0, 50, 100, 150, 200 and 250 mM) and cryopreserved using a straw freezing procedure. Thawed samples were analyzed for motility, viability, mitochondrial membrane potential (MMP), and acrosome integrity. In experiment 2, penetrability of spermatozoa cryopreserved with 100 mM glycerol or trehalose was examined. Replacement of cryoprotectant glycerol (100 mM) with trehalose had no effect on sperm viability, but replacing it with 100 mM trehalose improved motility, MMP and acrosome integrity significantly. Sperm motility and MMP were considerably higher in 100 mM trehalose, whereas the acrosome integrity was substantially higher in 100–250 mM trehalose. The in vitro penetration rate was also significantly higher in spermatozoa cryopreserved with trehalose (61.3%) than in those cryopreserved with glycerol (43.6%). In conclusion, 100 mM non-permeable trehalose can be used to replace glycerol, a permeating cryoprotectant, for maintenance of better post-thaw quality of boar spermatozoa.     

Birth of puppies after intrauterine and intratubal insemination with frozen-thawed canine semen

The present study was performed to assess the fertility of frozen-thawed dog semen prepared by freezing with 6% glycerol and thawing at 70℃ for 8 sec, and to evaluate the least number of post-thaw spermatozoa necessary to achieve pregnancy by intrauterine or intratubal artificial insemination. It was found that the pregnancy rate of intrauterine artificial insemination was 100% using 6% glycerol buffer and thawing at 70℃ for 8 sec with 5 × 10⁷ spermatozoa. Even though the pregnancy rate (80%) and the whelping rate (24.5%) in the 5 × 10⁶ spermatozoa inseminated group were lower than those of the 5 × 10⁷ spermatozoa group, conception was confirmed with 5 × 10⁶ spermatozoa. Although the pregnancy rate of intratubal insemination was low (20%) with 4 × 10⁶ spermatozoa, this study is the first report to show the pregnancy rate of intratubal insemination with frozen-thawed ejaculated canine semen. In order to improve the pregnancy rate with intratubal insemination of canine spermatozoa, it is necessary to investigate the optimal insemination site of the uterine tube, the appropriate number of sperm, and the direct effect of buffer on oocytes.     

Reduction of Oxidative Stress in Bovine Spermatozoa During Flow Cytometric Sorting

The goal of the study was to investigate the effect of antioxidant supplementation on the quality of frozen-thawed flow cytometrically sorted bull spermatozoa. Twelve ejaculates from two Holstein Friesian bulls were sorted according to the Beltsville Sperm Sexing Technology. Each ejaculate was divided into three parts and processed as (i) unsorted controls, (ii) according to a standard sorting protocol and (iii) in the presence of different antioxidants (S-AO). Cooling and freezing of the samples were performed in the same way for all three groups, except that antioxidants were added to the TRIS-egg-yolk freezing extender for those semen samples that were already sorted in the presence of antioxidants. The semen quality in frozen-thawed samples was determined by morphology analysis immediately after thawing, motility estimation in a thermo-resistance test after 0, 6, 12 and 24 h incubation at 37 degrees C and Fluorescein isothiocyanate conjugated PNA/propidium iodide (FITC-PNA/PI) staining after 0, 12 and 24 h of incubation at 37 degrees C. There was a significantly higher (p < 0.05) percentage of motile spermatozoa in S-AO samples in comparison to unsorted frozen-thawed control at 0, 6 and 24 h after thawing and compared with normally sorted samples at all times after thawing. The percentage of damaged acrosomes was significantly lower (p < 0.05) in S-AO samples than in the unsorted controls (20.8 +/- 6.9% vs 30.3 +/- 12.0%). The percentage of morphologically abnormal spermatozoa in this group was significantly lower (p < 0.05) than in the unsorted controls and normally sorted samples (25.8 +/- 5.2%, 36.0 +/- 12.5% and 35.1 +/- 7.4%, respectively). Analysis of frozen-thawed spermatozoa with FITC/PI revealed no significant difference in membrane integrity at 0 and 12 h after sorting, but after 24 h of incubation the S-AO samples had a significantly higher (p < 0.001) percentage of spermatozoa with intact membranes in comparison to unsorted controls and normally sorted semen (40.7 +/- 6.3%, 7.8 +/- 4.7% and 7.4 +/- 4.6%, respectively). The percentage of acrosome-reacted spermatozoa was significantly lower (p < 0.05) in the S-AO samples than in the unsorted controls (14.1 +/- 7.5%, 23.4 +/- 5.4% and 28.8 +/- 6.3% vs 25.9 +/- 14.4%, 38.5 +/- 16.7% and 79.8 +/- 4.1%, for 0, 12 and 24 h after thawing, respectively) and in comparison to normally sorted semen 24 h after thawing (67.3 +/- 10.0%). This study demonstrates the highly protective effects of antioxidants on the quality of flow cytometrically sorted frozen-thawed bull spermatozoa.     

Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen-thawed spermatozoa onto the uterotubal junction

REASONS FOR PERFORMING STUDY: To compensate for the wide variation in the freezability of stallion spermatozoa, it has become common veterinary practice to carry out repeated ultrasonography of the ovaries of oestrous mares in order to be able to inseminate them within 6-12 h of ovulation with a minimum of 300-500 x 10(6) frozen-thawed spermatozoa. Furthermore, in order to achieve satisfactory fertility, this requirement for relatively high numbers of spermatozoa currently limits our ability to exploit recently available artificial breeding technologies, such as sex-sorted semen, for which only 5-20 x 10(6) spermatozoa are available for insemination. OBJECTIVES: This study was designed to evaluate and compare the efficacy of hysteroscopic vs. conventional insemination when low numbers of spermatozoa are used at a single fixed time after administration of an ovulation-inducing agent. METHODS: In the present study, pregnancy rates were compared in 86 mares inseminated once only with low numbers of frozen-thawed spermatozoa (3-14 x 10(6)) at 32 h after treatment with human chorionic gonadotrophin (hCG), either conventionally into the body of the uterus or hysteroscopically by depositing a small volume of the inseminate directly onto the uterotubal papilla ipsilateral to the ovary containing the pre-ovulatory follicle. RESULTS: Pregnancy rates were similarly high in mares inseminated conventionally or hysteroscopically with 14 x 10(6) motile frozen-thawed spermatozoa (67% vs. 64%). However, when the insemination dose was reduced to 3 x 10(6) spermatozoa, the pregnancy rate was significantly higher in the mares inseminated hysteroscopically onto the uterotubal junction compared to those inseminated into the uterine body (47 vs. 15%, P < 0.05). CONCLUSIONS: When inseminating mares with <10 x 10(6) frozen-thawed stallion spermatozoa, hysteroscopic uterotubal junction deposition of the inseminate is the preferred method. POTENTIAL CLINICAL RELEVANCE: Satisfactory pregnancy rates are achievable after insemination of mares with frozen-thawed semen from fertile stallions 32 h after administration of human chorionic gonadotrophin (Chorulon). Furthermore, these results were obtained when mares were inseminated with 14 x 10(6) progressively motile frozen-thawed spermatozoa from 2 stallions of proven fertility.     

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.     

Effect of Catalase and Superoxide Dismutase on Motility, Viability and Acrosomal Integrity of Frozen-Thawed Cat Spermatozoa

The aim of this study was to evaluate the effect of antioxidant catalase (CAT) and superoxide dismutase (SOD) in semen extender on motility, viability and acrosomal integrity of frozen-thawed cat spermatozoa. Semen was collected by using an artificial vagina from five domestic cats (two ejaculates/cat). Spermatozoa were diluted in egg yolk Ttris-fructose citrate solution (EYT-FC) without glycerol and cooled at 4°C for 1 h, then diluted further with EYT-FC with glycerol (7% final concentration) and 400 IU/ml of CAT (treatment 1) or SOD (treatment 2) or without antioxidants (control). Before freezing using a styrofoam box, diluted spermatozoa filled in 0.25-ml straws were equilibrated for 1 h at 4°C. After thawing, spermatozoa were assessed for motility, viability and acrosomal integrity. Cryopreservation significantly impaired sperm motility, viability and acrosomal integrity (p   <   0.05). However, motility, viability and acrosomal integrity of frozen-thawed cat spermatozoa in the EYT-FC with CAT, SOD and without the antioxidants were not significantly different. The average percentages of spermatozoa motility after thawing compared between control, treatment 1 and treatment 2 group were 43.5 ± 3.2, 42 ± 4.1 and 38 ± 4.5; for viability: 44.8 ± 3.5, 50.6 ± 5.7 and 47.1 ± 4.1 and for acrosomal integrity: 45 ± 3.5, 44.9 ± 3.4 and 44.4 ± 3.3, respectively. In conclusion, adding CAT and SOD to EYT-FC did not improve motility, viability and acrosomal integrity in cryopreserved cat spermatozoa.     

Effect of platelet activating factor (PAF) supplementation in semen extender on viability and ATP content of cryopreserved canine spermatozoa

The aim of this study was to investigate the effect of platelet activating factor (PAF) on the quality characteristics of cryopreserved canine spermatozoa. Cryopreserved semen of 5 mixed-breed dogs was treated with different concentrations of exogenous PAF (1 x 10(-3) M, 1 x 10(-4) M, 1 x 10(-5) M and 1 x 10(-6) M) and examined at different time intervals (0, 30, 60 and 120 min). Cryopreserved semen treated without PAF was used as the control. Sperm quality was evaluated for motility (computer-assisted semen analysis, CASA), mitochondrial function (JC-1/PI assay) and plasma membrane integrity (SYBR-14/PI assay and Hoechst 33258). Also, ATP content of spermatozoa was determined using a bioluminescence assay. Treatment of cryopreserved semen with 1 x 10(-3) M PAF at 120 min of incubation resulted in significantly higher total sperm motility compared with the control. It was observed that PAF-improved total sperm motility was concurrent with enhanced sperm motility patterns after treatment of cryopreserved semen. Treatment of cryopreserved semen with PAF did not improve either sperm mitochondrial function or plasma membrane integrity, as monitored by different fluorescent membrane markers. Furthermore, ATP content of cryopreserved spermatozoa was significantly higher when PAF was used at a concentration of 1 x 10(-3) M compared with the control and other PAF treatments, regardless of the incubation time. The findings of this study indicated that treatment with 1 x 10(-3) M PAF at 120 min of incubation rendered better quality of cryopreserved canine semen, which was associated with improved sperm motility parameters and ATP content. It can be suggested that exogenous PAF addition is beneficial as a supplement for canine semen extender used for cryopreservation.     

Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws

Experiments were conducted to study the effect of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa after cryopreservation in straws. Semen (split ejaculate) in maxi-straws (6 mm o.d.) was frozen using a programmable freezing chamber. Three methods for in vitro sperm evaluation were used: motility (MOT), acrosome integrity (NAR) and flow cytometric analysis of sperm treated with carboxyfluorescein diacetate and propidium iodide to assess sperm plasma membrane integrity (PMI). No interactions were found among the three variables evaluated. Length of prefreeze exposure to glycerol, ranging from .5 min to 75 min, had no effect on post-thaw sperm viability. Exposure of sperm to a glycerol-containing extender medium at 5 degrees C gave improved post-thaw viability over that exposed at 0 degree C (P less than .05). Glycerol at a concentration of 3 or 4% resulted in maximum post-thaw MOT. Acrosome integrity values were greatest for 2 and 3% glycerol, whereas PMI was greatest when glycerol concentration was 4 to 6%. The primary cryoprotective effect of glycerol on boar semen may be extracellular. It is concluded that 3 or 4% glycerol gives maximum viability of frozen-thawed spermatozoa when the present methods are employed.     

Effect of incorporation of additives in tris-based egg yolk extender on buffalo (Bubalus bubalis) sperm tyrosine phosphorylation during cryopreservation

Phosphorylation of tyrosine residues on sperm protein is a known indicator of capacitation and a major intracellular signalling event. There is evidence that sperm cryopreservation promotes tyrosine phosphorylation and is associated with reduced fertility of spermatozoa. Under this study, cryoprotective role of different additives namely taurine, trehalose, catalase and 4-bromophenacyl bromide on buffalo sperm quality was evaluated. Buffalo semen was cryopreserved in tris-based egg yolk extender supplemented with additives like taurine (50 mm) or trehalose (100 mm) or 4-bromophenacyl bromide (200 μm) or catalase (100 U/ml) and used for assessment of levels of tyrosine phosphorylation in frozen-thawed spermatozoa. The results obtained were compared with the level of protein tyrosine phosphorylation of semen cryopreserved in tris-based egg yolk extender without additives. Proteins were extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random and analysed for tyrosine phospho-proteins using SDS-PAGE followed by immunoblotting. Monoclonal anti-phosphotyrosine antibody (Clone pT-154) was used as primary antibody followed by treatment with HRP-conjugated secondary antibody. Signals were detected on X-ray film using chemiluminescence. Nine proteins (p20, p30, p32, p38, p49, p56, p59, p72 and p86) were found to be tyrosine phosphorylated in cryopreserved spermatozoa. Supplementation of additives significantly (p<0.05) reduced the level of protein tyrosine phosphorylation in spermatozoa. Moreover, this study showed improved (p<0.05) post-thaw motility, viability and membrane integrity of spermatozoa on addition of these additives. The results obtained clearly indicate reduced level of capacitation like changes on supplementation of additives in terms of protein tyrosine phosphorylation.     

海藻糖对猪精液冷冻保存效果的影响

在传统的Tris-柠檬酸-葡萄糖稀释液基础上，分别添加25％、50％、75％、100％的海藻糖，研究不同浓度海藻糖对猪精液冷冻后精子质量的影响。结果表明，海藻糖相对于对照TCG稀释液能够显著改善和提高猪精液的冷冻效果，其最佳添加浓度为25％，冷冻-解冻后猪精子活力、活率、线粒体活性、质膜完整性以及顶体完整率均显著提高（P〈0．05），分别达到41．38％、46．34％、44．56％、43．51％和64．09％。海藻糖可以明显抑制精子获能，获能处理前精子获能率仅为3．68％，而获能处理后达到41．82％，有利于促进精子获能。精液稀释液中甘油的适宜添加浓度为2％，海藻糖只有与甘油共同作用，才能在冷冻-解冻过程更加有效地保护精子。猪精子活力、活率、线粒体活性、质膜完整率、顶体完整率等之间存在极显著的正相关关系（P〈0．01），而与获能处理前精子的获能率存在显著的负相关关系（P〈0．05）。     

Comparison of glycerol, lactamide, acetamide and dimethylsulfoxide as cryoprotectants of Japanese white rabbit spermatozoa

The rabbit is considered to be a valuable laboratory animal. We compared glycerol, lactamide, acetamide, and dimethylsulfoxide (DMSO) as cryoprotectants in egg-yolk diluent of ejaculated Japanese white rabbit spermatozoa for improvement of sperm cryopreservation methods. Rabbit semen was frozen with 1.0 M glycerol, lactamide, acetamide, or DMSO in plastic straws. Forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rate of forward progressive motile spermatozoa in lactamide (37.8 +/- 3.0%) was significantly (P<0.05) higher than in glycerol (17.0 +/- 3.3%). In addition, the rates of sperm plasma membrane integrity in lactamide and acetamide (35.9 +/- 3.3% and 30.2 +/- 3.0%, respectively) were significantly (P<0.05) higher than in glycerol (17.0 +/- 2.6%). The results indicate that 1.0 M lactamide and acetamide have higher cryoprotective effects than 1.0 M glycerol for cryopreservation of Japanese white rabbit spermatozoa.     

Cryopreservation of Stallion Spermatozoa with INRA96 and Glycerol

The aim of the present study was to improve success of cryopreservation of stallion spermatozoa. Semen from eleven stallions was collected and frozen in INRA 96 with two different concentrations of glycerol (3.5% and 6.0%) and compared with a control freezing process. The mean post-thaw motility for the eleven stallions of 57.93% (3.5% glycerol) and 66.50% (6.0% glycerol), which was statistically higher (P < 0.05) when compared with the mean post-thaw motility (39.7%) for semen in a control egg-yolk extender (Equipro^® CryoGuard™ Complete, Minitube). The Equipro^® CryoGuard™ Complete is a commercial semen freezing protocol that has been one of the standard processes used in our laboratory for freezing equine spermatozoa. INRA 96 with 6% added glycerol was used in the fertility trial as it provided the highest spermatozoa survival. To evaluate fertility of the frozen semen, eight mares were bred over two cycles with both fresh and frozen semen. The pregnancy rate of mares bred with frozen semen (55.6%) was not statistically different (P > 0.05) from the pregnancy rate of mares bred with fresh semen (55.6%). INRA 96 with 6.0% glycerol improved the survivability of stallion spermatozoa through the cryopreservation process, and subsequent fertility was not different (P > 0.05) from fresh, extended semen.     

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.     

Storage of Fresh Bovine Semen in a Diluent Based on the Ionic Composition of Cauda Epididymal Plasma

For artificial insemination (AI) in cattle, much lower insemination doses can be applied when fresh semen is used instead of frozen-thawed semen. However, a particular disadvantage of fresh semen is its limited shelf life. As bovine spermatozoa can be stored for several weeks in the cauda epididymis without negative effects on their fertilizing capacity, it is an interesting organ to serve as a model in order to prolong the shelf life of fresh semen. First, the storage capacity of a diluent [cauda epididymal plasma (CEP-1)] with the same ionic composition, pH and osmolarity as the bovine CEP was compared with a Tris diluent for extended preservation of fresh ejaculated bovine semen. Secondly, the ionic composition of the CEP-1 diluent was modified (CEP-2) and its storage capacity was compared with this of the CEP-1 and Tris diluent. Finally, the effect of addition of different polyols (sorbitol, glycerol, mannitol) and egg yolk concentrations (5, 10 and 20%) to the CEP-2 diluent was assessed. Sperm quality decreased rapidly in the CEP-1 diluent. The quality and especially progressive motility of spermatozoa stored in the CEP-2 diluent were better those in the CEP-1 and Tris diluent. No significant effects of different sugars or egg yolk concentrations on the quality of fresh bovine semen in the CEP-2 diluent were observed. In conclusion, the CEP-2 diluent with 10% egg yolk and 1 g/l sorbitol may be used for extended preservation of fresh bovine semen at 5 degrees C up to 6 days.     

黄芪多糖对猪精液冷冻保存效果的影响

为了评价黄芪多糖（Astragalus polysaccharides）对外来公猪精液冷冻保存的影响情况,为猪精液冷冻稀释液配方的改良提供理论依据,试验采集美系长白、大约克与杜洛克3种公猪的精液,用添加不同浓度（0、0.01%、0.02%、0.03%、0.04%、0.05%、0.06%）黄芪多糖的冷冻稀释液稀释,0.25 mL塑料细管分装并冷冻,测定解冻后精子活力、畸形率及顶体完整率,并进行相同浓度3种公猪之间和同种公猪不同浓度之间的比较。结果表明,在相同黄芪多糖浓度下,仅杜洛克公猪冻后精子顶体完整率在不添加黄芪多糖时显著高于大约克公猪（P<0.05）,其余均无显著差异（P>0.05）;每种公猪的最佳黄芪多糖添加浓度均为0.04%,在该浓度下精液冻后质量均显著优于对照组（P<0.05）;各种公猪最佳黄芪多糖添加浓度下的精液冻后质量指标之间均无显著差异（P>0.05）。总之,稀释液中添加0.04%黄芪多糖在长白、大约克与杜洛克3种公猪的精液冷冻都可以取得较好的效果。     

Fertilizing capacity of boar semen frozen in an extender supplemented with ostrich egg yolk lipoprotein fractions--a pilot study

A limited field trial was performed to evaluate the fertilizing capacity of boar spermatozoa frozen in an extender supplemented with lipoprotein fractions isolated from ostrich egg yolk (LPFo). Boar semen, diluted in an extender containing lactose with lyophilized lipoprotein fractions, glycerol and Orvus Es Paste (lactose-LPFo-G), was frozen using a controlled programmable freezer. Sperm characteristics, such as motility, plasma membrane and acrosome integrity, and mitochondrial function were monitored. Post-cervical artificial inseminations (post-CAIs) in multiparous sows (Polish Large White) were performed using the Soft & Quick catheter/cannula set. Sows were inseminated 2 to 3 times within one oestrus. Possible returns of sows to oestrus were determined from 21 to 30 days after post-CAIs. In this field trial, sows inseminated with 2 x 10(9) motile frozen-thawed spermatozoa resulted in pregnancy and farrowing rates of 75%, respectively. The average piglets born live was 10.5 +/- 0.4 (mean +/- SEM). The data of this study showed that post-CAI of boar semen frozen in LPFo-containing extender has the potential to provide acceptable fertility results. Further investigations are needed to elucidate the cause of variations in pregnancy/farrowing rate associated with frozen-thawed boar semen.