Motility of Fresh and Frozen-Thawed Stallion Sperm from Three Segments of the Epididymal Cauda and the Effect of Post-Thaw Seminal Plasma Addition on Motility

Preservation of epididymal spermatozoa is an advantageous method to preserve genetic material of endangered species or valuable breeding animals after sudden death and injuries. Despite lower pregnancy rates, fertilization with epididymal sperm has been proven successful. Variable sperm quality after cryopreservation among individual stallions and the usually terminal chance to preserve epididymal sperm are opportunities for the development of a freezing procedure suitable for the majority of stallions. To evaluate the effect of the preservation procedure, we analyzed the sperm motion characteristics after every step of processing. In addition, we investigated the influence of seminal plasma on motility of frozen-thawed semen. We compared three segments of the cauda epididymidis and harvested spermatozoa by retrograde flushing (most caudal part) and mincing (more cranial segments) to augment the number of spermatozoa. During processing, there were differences in sperm motion characteristics depending on the segment of the cauda epididymidis. Distinct increases in motility due to different extenders and the effect of seminal plasma suggest that motion characteristics of raw and frozen-thawed spermatozoa are strongly influenced by microenvironment and must be interpreted with caution.     

Comparison of Apoptotic Cells Between Cryopreserved Ejaculated Sperm and Epididymal Sperm in Stallions

The development of a reliable technique to freeze epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The aim of this study was to compare the apoptotic indices of sperm obtained from ejaculate, sperm recently recovered from the epididymides (EP), and sperm recovered from epididymides stored at 5°C for 24 hours (EP-stored). For the first category, two ejaculates from seven stallions were collected and then submitted to cryopreservation using an egg yolk-based extender. One week after the last semen collection, the stallions were submitted to bilateral orchiectomy, and sperm from one of the cauda epididymis was harvested immediately after castration (EP). The remaining testicle was stored in a passive refrigeration container at 5°C for 24 hours before the cauda epididymal sperm was harvested (EP-stored). Sperm harvesting from the epididymis for EP and EP-stored was performed by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender. The recovered sperm was then cryopreserved using the egg yolk-based extender. Sperm motility parameters were studied by computer-assisted semen analysis, and apoptosis was estimated by measuring caspase activity and membrane phospholipid translocation using epifluorescence microscopy. The samples were evaluated immediately (0 hour) and 8 hours after thawing. At 0 hour, no differences in sperm parameters were observed among the groups, but after 8 hours, significant statistical differences were observed in sperm motility parameters and plasma membrane integrity among the treatment groups. In addition, viable cells with no apoptotic signs were more prevalent in EP and EP-stored, suggesting that epididymal sperm is less sensitive to the cold shock caused by sperm cryopreservation.     

Artificial insemination of frozen epididymal sperm in beagle dogs

Freeze-storage of epididymal sperm is an important technique for the preservation of gametes in animals, including those becoming extinct. We froze canine sperm recovered from the cauda epididymis and investigated the fertility. The qualities of sperm from the cauda epididymis before freezing were: mean sperm motility, 89.4 +/- 1.6 (SE) %; sperm viability, 89.1 +/- 1.1%; and these were significantly higher than those of sperm from the caput-corpus epididymis (P<0.01, P<0.05). The number of sperm recovered from both cauda epididymides varied among animals: 6.3-122.3 x 10(7), mean 61.5 +/- 10.0 x 10(7). Freezing was used only for sperm recovered from the cauda epididymis. The sperm motility and viability after thawing were 19.5 +/- 2.5% and 53.1 +/- 3.3%, respectively. These were slightly lower than those of frozen-thawed ejaculated sperm, but the differences were not significant. When 2 x 10(8), 3 x 10(8), or 4 x 10 (8) sperm were inseminated in the unilateral uterus, only one animal inseminated with 3 x 10(8) sperm was fertilized (1/16, 6.3%). When 1 x 10(8) sperm were inseminated in the bilateral uterine tubes, one of six animals (16.7%) was fertilized. Therefore, although the qualities of epididymal sperm after thawing were similar to those of ejaculated sperm, the conception rate obtained with frozen-thawed epididymal sperm was low in beagle dogs. It is necessary to investigate the differences in damage between epididymal sperm after thawing and ejaculated sperm and to develop a method for improving the conception rate.     

A comparison of electroejaculation and epididymal sperm collection techniques in stallions

The purpose of this study was to evaluate 2 methods of semen collection that could be used as terminal procedures in stallions with irreparable conditions, such as fractures or colic. Electroejaculation was attempted under general anesthesia. Forty-eight hours later, the ponies were castrated and 2 different epididymal sperm collection techniques were attempted by using a flushing or floating method. Additionally, the effect of supplemental seminal plasma was evaluated. Experimentally, electroejaculation was found to be a safe but ineffective method of terminal semen collection. Viable sperm cells were successfully recovered with both types of epididymal collection. The flotation method was least cumbersome and showed a tendency to be superior to flushing in terms of sperm motility and percentage of cells passing through glass wool/sephadex filtration, although differences did not reach significance. The addition of seminal plasma to epididymal spermatozoa prior to cryopreservation was of no value. In conclusion, either method of epididymal sperm collection is an acceptable method of terminal semen collection.     

Lectin-binding characteristics and capacitation of canine epididymal spermatozoa

Cross sections of the testes and the caput, corpus and cauda epididymides removed from 12 dogs were stamped on glass slides, and the sperm on the slides were stained with 6 different FITC-lectins (Con A, DBA, PNA, PSA, SBA, and WGA) to examine the characteristics of the surface glycoproteins (GPs) on canine epididymal sperm. The corpus epididymal sperm were washed three times by centrifugation, and their lectin-binding characteristics were investigated. The washed sperm from the corpus and cauda epididymides were incubated for 24 hr, and the fertilizing capacity of the sperm was evaluated by calculating the percentages of actively motile sperm (%MO), hyperactivated sperm (%HA), and acrosome-reacted sperm (%AR), and the number of canine zona-pellucida (ZP)-binding sperm. The testicular sperm did not stain with SBA lectin, but the SBA lectin fluorescence was observed on the surface of the entire heads of the caput epididymal sperm. Although all of the entire heads or acrosomal regions of the corpus epididymal sperm stained with all 6 FITC-lectins, the heads and acrosomal regions of the cauda epididymal sperm did not stain with DBA or SBA lectins. Washing the sperm from the corpus epididymis resulted in loss of the fluorescence of the FITC-DBA and -SBA lectins. The mean %MO, %HA, %AR, and ZP-binding number of the cauda epididymal sperm after 24 hr of incubation were higher than the values for the corpus epididymal sperm. All of the mean values for the washed sperm from the corpus and cauda epididymides were higher than the values for the unwashed sperm from the corpus and cauda, and with the exception of %AR, the values from the washed sperm from the corpus epididymis were significantly higher (P<0.05, 0.01). The results indicate that DBA- and SBA-lectin-binding GPs on the surface of canine epididymal sperm are associated with the fertilizing capacity and may be decapacitation factors.     

Effect of Storage Time and Temperature of Equine Epididymis on the Viability,Motion Parameters,and Freezability of Epididymal Sperm

The recovery of sperm from the epididymal cauda may be the last chance to obtain genetic material when sudden death or serious injuries occur in valuable stallions. However, the lack of technical knowledge regarding the storage and transportation of the epididymis often prevents the preservation of the sperm. Therefore, the aim of this study was to compare sperm parameters of sperm obtained immediately after orchiectomy with sperm recovered from epididymal cauda at different times after storage at 5°C and at room temperature (RT). For that, 48 stallions of different breeds were used. In group 1 (control group), eight stallions were used, and the harvest of the epididymal sperm was performed immediately after orchiectomy. In group 2, 40 stallions were used, which were divided into five groups according to the storage time of the epididymis after orchiectomy (6, 12, 18, 24, or 30 hours), making a total of eight stallions per group. One epididymis of each stallion was stored at 5°C, and the contralateral epididymis was stored at RT, both for the same period. The sperm parameters of total motility, progressive motility, progressive linear velocity, curvilinear velocity, percentage of rapid sperm, and plasma membrane integrity were evaluated in all the groups after sperm recovery, resuspension in a sperm freezing diluent, and thawing. In conclusion, the storage of the testis-epididymis complex at 5°C provided better preservation of epididymal sperm than the storage at RT, and regardless of the temperature, the progressive motility is the sperm parameter that is most sensitive to storage time.     

B‐mode Ultrasound and Grey‐Scale Analysis of the Epididymis in Boars,and the Relationship to Semen Parameters

This study investigated the epididymis of mature boars (n = 10) by means of B‐mode ultrasound and grey‐scale analysis (GSA) for echogenicity (EG) determination using an ultrasound unit HS 1500V, a linear transducer (frequencies 7.5–9.0 MHz), and standardized unit settings. All boars had their epididymal caput, corpus and cauda scanned six times before and after semen collection, respectively, at weekly intervals. Semen was subjected to spermatological examination including volume, total and forward motility, sperm abnormalities, as well as total sperm count and concentration. The caput and corpus both had a homogeneous fine echotexture. The cauda was homogeneous too but had a marbled echotex‐ture. Echogenicity before and after semen collection was caput > corpus > cauda, respectively (p < 0.001). Echogenicity was higher before than after semen collection for all parts of the epididymis, respectively (p < 0.001). Echogenicity of the caput correlated slightly positively with the total sperm count pre‐collection (r = 0.301; p = 0.020) and with ejaculate volume pre‐ and post‐collection (r = 0.302 vs 0.306; p = 0.017 vs 0.019), and slightly negatively with sperm concentration post‐collection (r = ?0.275; p = 0.034). No relationship was found for EG of the corpus and cauda for any of the ejaculate parameters. In conclusion, B‐mode ultrasound and GSA proved feasible for imaging the epididymis in boars. Single relationships between EG and ejaculate parameters were found and deserve further investigation.     

Morphological and acrosomal changes of canine spermatozoa during epididymal transit

Background

During epididymal transit, functional and structural modifications leading to full maturation enable male gametes to reach, recognize and fertilize the oocytes. In dogs, little is known on the modifications of spermatozoa during the passage in the epididymis. The aim of this study was to describe the motility, morphology and acrosomal patterns of canine spermatozoa retrieved from the epididymis caput, corpus and cauda.

Results

After the dilution required for the collection of epididymal content, sperm motility was significantly higher (P <0.0001) in the cauda compared to corpus and caput.Proportions of spermatozoa with normal morphology were significantly higher in corpus (P =0.02) and cauda (P <0.0001) compared to caput. Overall morphological abnormalities of the head and neck/midpiece were similar in the three different epididymal regions. A significantly increased prevalence of tail defects, mainly represented by single bent tails, was observed in the corpus compared to caput (P <0.0001) and cauda (P =0.006).Numbers of immature sperm with cytoplasmic droplets decreased from the proximal to the distal region of the epididymis. Particularly, proximal cytoplasmic droplets were more frequently found in spermatozoa collected from the caput epididymis than in the corpus (P <0.0001) and in the cauda (P <0.0001), whereas the occurrence of distal cytoplasmic droplets was higher in the corpus than in the caput (P =0.0003) and in the cauda (P <0.05).Significantly higher proportions of spermatozoa with intact acrosomes were retrieved from the cauda epididymis than from the caput (P =0.03) and the corpus (P =0.008). This difference was mainly due to a lower proportion of spermatozoa with abnormal acrosomes (mainly swollen acrosomes) rather than with absent acrosomes.

Conclusions

Canine spermatozoa undergo several modifications in the epididymis. The acquisition of progressive motility, migration of the cytoplasmic droplet and acrosomal reshaping lead to mature spermatozoa which are then stored in the cauda epididymis. From this site, spermatozoa can be retrieved and used in assisted reproductive techniques as a valuable tool for propagating genetic traits of high value individuals that dies accidentally or undergoes orchiectomy for medical purposes. Further investigations should be also focused on the potential use of spermatozoa recovered from other epididymal regions.     

Assessment of Selected Quality Parameters of Epididymal Cat (Felis catus s. domestica,L. 1758) Sperm Using Flow Cytometry Method and Computer Assisted Sperm Analyser

The improvement of biotechnical methods connected with fast and precise semen quality assessment and its utilization in assisted reproductive techniques is an urgent necessity in felids. The aim of this study was to evaluate some quality parameters (i.e. the viability and share of cells with intact plasma membrane) of epididymal sperm of cats using the flow cytometry method and computer‐assisted sperm analysis (CASA) examination. The material consisted of epididymal spermatozoa flushed from 22 pairs of epididymes after routine neutering procedures obtained from domestic cats aged between 8 and 36 months. The epididymes were cut and incubated with an extender without egg yolk. The samples were assessed for sperm viability (Live/Dead Sperm Viability Kit^®), percentage of subtle membrane changes (Apoptosis Detection Kit^®) and motility using FACScalibur flow cytometer and assisted sperm analyser htm ivos version 12.2. The flow cytometry method revealed 71.3% and 84.4% of live sperm using Live/Dead Sperm Viability Kit and Apoptosis Detection Kit respectively. The population of early‐apoptotic and late‐apoptotic sperm were 0.8% and 1.1% respectively. The CASA examination found 51.5% of motile sperm. However, the motility examination under light microscope revealed 69.5% of motile sperm. The data revealed an indistinctive per cent of apoptotic cells and 18.9% and 15.6% of dead cells using Live/Dead Sperm Viability Kit and Apoptosis Detection Kit, respectively, which indicate that the sperm obtained after flushing the epididymis possess potential properties for further assisted reproduction techniques.     

Tris–Egg Yolk–Glycerol (TEY) Extender Developed for Freezing Dog Semen is a Good Option to Cryopreserve Bovine Epididymal Sperm Cells

Cryopreservation of epididymal spermatozoa is often performed after shipping the excised testis–epididymis complexes, under refrigeration, to a specialized laboratory. However, epididymal spermatozoa can be collected immediately after excision of the epididymis and sent extended and refrigerated to a laboratory for cryopreservation. In this experiment, we evaluated the effect of both methods of cold storage bovine epididymal spermatozoa as well as of two different extenders on spermatozoa characteristics after freeze–thawing. For that, spermatozoa collected from the caudae epididymis of 19 bulls were extended and cryopreserved in either AndroMed^® or a Tris–egg yolk (TEY)‐based extender. Cryopreservation of sperm cells was performed immediately after castration (Group A, n = 9) or after cold storage for 24 h diluted in the two extenders and (Group B, n = 9) and also after cold storage for 24 h within the whole epididymis (Group C, n = 10). Sperm subjective progressive motility (light microscopy), plasma membrane integrity (hypoosmotic swelling test) and sperm viability (eosin–nigrosin) were evaluated. In vitro fertilization and culture (IVF) was performed to assess the blastocyst rate. No differences (p > 0.05) were observed on post‐thaw sperm parameters between samples from Group A, B and C. TEY extended samples presented a higher (p < 0.01) percentage of progressive motile and live sperm, than those extended in AndroMed^®. Blastocyst rate after IVF differed only (p < 0.05) between the reference group (IVF performed with frozen semen with known in vitro fertility) and Group A extended in AndroMed^®. We conclude that when cryopreservation facilities are distant from the collection site, bovine epididymal sperm can be shipped chilled overnight either within the epididymal tail or after dilution without deleterious effect on post‐thaw sperm quality. TEY extender was more suitable for cold storage and freezing bovine epididymal sperm, than the commercial extender AndroMed®.     

Influence of recovery methods and extenders on bull epididymal spermatozoa quality

The aim of this study was to compare the effects of two extraction methods in combination with two different extenders in bull epididymal sperm collection. Testes from 23 sexually mature Limousine bulls were collected at the abattoir. Epididymal sperm recovery was performed using both the float-up (FL) and the retrograde flushing (RF) technique. Within extraction methods, half testes were processed with a Tris egg yolk extender and half with a Tris egg yolk-free extender. Sperm concentration, motility, viability and morphology were evaluated. Sperm concentration was not significantly different between methods. Flushing technique was significantly better than the FL method in terms of sperm quality, considering total motility (80.3 ± 2.3% vs 71.6 ± 2.0%, p < 0.001, respectively) and viability (84.5 ± 1.5% vs 77.2 ± 1.3%, p < 0.001, respectively). Egg yolk influenced positively motility and morphology in the FL method, whereas decreased viability in flushed samples. Results suggest the use of the RF technique to collect cattle epididymal sperm.     

Comparison of DNA Fragmentation Assay in Frozen‐Thawed Cat Epididymal Sperm

DNA fragmentation of frozen‐thawed feline epididymal sperm from corpus and cauda regions was evaluated by three different techniques. The DNA fragmentation index (DFI) was compared between techniques: the sperm chromatin structural assay (SCSA^®), acridine orange staining techniques (AOT) and the sperm chromatin dispersion (SCD). There were significant differences in DFI among the techniques (p < 0.05) with no correlations. Only DFI values obtained from SCD revealed a significantly higher DFI in corpus compared with cauda spermatozoa (p < 0.05). The discrepancy between techniques might be due to the sensitivity of each technique, differences in severity of DNA damaged that can be detected. The difference in DFI between epididymal regions from SCD technique might indicate different maturational stages of spermatozoa, with less chromatin condensation of spermatozoa in corpus compared with cauda epididymis.     

Comparison of fertility on intrauterine insemination between cryopreserved ejaculated and cauda epididymal sperm in dogs

The fertility was compared between ejaculated and cauda epididymal sperm sensitized with prostatic fluid in dog after freeze-thawing using the fertility of ova from the contralateral ovary after injection (2 × 10(8) sperm) into dog uterus on the unilateral ovariectomized side, on the basis of the presence or absence of conception. No significant difference was observed in sperm quality after freeze-thawing between the two groups and conception rates were equivalent and low. Therefore, to achieve a high fertility by intrauterine insemination of canine frozen-thawed ejaculated and cauda epididymal sperm, intrauterine insemination on both sides is recommended, rather than insemination with a lot of sperm of the uterine horn on one side.     

不同繁殖季节成年牦牛附睾组织特征比较

为比较不同繁殖季节成年牦牛附睾组织结构特征变化,应用苏木精-曙红常规染色、Masson's和Gomori's特殊组织化学染色方法比较不同繁殖季节牦牛（6头繁殖期成年牦牛和9头繁殖间期成年牦牛）附睾的组织结构特点并用IPP图像分析软件进行定量分析。结果显示,与繁殖间期相比,繁殖期附睾尾间质胶原纤维和网状纤维较附睾头及附睾体明显增多;附睾头和附睾体柱状上皮厚度显著增加（P<0.05）,附睾头纤毛长度增加显著（P<0.05）;附睾头和附睾尾管腔内径及外径显著增高（P<0.05）;但是附睾尾管腔外径繁殖期与繁殖间期相比差异不显著（P>0.05）。本研究结果表明,成年牦牛附睾管外围网状纤维与胶原纤维分布一致,二者在附睾尾较为丰富,可能与其较强的收缩能力及精子运输有关;柱状纤毛上皮高度及纤毛长度、管腔内径与外径的变化与其所处的不同繁殖季节密切相关,附睾管腔的膨大和回缩亦可能是高原地区季节性繁殖的哺乳动物中一种普遍存在的现象。     

Histological Characteristics of Adult Yak Epididymis in Different Breeding Seasons

To compare the histological changes of the adult yak's epididymis in different breeding seasons,six adult yak testis in breeding season and nine adult yak testis in breeding interval were collected for structure investigation by HE staining,Masson's and Gomori's histochemistry methods,and IPP (Image-Pro Plus) statistics method was used to quantitative statistics.The results showed that comparing with the adult yak in the breeding interval,the epididymis ducts of adult yak in the breeding season were covered with the columnar ciliated epithelium.The collagen and reticular fiber in cauda epididymis were obviously more abundant than caput and corpus epididymitis.And the thickness of columnar epithelium cells in caput and corpus epididymitis,the length of the cilia in caput,and also the internal and external lumen diameter of caput and corpus epididymitis were all significantly increased in the breeding season (P<0.05),but the external lumen diameter of the cauda epididymis had no significant differences (P>0.05).In conclusion,the research showed that the distribution of collagen and reticular fiber in adult yak's epididymis interstitial were similar,and they were more rich in cauda epididymis,which might relate to the capacity and the sperm transport;The changes of the epithelial thickness,the length of cilia,the internal and external lumen diameter were close related to the different breeding seasons,and it might be a common phenomenon in plateau mammals that the enlargement and reduction of the epididymal duct in different breeding seasons.     

Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours)

Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post‐mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer‐assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3′3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time.     

牦牛GPX5基因CDS区的克隆和表达研究

GPX5作为谷胱甘肽过氧化物酶的成员之一,表达于牦牛附睾之中,其主要作用为抗氧化应激。本实验旨在通过分子技术对牦牛GPX5基因进行克隆,分析其生物信息学的特征和附睾组织的表达特点。结果显示:牦牛GPX5基因CDS区有630 bp、209个氨基酸被编码,所编码蛋白带正电,具有亲水性且不稳定,无跨膜域却存在信号肽;GPX5基因在附睾头的表达高于附睾的颈和尾(P<0.01)。牦牛附睾中的GPX5能有效抵抗氧化应激以保护精子。     

水牛附睾不同部位精子超微结构及荧光标记差异分析

哺乳动物精子经过附睾成熟后才能获得运动及受精的能力,为解释水牛精子在附睾中的成熟过程,本研究选用性成熟期的沼泽型水牛附睾,利用乙烯吡咯烷酮包裹的硅胶微小颗粒（Percoll）梯度离心纯化分别提取附睾头、体和尾部精子,应用计算机辅助精子分析系统（CASA）检测精子活力,透射电镜观察附睾不同部位精子的超微结构,对精子进行荧光标记后,利用流式细胞仪和荧光显微镜观察检测不同部位精子质膜完整率、线粒体鞘膜电位和顶体差异。结果表明,Percoll分离得到附睾头、体和尾3部位精子的纯度达95%,不同部位精子活力分别为8.35%、20.21%和65.60%;附睾不同部位精子都存在着结构完整的精子以及相同的畸形类型,附睾尾部精子线粒体鞘高膜电位比率最高,精子质膜完整率从附睾头部到尾部逐渐升高,精子顶体完整率从附睾头部到尾部逐渐升高。本研究直观地展示了水牛附睾不同部位精子特征以及差异,为研究水牛精子成熟机理提供理论依据。     

Evaluation of membrane integrity of canine epididymal spermatozoa by short hypoosmotic swelling test with ultrapure water

The effectiveness of the water test, short hypoosmotic swelling test with ultrapure water was examined in canine epididymal spermatozoa to evaluate tail membrane integrity. Spermatozoa during epididymal transit were also characterized. Sperm suspension obtained from cauda epididymis was diluted 1:4 with ultrapure water, and incubated for 5 min. The percentage of swollen spermatozoa in the water test was significantly correlated with both the sperm motility and the swelling value obtained by the conventional hypoosmotic swelling test. Canine spermatozoa collected from the caput epididymis were not motile, but revealed membrane integrity in a water test. The water test can be used as a simple and short hypoosmotic swelling test to evaluate the tail membrane integrity of canine epididymal spermatozoa.