Search

For the preservation of endangered felid species, epididymal sperm may be received from valuable individuals after castration or death and they need to be cryopreserved for storage. However, pregnancy rates with epididymal or cryopreserved sperm are lower than with ejaculated and non-frozen semen even if insemination is surgically performed into the oviduct. To investigate whether equilibration, the first step of the cryopreservation procedure, has an impact on sperm-oviduct binding, we generated oviduct epithelial cell vesicles from isthmus segments of preovulatory domestic cats. Binding assays were performed with epididymal sperm in a cell culture medium (M199) without supplements, or after cooling to 15 °C in a freezing extender (TestG), supplemented with glycerol and the water-soluble fraction of hen's egg yolk mainly comprising LDL. The sperm-oviduct binding was assessed both quantitatively and qualitatively (head or tail binding of sperm with active or inactive mitochondria). Most of the bound sperm prepared in M199 had active mitochondria and were attached to the vesicles by their heads. In equilibrated samples, the proportion of bound sperm with active mitochondria and the proportion of head-bound spermatozoa were reduced. The total motility of the sperm after 1 h of incubation in the absence or presence of vesicles were also affected by the preparation (higher in equilibrated) and the incubation (lower in co-incubated), while mitochondrial activity was influenced just by the preparation. Obviously, LDL has a beneficial effect on sperm motility, but we suggest that it interferes with the molecular sperm-oviduct crosstalk and causes a reduced binding of "good" sperm., Competing Interests: Declaration of competing interest There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Sperm maturation in the epididymis is a tightly regulated process which involves secretion and addition of a variety of proteins onto the sperm surface. The molecular mechanisms governing these processes has gained interest in the last decade. In vitro model systems to study the role of epididymal proteins in sperm maturation and other physiological process are very important. Isolation of epididymal cells, culture of epididymal explants and generation of immortalized cells were standardized to be used as in vitro models to study epididymal function. However, isolation and maintenance of primary cultures of epididymal epithelial cells seems to be the best option because of its closeness to the in vivo conditions. Though structural and morphological characterization of primary cultures of epididymal epithelial cells were carried out, the same were not conducted at the molecular level. In this study, we isolated adult rat epididymal primary epithelial cells (EPECs) and characterized them for their purity and cell specific expression of molecular markers. Isolated EPECs exhibited normal cell morphology and were sub cultured and maintained up to 3 weeks. EPECs expressed the epithelial marker, E-cadherin and their purity was estimated to be 73% using flow cytometry. EPECs abundantly expressed CRISP1, Urp1a, Pate-F, Crisp1, Ar and Spag11e, markers of epididymal cells and were negative for Urp1b and Pate, markers negative for epididymis. Results of our study provide a systematic characterization of EPECs at the molecular level and thus a refinement to the previously reported characterization methods., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Germplasm banking is a key technology enabling the ex-situ conservation of wild species. However, cryopreservation protocols must be tested to assure the applicability of the banked material. The objective of this study was defining a range of parameters for the composition of a semen extender for Cantabrian chamois epididymal spermatozoa (post-mortem collection). The freezing extender was based in a TES-Tris-fructose buffer, modifying its composition in three experiments: Osmolality of the buffer (320, 380 or 430 mOsm/kg, 8% glycerol, 15% egg yolk), glycerol (4 or 8%, 430 mOsm/kg, 15% egg yolk), egg yolk (10 or 15%, 430 mOsm/kg, 4% glycerol). Sperm was extended at 100 mill. spermatozoa/ml, cooled at 5 °C and frozen at -20 °C/min. Sperm quality was assessed pre and post-thawing (CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). Freezability was good overall, with total motility of 65.5% ± 2.4 initial and 55.8% ± 2.4 post-thawing. The extenders affected the post-thaw sperm quality marginally. Whereas osmolalities and glycerol concentrations seemed not to differ, 430 mOsm/kg and 4% glycerol might be preferred. Egg yolk concentrations only differed on sperm velocity (VCL: 84.0 ± 6.7 μm/s in 10% vs. 70.7 ± 6.2 μm/s in 15%, P < 0.05). Our results suggest a good cryotolerance of chamois epididymal spermatozoa, with a preferred extender composition of hyperosmotic buffer, glycerol in the 4% range and lower egg yolk (10% range) than other ruminants., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The study represents a comparison of cryopreservation of domestic cat epididymal spermatozoa with two commercially available freezing media: CaniPlus Freeze (CPF) and SpermFreeze (SF). The viability of nonfrozen spermatozoa evaluated by the VitalScreen test was 68.7 ± 3.0%. These figures were lower for the frozen-thawed spermatozoa: 51.2 ± 6.3% for CPF group and 54.4 ± 3.1% for SF group. The motility of nonfrozen spermatozoa was 57.2 ± 4.5%. These figures were reduced in both frozen-thawed groups; however, there was no significant difference in these parameters between CPF (30.8 ± 7.1%) and SF (27.4 ± 8.1%) groups. The percentage of nonprogressively moving motile spermatozoa after freezing-thawing was decreased in both frozen-thawed groups (23.5 ± 5.9 and 12.0 ± 2.4 for CPF and SF frozen correspondingly) as compared with nonfrozen controls (42.1 ± 4.1%). Morphology of spermatozoa was assessed by light microscopy. The mean percentages of normal spermatozoa were 28.5 ± 4.1% for nonfrozen group, 26.0 ± 2.3% for CPF frozen group, and 23.9 ± 1.9% for SF frozen group. The most frequent anomalies in all the three groups were flagella and combined defects. In vitro fertilization (IVF) of domestic cat oocytes with nonfrozen and frozen-thawed spermatozoa produced developing embryos. The percentage of in-vitro-derived embryos was 43.6% after using nonfrozen spermatozoa. Frozen-thawed spermatozoa developed at a similar rate (44.0%) after using SF. However, the rate of embryo development was lower (20.1%) when CPF was used. The in-vitro-derived embryos in the nonfrozen group consisted of 46.9 ± 2.5 cells after 5-day culturing. After cryopreservation with SF and CPF the cell numbers per embryo were 39.9 ± 2.7 and 31.8 ± 3.4 correspondingly. In CPF group these numbers were lower than in nonfrozen controls. Cryopreservation of spermatozoa with either of two freezing media led to a decrease in post-thaw viability and motility of spermatozoa but did not affect the rate or spectrum of their morphological anomalies. The use of CPF, but not SF led to a decrease of sperm fertilizing abilities., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In this study, we successfully described for the first time a vitrification of epididymal Iberian ibex spermatozoa. Spermatozoa from epididymis were obtained from 15 Iberian ibex. The right epididymis' semen sample was vitrified and the left one was frozen. After thawing/warming, samples were selected by density gradient. Sperm characteristics from each treatment were evaluated. To test the spermatozoa fertilization ability, heterologous IVF was carried out using bovine oocytes. Despite of the observation of a decrease of about 40% for motility sperm between pre-freezing and post-thawing (75.0 ± 5.2 and 45.0 ± 6.0) and pre-vitrification and post-warming (78.2 ± 5.2 and 33.9 ± 6.2) (P < .05), after the washing, an improvement of sperm motility was found when using the vitrification treatment compared to frozen-thawed. Heterologous IVF showed that Iberian Ibex spermatozoa, either frozen-thawed or vitrified-warmed, were equally capable of penetrating ZP intact bovine oocytes, leading to pronuclear formation (%) and hybrid embryo cleavage (%), (31.3 ± 27.2 and 45.1 ± 24.4, respectively). As expected, in the homologous IVF group, higher percentages of penetration, pronuclei formation and cleavage were found compared to heterologous groups using Iberian ibex frozen and vitrified sperm (P < 0,5). The highest pronuclei formation was found after 20 h post insemination in both heterologous IVF groups (30.2 ± 6.7 and 31.7 ± 21.5 thawed and vitrified group). Consequently, the cleavage rate (48 h) followed the same results to homologous and thawed and vitrified groups (76.1 ± 15.9; 31.3 ± 27.2 and 45.1 ± 24.4, respectively) (P < .05). In conclusion, Iberian ibex sperm vitrification is a promising and useful alternative to conventional methods resulting in good quality spermatozoa post-thaw, and an adequate in vitro fertilizing ability., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mammalian spermatozoa gradually mature and acquire fertility during the transition from the testis to the caput and cauda epididymis, after which they are stored at the tail of the epididymis and the ampulla of vas deferens. During ejaculation, mixing of spermatozoa with the secretions of accessory sex glands leads to their dilution and changes in their function. Although remarkable progress has been made toward the understanding of changes in spermatozoa biochemistry and function before and after ejaculation, it is unknown whether microRNAs (miRNAs) are involved in regulating the function of spermatozoa during the transition between the cauda epididymis and ejaculation. In this study, 48 miRNAs were selected for analysis on the basis of their potential involvement in spermatogenesis, sperm maturation, and quality parameters markers. The differential expression levels of these 48 miRNAs between the caudal epididymis and fresh ejaculates of boar spermatozoa were determined. We found that 15 miRNAs were significantly differentially expressed (eight downregulated and seven upregulated) between boar cauda epididymal and fresh spermatozoa. Five miRNAs hypothesized to be involved in sperm apoptosis were further tested to demonstrate their influence over the expression of their target mRNAs using quantitative reverse-transcription polymerase chain reaction. Together, our findings suggest that these differentially expressed miRNAs are associated with the functional regulation of spermatozoa between cauda epididymis and ejaculation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Epididymal sperm preservation offers a potential for rescuing genetic material from endangered or valuable animals after injury or death. Spermatozoa from corpus, as well as from cauda, have the capability to be motile and to undergo capacitation and can thus potentially be preserved for assisted reproductive technologies. In the present study, feline frozen-thawed epididymal spermatozoa from corpus and cauda regions were investigated for their ability to fertilize homologous oocytes and further embryo development in vitro. Epididymal spermatozoa from corpus and cauda of seven cats were cryopreserved and used for IVF. Cumulus-oocyte complexes (n = 419) were obtained from female cats after routine spaying. Frozen-thawed corpus epididymal spermatozoa showed similar properties of acrosome integrity, membrane integrity, and chromatin integrity as frozen-thawed spermatozoa from cauda except corpus spermatozoa showed lower motility (P < 0.05). The fertilizing capacity of frozen-thawed corpus epididymal spermatozoa was confirmed by similar number of embryos developing to the two- and four-cell stages compared with sperm from cauda (32.03% vs. 33.33%). However, oocytes fertilized with corpus spermatozoa had lower potential to develop to the blastocyst stage (6.79%) and had lower cell numbers compared to oocytes fertilized with cauda spermatozoa (14.08%). In conclusion, spermatozoa from corpus epididymis had a similar capability to fertilize homologous oocytes in vitro as sperm from cauda but resulted in fewer embryos developing to the blastocyst stage compared to spermatozoa from the cauda., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aims of this study were to compare the viability and in vivo and in vitro fertilization potential post-thaw sperm collected at different times postorchiectomy from bull epididymides (EP) at 18 °C to 20 °C, with those of semen collected by electroejaculation (EJ) from the same bulls. Semen samples were collected by EJ from 10 Zebu bulls and cryopreserved. A week later 20 epididymides from these bulls were obtained by orchiectomy and randomly divided into five groups (G) to be maintained at ambient temperature for 6, 12, 18, 24, and 30 hours before sperm recovery by retrograde flow. The sperm were cryopreserved, and post-thaw parameters were determined by both computer-assisted sperm analysis and morphologic analysis. In vitro fertilization of oocytes was performed to assess the cleavage rate, blastocyst rate, total number of cells, and hatching rate of embryos. The G30 sperm samples were also used for fixed time artificial insemination (FTAI) of Zebu heifers (n = 10). The results of post-thaw sperm viability showed that total and progressive motility and plasma membrane integrity were lower in sperm in which cryopreservation was delayed for 30 hours, showing a negative correlation of these parameters with delay before cryopreservation. In all groups, it was possible to obtain viable embryos, and embryos from G6 samples had more cells than the other groups. The greatest embryo production rates were observed in G6, G12 and G18 (27.2 to 32.2%) and it was significantly lower in G24 and G30 samples. For EJ, many individual variations were observed in embryo production potential between bulls. G30 samples, with only 5.2% of post-thaw progressive motility, were able to fertilize and produced a pregnancy. To the authors' knowledge, this is the first time in vitro embryos up to 8 days of development and a pregnancy after FTAI have been produced with sperm from bull epididymides that had been stored at 18 °C to 20 °C for up to 30 hours., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The present study evaluated functional aspects of binder of sperm 1 (BSP1) in the bovine species. In a first experiment, cumulus-oocyte complexes (n = 1274) were incubated with frozen-thawed ejaculated sperm (18 hours) in Fert-TALP medium containing: heparin, 10, 20, or 40 μg/mL BSP1. Heparin followed by gelatin affinity chromatography was used for purification of BSP1 from bovine seminal vesicle fluid. With ejaculated sperm, cleavage rates were similar when Fert-TALP medium was incubated with heparin (74.1 ± 2.7%), 10 μg/mL BSP1 (77.8 ± 3.1%), or 20 μg/mL BSP1 (74 ± 2.0%). Day-7 blastocyst rates were equivalent after incubations with heparin (40.8 ± 5.0%) and 10 μg/mL BSP1 (34.1 ± 4.4%), but reduced after 20 μg/mL BSP1 (22.4 ± 2.9%) and 40 μg/mL BSP1 (19.3 ± 4.1%; P < 0.05). In the second experiment, cumulus-oocyte complexes (n = 1213) were incubated with frozen-thawed cauda epididymal sperm (18 hours) in Fert-TALP medium containing: no heparin, heparin, 10, 20, or 40 μg/mL. Cleavage and blastocyst rates were similar after treatments with heparin (68.5 ± 1.3% and 24.7 ± 3.2%, respectively) or without heparin (65.5 ± 1.8% and 27.3 ± 1.6%, respectively). Cleavage was higher after treatment with any BSP1 concentrations (74.2 ± 2.7%-79.0 ± 1.1%) than without heparin (P < 0.05). Also, cleavage was better after Fert-TALP medium incubation with 40 μg/mL BSP1 (79.0 ± 1.1%) than with heparin (68.5 ± 1.3%; P < 0.05). Embryo development was higher (P < 0.05) after treatment with 20 μg/mL BSP1 (35.6 ± 2.5%) and 40 μg/mL (41.1 ± 2%) than after incubations with heparin (24.7 ± 3.2%) or without heparin (27.3 ± 1.6%). Interestingly, BSP1 did not cause reductions in blastocyst rates after fertilization with epididymal sperm, as observed with ejaculated sperm. On the basis of immunocytochemistry, there was BSP1 binding to frozen-thawed ejaculated but not to epididymal sperm. Also, anti-BSP1 reaction remained on ejaculated sperm (as expected) and appeared on epididymal sperm after incubation with purified BSP1. Acrosome reaction of ejaculated and epididymal sperm was induced after incubation with purified BSP1 as well, indicating an effect of BSP1 on capacitation. In conclusion, purified BSP1 from bull seminal vesicles was able to bind to and induce capacitation of ejaculated and epididymal sperm. Also, BSP1 added to fertilization media and allowed proper cleavage and embryo development, with the effects being modulated by previous exposure or not of spermatozoa to seminal plasma., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Epididymal sperm preservation can be used to avoid the total loss of genetic material in threatened species. Spermatozoa from the corpus, as from the cauda, are motile and can undergo capacitation. Thus, they can potentially be preserved for assisted reproductive technologies. However, cryopreservation of spermatozoa has a direct detrimental effect on sperm quality. The aim of this study was to compare the chromatin stability and the survival rate of spermatozoa from the corpus and cauda epididymis after cryopreservation. Epididymal spermatozoa were collected and cryopreserved from the corpus and cauda of 12 domestic cats. Sperm motility, progressive motility, membrane integrity, acrosome integrity, and DNA integrity were evaluated before and after freezing thawing. The average total number of spermatozoa collected from the corpus was lower (10.2 × 10(6) ± 7.4) than that from the cauda epididymis (24.9 × 10(6) ± 14.4; P = 0.005). The percentage of spermatozoa with intact DNA did not differ significantly whether it was collected from the corpus or cauda regions and did not decrease after freezing thawing in either region. However, motility of spermatozoa from both regions was affected by the freezing thawing process with a significant decline in motility after thaw compared with fresh spermatozoa. A significant difference in the percentage of motile sperm between the corpus and cauda was observed after the freezing thawing process (P < 0.001). Although sperm motility was lower in postthaw spermatozoa from the corpus epididymidis than from the cauda, the rate of the reduction did not differ between regions. This study indicates that the cryopreservation process does not have a negative effect on chromatin stability of feline epididymal spermatozoa. Spermatozoa from the corpus region have a similar freezability as spermatozoa from the cauda region. Therefore, preservation of spermatozoa from the corpus and the cauda epididymidis might be of value in preserving genetic material from endangered or valuable felids., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Equine spermatozoa from the cauda epididymis were previously collected and frozen, and the fertility was assessed. Most studies were performed on healthy stallions that had undergone routine castration or on the epididymis collected at the abattoir, but there are no studies on the quality of epididymal semen in subjects which have died from colic or which underwent intensive care. The present study was designed to verify whether a severe illness could affect epididymal semen quality and freezability in the stallion. Therefore, epididymal semen characteristics during the freezing process in stallions which had died from colic and in healthy stallions submitted to elective castration were compared. Five stallions that had died from colic (ill stallions [ISs]) and seven stallions that had undergone elective castration (healthy stallions) were castrated, and cauda epididymis spermatozoa were collected and processed. Sperm quality was tested after collection, after washing procedures, at the end of the equilibration (5 °C for 75 minutes), and after freezing/thawing. Sperm quality was measured by objective motility characteristics, membrane and acrosome integrity, and mitochondrial activity. After collection, sperm in ISs showed low kinetic parameters (total motility: 17.3 ± 3%, progressive motility: 6 ± 1%, average path velocity: 57.4 ± 35.4 μm/s, straightness: 74.2%) compared with healthy stallions (total motility: 90.8 ± 3.7%, progressive motility: 70 ± 4%, average path velocity: 118.1 ± 12.6 μm/s, straightness: 82.4%) but demonstrated similar membrane and acrosome integrity (85 ± 2.8% vs. 87.6 ± 3.1%). Sperm kinetic parameters increased after washing procedures and cooling in ISs, reaching comparable values after equilibration (5 °C for 75 minutes) and freezing/thawing. The data reported in this study suggest that the quality of the equine epididymal spermatozoa cryopreserved in stallions that had died from colic was similar to that reported in epididymal sperm after elective castration and was also similar to the data reported in literature for cryopreserved equine semen., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study was designed to describe and compare basic semen characteristics and sperm motility parameters obtained via computer-assisted sperm analysis (CASA) in feline semen collected from the urethra and epididymis, on the basis of large, unselected population of domestic cats. The semen collected from 214 males was subjected for routine semen assessment and CASA evaluation. Semen collected by urethral catheterization (CT) and by epididymal slicing (EP) has comparable characteristics according to total sperm count (47.7 ± 42.1 and 52.9 ± 45.0), subjective motility (71.1 ± 17.0 and 69.3 ± 13.9), viability (74.9 ± 13.4 and 76.7 ± 10.6), and morphology (52.6 ± 19.0 and 47.2 ± 17.4). The study of a large feline population confirmed a high incidence of teratospermy in cats, which negatively affects sperm motility parameters assessed by CASA. A lack of a correlation between CT and EP semen for total sperm count and viability, as well as occasional gross differences between the morphology of CT and EP semen of the same cat suggests that many factors may affect sperm cells, and the fertility and/or infertility of patients should not be assessed after examining only one sample. Additionally, technical problems with assessment of EP samples (understated results) suggest that CT semen is more appropriate for an analysis by CASA than EP., (Copyright © 2015 Elsevier Inc. All rights reserved.)

High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The aim of the study was to assess the effect of long exposure to summer circadian heat stress cycles on sperm parameters and the motile subpopulation structure of epididymal sperm cells from rabbit bucks. Twelve White New Zealand rabbit bucks were exposed to a daily constant temperature of the thermoneutral zone (from 18 °C to 22 °C; control group) or exposed to a summer circadian heat stress cycles (30 °C, 3 h/day; heat stress group). Spermatozoa were flushed from the epididymis and assessed for sperm quality parameters at recovery. Sperm total motility and progressivity were negatively affected by high temperatures (P < 0.05), as were also specific motility parameters (curvilinear velocity, linear velocity, mean velocity, straightness coefficient, linearity coefficient, wobble coefficient, and frequency of head displacement; P < 0.05, but not the mean amplitude of lateral head displacement). Heat stress significantly increased the percentage of less-motile sperm subpopulations, although the percentage of the high-motile subpopulation was maintained, which is consistent with the fact that no effect was detected on fertility rates. However, prolificacy was reduced in females submitted to heat stress when inseminated by control bucks. In conclusion, our results suggest that environmental high temperatures are linked to changes in the proportion of motile sperm subpopulations of the epididymis, although fertility is still preserved despite the detrimental effects of heat stress. On the other hand, prolificacy seems to be affected by the negative effects of high temperatures, especially by altering female reproduction., (Copyright © 2015 Elsevier Inc. All rights reserved.)

From many endangered or threatened species which are expected to profit from assisted reproduction techniques, mainly epididymal sperm of dead or freshly castrated males are available. These sperm had contact to epididymal secretion products but not to seminal fluid components. Notably, products of accessory sex glands have been shown in domestic animals to condition sperm for fertilization, in particular by mediating sperm-oviduct interaction. We report for the first time that motile epididymal sperm from domestic cats are able to bind to fresh oviduct epithelial cell explants from preovulatory females (median [min, max] of 10 [8, 16] and 10 [8, 17] sperm per 0.01 mm(2) explant surface from both isthmic and ampullar regions, respectively). More sperm attach to the explants when epididymal sperm were preincubated for 30 minutes with seminal fluid separated from electroejaculates of mature tomcats (median [min, max] of 17 [13, 25] and 16 [12, 21] sperm per 0.01 mm(2) explant surface from isthmus and ampulla, respectively). The proportion of bound sperm increased from a median of 54% to 62% by seminal fluid treatment. Sperm-oviduct binding could be facilitated by the decelerated sperm motion which was observed in seminal fluid-treated samples or supported by seminal fluid proteins newly attached to the sperm surface. Seminal fluid had no effect on the proportion of sperm with active mitochondria. Extent and pattern of sperm interaction in vitro were independent of explant origin from isthmus or ampulla. Sperm were attached to both cilia and microvilli of the main epithelial cell types present in all explants. In contrast to published sperm-binding studies with porcine and bovine oviduct explants where predominantly the anterior head region of sperm was attached to ciliated cells, the tails of some cat sperm were firmly stuck to the oviduct cell surfaces, whereas the heads were wobbling. Whether this response is a preliminary step toward phagocytosis or a precondition to capacitation and fertilization remains to be determined. In conclusion, treatment of epididymal sperm with seminal fluid or particular protein components should be considered in future investigations for its potential to improve the outcome of artificial insemination in felids., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Sperm samples may be used for assisted reproductive technologies (e.g., farmed or endangered species) or as a source of haploid DNA or sperm-specific RNA. When ejaculated spermatozoa are not available or are very difficult to obtain, as is the case for most wild endangered species, the epididymides of dead animals (e.g., animals that have been found dead, shot by hunters or poachers, or that that require euthanasia in zoological collections) can be used as a source of sperm. Such epididymal sperm samples are usually contaminated with cellular debris, erythrocytes, leukocytes, and sometimes also bacteria. These contaminants may be sources of reactive oxygen species that damage spermatozoa during freezing or contribute undesired genetic material from diploid cells. We used single-layer centrifugation through a colloid formulation, Androcoll-C, to successfully separate wolf epididymal spermatozoa from contaminating cells and cellular debris in epididymal samples harvested from carcasses. Such a procedure may potentially be applied to epididymal sperm samples from other species., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Ubiquitin is a 8.5-kDa peptide that tags other proteins for proteasomal degradation. It has been proposed that ubiquitination might be responsible for the elimination of defective spermatozoa during transit through the epididymis in humans and cattle, but its exact biological function in seminal plasma has not yet been clarified. In the domestic cat (Felis catus), the percentage of immature, unviable, and abnormal spermatozoa decreases during the epididymal transit, indicating the existence of a mechanism that removes defective spermatozoa. Magnetic cell separation techniques, based on the use of magnetic beads coated with anti-ubiquitin antibodies, may allow the selective capture of ubiquitinated spermatozoa from semen, thus contributing to the identification of a potential correlation between semen quality and ubiquitination process. Moreover, the selective identification of all the ubiquitinated proteins in different epididymal regions could give a better understanding of the ubiquitin role in feline sperm maturation. The aims of this study were as follows: (1) to verify the possibility of separating ubiquitinated spermatozoa with magnetic ubiquitin beads and identify the morphological and acrosomal differences between whole sample and unbound gametes, (2) to characterize all the ubiquitinated proteins in spermatozoa retrieved in the three epididymal regions by a proteomic approach. The data indicated the presence of ubiquitinated proteins in cat epididymal semen. However, a correlation between abnormal and ubiquitinated spermatozoa has not been found, and ubiquitin cannot be considered as a biomarker of quality of epididymal feline spermatozoa. To the author's knowledge, this is the first identification of all the ubiquitinated proteins of cat spermatozoa collected from different epididymal regions. The proteomic pattern allows a further characterization of cat epididymal semen and represents a contribute to a better understanding of the ubiquitin role in feline sperm maturation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Two experiments were conducted to study the effect of shortening the equilibration time with the cryoprotectant glycerol before freezing epididymal sperm recovered postmortem from Iberian ibex. In the first experiment, the standard equilibration time of 3 hours was compared with 2 hours, and subjective sperm motility and quality of movement were greater (P < 0.05) in the latter group. In the second experiment, reducing the equilibration time from 2 hours to 15 minutes did not affect sperm motility (evaluated subjectively and objectively), viability, acrosomal integrity, or membrane functional integrity. In conclusion, shortening the equilibration time can be used as a technique to simplify the cryopreservation process and this provides practical advantages under field conditions., (Copyright © 2014 Elsevier Inc. All rights reserved.)

It is important to understand the proliferative activity of the different structures of the male reproductive apparatus in livestock species, such as Sus domesticus, to ensure reproductive efficiency. The main aims of this study were (a) to evaluate the proliferative activity of the spermatogonia in the different stages of the seminiferous cycle and (b) to study the cell proliferation in the epididymal epithelium in each region, identifying the different cells involved. For this, the testes and epididymis of three healthy, sexually mature Sus domesticus boars were used. The organs were processed for light microscopy, and immunohistochemical techniques were used to detect proliferating cell nuclear antigen. The cells immunostaining positively and negatively for proliferating cell nuclear antigen were counted and several parameters and indexes were calculated to evaluate the proliferation in both epithelia, taking into account the stage of the seminiferous epithelium cycle, and, in the case of the epididymal epithelium, the different regions and cells are the same. Finally, a contrast analysis of equality between pairs of means was carried out followed by a least significant differences test, in which differences were considered significant at P < 0.05. In the seminiferous epithelium, the greatest total number of spermatogonia and proliferating spermatogonia was observed in the postmeiotic stages (mainly VII and VIII). The proliferation index of the spermatogonia increased from the meiotic to postmeiotic stages. As regards the epididymal epithelium, the total proliferation index was higher in the caput. In each region, the clear and principal cells showed the highest proliferation index with respect to the total number of cells counted, whereas the proliferation index of each cell with respect to the same type was higher in the clear cells, followed by the narrow and principal cells. In conclusion, the proliferative activity of spermatogonia in the seminiferous epithelium of Sus domesticus is stage-dependent, and mainly occurs in the postmeiotic stages. In the epididymal epithelium, proliferative activity takes place in several cell types and is dependent on the anatomical region of the epididymis. We think that these results may be of importance for understanding the pathologic or reproductive processes in which cell proliferation is involved in the male reproductive system., (Copyright © 2014 Elsevier Inc. All rights reserved.)

High temperatures have negative effects on sperm quality leading to temporary or permanent sterility. The study tried to confirm the harmful effects of high temperatures on epididymal sperm cells in comparison with other temperatures (scrotal, environmental, and refrigeration temperatures), the main objective was the assessment of the addition of retinol as an antioxidant agent to improve sperm quality parameters. Testes from 10 bulls were collected from a slaughterhouse. Sperm cells were flushed from the cauda epididymis and deferent duct and assessed for sperm quality parameters at recovery. Afterward, sperm cell samples were exposed to one of four different temperatures (4 °C, 22 °C, 32 °C, and 41.5 °C for 3 hours) in presence or absence of retinol in the storage extender. Percentages of viability and morphologic abnormalities were determined using eosin-nigrosin staining. Acrosome integrity and sperm plasma membrane integrity were assessed by fluorescence Pisum sativum agglutinin lectin (FITC-PSA) staining and the hypo-osmotic swelling test, respectively. Total and progressive motility were analyzed by computer-assisted sperm analysis. Sperm quality parameters were mainly affected by high temperatures (41.5 °C). The addition of all-trans-retinol to the storage extender did not show any effect on sperm quality parameters. However, the percentage of sperm cells with altered acrosome was significantly reduced when retinol was present in the extender under heat stress conditions (41.5 °C). In conclusion, retinol might stabilize sperm acrosomal membrane in situations of oxidative stress because of high temperatures., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The phenomenon of teratozoospermia in felids is not fully understood. In this study, we investigated the testicular androgen:estrogen balance in domestic cats and correlated these data with epididymal sperm morphology and the degree of spermatogenic activity. During spring and summer, testes and blood samples were obtained from 37 mixed-breed domestic cats (12 to 48 mo). The epididymal sperm were harvested and evaluated for sperm counts, motility, and morphology. Distal cytoplasmic droplets were not considered a defect, and samples were considered normozoospermic if they contained more than 60% normal sperm (N = 25) or teratozoospermic if they contained less than 45% normal sperm (N = 12). The testicular and serum concentrations of testosterone (T) and 17β-estradiol (E2) were determined with an enzyme immunoassay. The gonadosomatic index and epididymal sperm numbers and motility did not differ between groups. The percentage of normal sperm was higher in normozoospermic (74.3 ± 2.0, mean ± SEM) than in teratozoospermic samples (43.1 ± 1.4). The most prevalent sperm defects in the teratozoospermic group were abnormal acrosomes (9.7 ± 2.0) and bent midpieces (12.2 ± 2.0) or tails (24.0 ± 2.7) with cytoplasmic droplets. Histomorphometric data were similar between groups, although there was a lower Leydig cell nuclear volume in teratozoospermic samples. Normozoospermic samples contained a higher percentage of haploid cells and had a higher index of total spermatogenic transformation than teratozoospermic samples. Serum concentrations of T (0.5 ± 0.1 vs. 0.8 ± 0.4 ng/mL) and E2 (9.5 ± 1.2 vs. 11.4 ± 2.3 pg/mL) and testicular T concentrations (471.6 ± 65.3 vs. 313.4 ± 57.6 ng/g) were similar between groups. However, compared with normozoospermic samples, teratozoospermic samples had higher testicular E2 concentrations (8.5 ± 3.6 vs. 5.4 ± 0.5 ng/g) and a lower T:E2 ratio (31.8 ± 4.1 vs. 87.2 ± 11.6). There were significant correlations between testicular E2 values and percentages of normal sperm (r = -0.55) as well as those with primary sperm defects (r = 0.58) or abnormal acrosomes (r = 0.64). The T:E2 ratio was also correlated with meiotic index (r = 0.45) and percentage of normal sperm (r = 0.58). In conclusion, a high testicular E2 concentration and a reduced T:E2 ratio were significantly associated with higher ratios of abnormal sperm types, suggesting that the balance between androgens and estrogens is an important endocrine component in the genesis of teratozoospermia in felids., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The need for information on the reproductive physiology of different wildlife species is important for ex situ conservation using such methods as in vitro fertilization (IVF). Information on species reproductive physiology and evaluation of sperm quality using accurate, objective, repeatable methods, such as computer-assisted sperm analysis (CASA) for ex situ conservation has become a priority. The aim of this study was to evaluate motility patterns of antelope epididymal spermatozoa incubated for 4 h under conditions that support bovine IVF using CASA. Cauda epididymal spermatozoa were collected postmortem from testicles of springbok (N=38), impala (N=26), and blesbok (N=42), and cryopreserved in biladyl containing 7% glycerol. Spermatozoa were thawed and incubated in Capacitation media and modified Tyrode lactate (m-TL) IVF media using a protocol developed for domestic cattle IVF. The study evaluates 14 motility characteristics of the antelope epididymal sperm at six time points using CASA. Species differences in CASA parameters evaluated under similar conditions were observed. Several differences in individual motility parameters at the time points were reported for each species. Epididymal sperm of the different antelope species responded differently to capacitation agents exhibiting variations in hyperactivity. Motility parameters that describe the vigor of sperm decreased over time. Spermatozoa from the different antelope species have different physiological and optimal capacitation and in vitro culture requirements. The interspecies comparison of kinematic parameters of spermatozoa between the antelopes over several end points contributes to comparative sperm physiology which forms an important step in the development of species specific assisted reproductive techniques (ARTs) for ex situ conservation of these species., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The safety of testicular fine needle aspiration (FNA) has been proven in dogs but has not been fully established in men, while studies in rats have given contradictory results. Furthermore, the extent of damage inflicted by multiple punctures is unknown. The aim of this study was to determine the impact of FNA and of the number of punctures on the feline testis with clinical, gross anatomy and histological examinations. Twenty-seven sexually mature healthy laboratory Domestic Shorthair cats were randomly assigned to two groups: 5 cats in which no FNA was performed (control group), and 22 cats which had their left and right testis punctured with a 26 ga needle towards 3 and 8 directions, respectively (experimental group). Two cats at a time were orchiectomized 5 or 30 min, 1, 2, 4, 7 or 14 days or 1, 2, 3 or 4 mo post-aspiration. The cats of the control group were also orchiectomized. During the first week post-aspiration clinical examination revealed vaginal cavity hematoma (8/44 testes), while the histological findings were focal hemorrhagic areas (20/24 testes), erythrocytes inside the seminiferous tubules' lumen (9/24 testes), and germinal cell degeneration in <1.94% of the seminiferous tubules (15/24 testes). After the first week the histological findings were germinal cell degeneration in <2.14% of the seminiferous tubules (19/20 testes) and enlargement of the lumen of <5.16% of the seminiferous tubules (7/20 testes). The germinal epithelium and interstitium had an overall normal appearance. No significant differences were observed between the left and right testis. The results of the study indicate that testicular FNA should be considered a safe procedure in the cat when up to 8 punctures are performed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The buffalo is one of the few domestic animals that has a seasonal mating cycle, influenced by the photoperiod. It is known that the photoperiod regulates gonadal function probably via the pineal and/or hypothalamus-pituitary axis. Moreover, the hypothalamus (melatonin) and gonads influence the production of the signaling transmitter nitric oxide (NO), suggesting that the NO may have an important role in the regulation of gonadotropin-releasing hormone secretion. This further suggests the hypothesis that NO in the epididymis has an important role in the maturation of spermatozoa and their motility and posterior fertilization capacity. The aim of the present study is to investigate the seasonal variations in the morphology of the epididymis by means histochemical and immunocytochemical techniques. We used the NADPH-d, nitric oxide synthase (NOS) I and NOS III to clarify the relationship between epididymis function and NO signaling activity. The results of this work show that NO is present in the caput of epididymis during short photoperiods, i.e., periods of maximum gonadal activity (winter) and absent during long photoperiods, i.e., periods of gonadal regression according to the previously described role of NO in spermatozoa capacitation and motility in the caput epididymis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In mammals, the epididymis has numerous interrelated functions including absorptive and secretory activity that affect luminal environment and cell membrane, and the maturation and storage of sperm. Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymis and the motility of epididymal spermatozoa should be a balance between the maturation of flagellum and the inhibition of the flagellar machinery. In this study maturational change in sperm characteristics were evaluated in the epididymis of donkey. Spermatozoa collected from four portions of the epididymis (head, cranial corpus, caudal corpus, tail) were compared before and after ejaculation for viability, mitochondrial activity, kinetic parameters, and morphology. A significant increase in the mitochondrial activity along the epididymis was reported, suggesting a possible involvement in the motion mechanism. This should be corroborated by the significant correlation between mitochondrial activity and the total and progressive motility and the increase in velocities of spermatozoa recorded by computer-assisted sperm analysis. The percentage of most of the abnormal spermatozoa were similar in all tracts, with a great variability between jackasses. Only the bent midpiece percentage decreased significantly along epididymis. A significant increase in the percentage of distal cytoplasmic droplets (DCD), and a simultaneous decrease in the proximal cytoplasmic droplets (PCD), was found. The DCD fell down after ejaculation suggesting the late loss of the cytoplasmic residual (DCD) in the donkey, as hypothesized in the stallion. Because the prevalence of PCD were similar in both tail epididymal and ejaculated spermatozoa, a defect of the maturative process in the PCD sperm should be speculated., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mammalian spermatozoa acquire functionality during epididymal maturation and ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to investigate the impact of epididymal maturation, ejaculation and capacitation on phosphotyrosine content of sperm proteins. Western blot, immunocytochemical and flow cytometry analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues of the sperm head followed by a subtle increase after in vitro capacitation. As cells pass from caput to cauda epididymis, tyrosine phosphorylation becomes confined to a triangular band over the posterior part of midacrosome region, whereas in vitro capacitation causes a spread labeling over the whole head. Different bands with phosphotyrosine residues were detected during epididymal maturation and after in vitro capacitation: 1) 93, 66 and 45 kDa bands with specific phosphotyrosine expression in immature spermatozoa; 2) 76, 23 and 12 kDa bands with specific phosphotyrosine expression in mature spermatozoa, being significantly increased in their expression after in vitro capacitation; 3) 49, 40, 37, 30, 26 and 25 kDa constitutive bands that increased their phosphotyrosine expression after maturation and/or in vitro capacitation; and 4) 28 and 20 kDa bands with a specific phosphotyrosine expression in in vitro capacitated spermatozoa. These results provided integral novel data of expression and location of phosphotyrosine residues during epididymal maturation, ejaculation and in vitro capacitation of boar spermatozoa. Two new constitutive proteins bands of 26 and 25 kDa with phosphotyrosine residues were also identified., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The objective was to examine, in vitro, the motility, acrosomal proteolytic activity (APA), and penetrating ability of fowl sperm recovered from the testis and epididymis, as well as the proximal, middle, and distal vas deferens, to assess the potential fertilizing ability of sperm as a function of maturation. A motile sperm separation technique was used to estimate sperm motility with Accudenz, a gelatin slide technique was used to measure the diameter of the halo around the acrosome of individual sperm as an indication of APA, and a sperm-inner perivitelline layer (IPL) interaction assay was done to estimate the number of hole formations as an indication of sperm penetration into the IPL. Sperm in the testis exhibited the least motility, produced the smallest halos, and created the least number of holes per 0.25 mm(2). Motility, diameter of the halo, and number of holes increased gradually (P < 0.05) from the epididymis to the distal vas deferens and were markedly different (P < 0.05) between testicular and deferent duct sperm. Based on these in vitro experimental findings, we inferred that fowl sperm undergo a gradual process of maturational changes in motility, APA, and penetrability as a means of acquiring potential fertility during their passage throughout the male genital tract., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Testicles were isolated from thirty five apparently healthy dromedary camels (Camelus dromedarius), aged between 5 to 18 years, in a local slaughterhouse during the rutting season. Epididymal fluid was collected from one epididymis for determination of twelve biochemical and antioxidant parameters using ELISA commercial kits. Spermatozoa were harvested from each region of the other epididymis (head, body and tail) and stored in SHOTOR®, Green buffer® + 20% egg yolk and INRA-96® extenders at 5 and 30 °C. Results revealed that, in the epididymal fluid, concentrations of testosterone, glucose, albumin, total protein, cholesterol, fatty acids, iron, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were 5.19 ± 1.69 ng/mL, 3.10 ± 0.41 mmol/L, 6.26 ± 1.26 g/dL, 0.50 ± 0.07 mg/dL, 1.74 ± 0.09 mmol/L, 6.62 ± 0.81 nmol/ul, 926.20 ± 100.18 ug/dL, 51.17 ± 7.74 mIU/ml, and 143.16 ± 18.67 mIU/ml, respectively. The antioxidants activities of catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD) in the epididymal fluid were 121.55 ± 6.57 nmol/min/ml, 59.35 ± 10.98 nmol/min/ml and 0.18 ± 0.03 U/ml, respectively. Epididymal sperm motility and concentration were higher (P < 0.05) in the body and tail than the head. The viability indices of total and forward sperm motility, at 5 and 30 °C, obtained from the tail region were superior (P < 0.05) in both SHOTOR® and INRA-96® extenders than Green buffer extender. It may be concluded that INRA-96® extender is the best for storing dromedary epididymal spermatozoa at 5 and 30 °C., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

The objective was to compare the use of powdered coconut water (ACP-109c; ACP Biotecnologia, Fortaleza, CE, Brazil) and Tris extenders for recovery and cryopreservation of epididymal sperm from agouti. The caudae epididymus and proximal ductus deferens from 10 sexually mature agoutis were subjected to retrograde washing using ACP-109c (ACP Biotecnologia) or Tris. Epididymal sperm were evaluated for motility, vigor, sperm viability, membrane integrity, and morphology. Samples were centrifuged, and extended in the same diluents plus egg yolk (20%) and glycerol (6%), frozen in liquid nitrogen, and subsequently thawed at 37°C for 1 min, followed by re-evaluation of sperm characteristics. The two extenders were similarly efficient for epididymal recovery, with regard to the number and quality of sperm recovered. However, for both extenders, sperm quality decreased (P < 0.05) after centrifugation and dilution. After sperm cryopreservation and thawing, there were (mean ± SEM) 26.5 ± 2.6% motile sperm with 2.6 ± 0.2 vigor in the ACP-109c (ACP Biotecnologia) group, which was significantly better than 9.7 ± 2.6% motile sperm with 1.2 ± 0.3 vigor in Tris. In conclusion, agouti epididymal sperm were successfully recovered using either ACP-109c (ACP Biotecnologia) or Tris extenders; however, ACP-109c (ACP Biotecnologia) was a significantly better extender for processing and cryopreserving these sperm., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Recovering and storing sperm from the epididymides of males of rare felidae is useful for preserving the species. The objective of the present study was to determine pregnancy rates following artificial insemination (AI) of frozen-thawed epididymal sperm, which were cryopreserved following low-temperature storage of the epididymides. In this study, these sperm were used for unilateral intrauterine AI (UIUAI) or unilateral intratubal AI (UITAI) using 40 × 10(6) and 10 × 10(6) sperm, respectively. The caudal epididymides of 17 cats were stored at 4 °C for 24 h after castration. Artificial insemination of seven female cats was performed on Days 3 or 4 (start of estrus = Day 1) by UIUAI, 20 h after injection of 100 IU hCG to induce ovulation. Furthermore, UITAI at 24 h (UITAI-24) or 30 h (UITAI-30) after hCG were also done (five cats per group). It was noteworthy that AI by UIUAI and UITAI-24 was performed before ovulation, whereas AI by UITAI-30 was performed after ovulation. Pregnancy rates were 28.6% (2/7) by UIUAI, 80% (4/5) by UITAI-24, and 20% (1/5) by UITAI-30. Litter size was one or two by UIUAI, and one to four by UITAI. Spontaneous abortion occurred on Days 25-30 of pregnancy in one of the two female cats pregnant following UIUAI, and in two of five female cats pregnant following UITAI. Based on the high pregnancy rate obtained with 10 × 10(6) sperm in the UITAI-24 group (AI performed before ovulation), we concluded that this was the most appropriate method for AI with frozen-thawed epididymal sperm after initial low-temperature storage of epididymides., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The objective was to evaluate the suitability of an animal protein-free semen extender for cryopreservation of epididymal sperm from the two subspecies of North American bison: plains (Bison bison bison) and wood (Bison bison athabascae) bison. Both cauda epididymides (from six plains and five wood bison) were minced and incubated in Sp-TALPH buffer for approximately 2 h at 37 °C to release actively motile sperm. Sperm suspensions were filtered, centrifuged and the sperm pellet from each bull was divided into two fractions and diluted either in egg yolk containing extender, Triladyl, or in an animal protein-free extender, Andromed, and equilibrated for 20 min at 37 °C. Thereafter, samples were chilled and cryopreserved. Frozen-thawed sperm were evaluated for motility (computer assisted sperm analysis), viability (SYBR 14 and propidium iodide), acrosome integrity (FITC conjugated PSA), cryocapacitation (tyrosine phosphorylation of sperm proteins as a biomarker), and fertilizing ability (in a heterologous IVF system). There was no significant difference for progressive motility, viability, and acrosome integrity between the two extenders for plains bison (36.8 ± 9.0, 60.5 ± 17.4, and 77.3 ± 4.6%; overall mean ± SD) as well as for wood bison (11.7 ± 8.1, 13.7 ± 5.6, and 73.4 ± 4.2%). Levels of tyrosine phosphorylation did not differ for sperm preserved in the two extenders for both subspecies, although an inter-bull variability in the response to tyrosine phosphorylation between extenders was suggested for plains bison. Fertilization percent did not differ significantly between extenders for plains bison (84.16 ± 9.92%, overall mean ± SD) and for wood bison (59.53 ± 19.99%). In conclusion, in the absence of significant difference between extenders in post-thaw sperm characteristics, we inferred that Andromed (animal protein-free) was suitable for cryopreservation of epididymal sperm from North American bison., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cryopreservation of sperm from tree shrews, which are considered primitive primates, would enhance genetic management and breeding programs. Epididymal sperm were surgically harvested from male tree shrews, cryopreserved in two Tes-Tris-based cryodiluents, and used in four experiments. In Experiment 1, there were no significant differences in motility and acrosome integrity among five concentrations of egg yolk in TTE after cooling to 4 °C. However, sperm frozen in TTE containing 20% egg yolk at -172 °C/min had better (P < 0.05) post-thaw motility and acrosome integrity. In Experiment 2, sperm held for 10 min prior to storage in liquid nitrogen had greater motility than those held for 5 or 15 min (P < 0.05), but acrosome integrity was not different (P > 0.05) among treatments. In Experiment 3, sperm frozen in TTE diluent had higher (P < 0.05) motility and acrosome integrity than those in TEST diluent. In Experiment 4, there were no differences (P > 0.05) in the fertilization rate of oocytes and the proportion of tree shrews yielding fertilized oocytes, following AI with fresh versus frozen sperm. In conclusion, tree shrew epididymal sperm were successfully cryopreserved, as assessed by post-thaw motility, acrosome integrity, and fertilizing ability., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The objective of the present study was to evaluate the ability of rosemary to protect epididymal boar spermatozoa from freeze-thaw damage. Testis from eight boars were collected at the slaughterhouse in two trials. In the laboratory, sperm from epididymis were recovered by flushing and cryopreserved in lactose-egg yolk solution supplemented with various concentrations (low; medium; high) of rosemary. After thawing, total motility, viability, acrosome integrity, response to hypoosmotic swelling test (HOST) and malonaldehide (MDA) concentration were assessed. The results showed that there was an increase in motility at 1, 2 and 3 h in the presence of rosemary. The addition of this herb provided a significant beneficial effect on viability at 2 h of incubation, compared to the control group. Conversely, acrosome status was not affected by any extender. Higher concentration of rosemary produced significant improvement in percentages of positive HOST at 0 and 1 h, whereas no impact was observed at the end of incubation. Considering membrane lipid peroxidation, a greater decrease in MDA production was observed when rosemary content was raised. Rosemary-enriched freezing extender improved the post-thaw epididymis boar spermatozoa quality, showing a significant correlation between rosemary concentration and concentration of MDA. Further studies are needed to define the active component in rosemary that prevents peroxidation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The objective was to establish a protocol for the collection and evaluation of epididymal sperm in agoutis. Eight males (1-2 y old) underwent left orchidectomy and epididymal sperma were collected by retrograde flush. Average values were flush volume 32 μL, pH 6.9, sperm concentration 748 x 10(6) sperm/mL, with motility 86.5% and vigor 4.6. Viable sperm were present in all flush samples; 66% of sperm were alive, and 41.9% of sperm responded positively to the hypoosmotic test (using distilled water). There were 21.1% morphologically abnormal sperm, of which 2.0 and 19.1% were primary and secondary defects, respectively. The acrosome was intact in 99.5% of sperm. The sperm head was 4.89 ± 0.41 μm long and 3.13 ± 0.35 μm wide, with an area of 13.01 ± 2.01 μm(2). Midpieces were 5.33 ± 0.44 μm long and 0.98 ± 0.13 wide, sperm tails were 29.91 ± 2.29 μm, and overall sperm length was 40.12 ± 2.44 μm. In conclusion, epididymal sperm collection from agoutis was satisfactory; the collected sperm has the potential to be stored, facilitating development of other reproductive biotechnologies for this species., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This work was designed to study how this ability is affected by different sperm treatments routinely used for in vitro fertilization (IVF) assay. In this study, boar sperm samples from epididymal or ejaculated origin were processed by three different methods: left unwashed (NW group), washed in Dulbecco's phosphate-buffered saline supplemented with 0.1% BSA (BSA group), and washed on a Percoll(®) gradient (PERCOLL group). After preparation of semen samples, changes in motility patterns were studied by CASA, calcium uptake by spectrofluorimetry, and ROS generation, spontaneous acrosome reaction, and lipid disorder by means of flow cytometry. Finally IVF assays were also performed with the different semen samples and penetrability results evaluated at 2 and 4 h post insemination (hpi). Independently of the sperm treatment, epididymal spermatozoa showed higher values of progressive motility, percentage of live cells with low lipid disorder, and penetration ability at 4 hpi than the corresponding ejaculated spermatozoa. Ejaculated spermatozoa showed higher levels of calcium uptake, ROS generation and percentage of spontaneous acrosome reaction than epididymal sperm. Regarding sperm treatments, PERCOLL group showed the highest values for some motility parameters (linearity of the curvilinear trajectory, straightness, and average path velocity/curvilinear velocity), ROS generation and penetration ability at 2 and 4 hpi; however this same group showed the lowest values for sperm curvilinear velocity and lateral head displacement. From all experimental groups, ejaculated-PERCOLL-treated spermatozoa showed the highest fertilization ability after 2 hpi. Results suggest that capacitation pathways can be regulated by suitable treatments making the ejaculated sperm able to reach capacitation and fertilize oocytes in similar levels than epididymal spermatozoa, although most of the studied capacitation-associated changes do not correlate with this ability., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Knowledge concerning reproduction in common hippopotamus is scarce and in particular very little is known about male reproductive physiology and sperm cryopreservation. Testes were obtained from nine castrated bulls and sperm extracted from the epididymides of eight of these individuals. Mean ± SEM values of reproductive parameters were: testicular weight (including epididymis and tunicas)--275.9 ± 54.1 g, total sperm motility--88.1 ± 4.2%, total cells extracted--11.0 ± 3.6 × 10(9), intact acrosome--87.7 ± 1.8%, intact sperm morphology--51.6 ± 4.1%, and, for 3 individuals, hypoosmotic swelling test for membrane integrity-83.3 ± 1.8%. Chilled storage extenders tested were Berliner Cryomedium (BC), Biladyl(®), modification of Kenney modified Tyrode's medium (KMT), and Human Sperm Refrigeration Medium (HSRM). Extender had significant effect on post-dilution motility and motility and intact morphology after 4h and 24h at 4°C (P ≤ 0.007 for all). Berliner Cryomedium and HSRM were superior to Biladyl(®) and KMT. Freezing extenders tested were BC with either 6% dimethyl sulfoxide (Me(2)SO), or 5%, 7%, or 10% glycerol. Post-thaw motility was < 5% in 3/7 bulls in all extenders. When frozen in BC with 6% Me(2)SO, one bull had 15% post-thaw motility and 3/7 had 20 to 60%. In glycerol, 3/7 had 15-30% post-thaw motility in 5%, 2/7 in 7%, and 1/7 in 10%. The extender had significant effect on post-chilling motility (P = 0.008), post-thaw morphology (P = 0.016), and motility 30 min after thawing (P = 0.015). Berliner Cryomedium with 6% Me(2)SO or 7% glycerol were the freezing extenders of choice. Information obtained in this study allows initiation of cryobanking of sperm from the common hippopotamus which is of particular importance for genetically valuable individuals., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The collection of semen from tomcats by urethral catheterization (CT) after medetomidine administration offers a novel and easy approach to obtain good quality sperm for in vitro fertilization. This study was designed to compare the sperm quality parameters and in vitro fertilizing capacity of CT spermatozoa with those of spermatozoa retrieved after epididymal slicing (EP). Semen was collected in seventeen adult cats by urethral catheterization, after which the cat was orchiectomized. Motility, morphology, plasma membrane integrity, acrosomal status, and in vitro fertilizing capacity of both fresh CT and EP samples were evaluated. The results showed that both total and progressive motility, as well as the percentage of normal spermatozoa, were higher for EP sperm than for CT sperm (P<0.01). Epididymal sperm had a lower percentage of spermatozoa with an intact acrosome (P<0.01), while CT sperm contained more spermatozoa with tail abnormalities (P<0.01). Other morphological parameters, as well as plasma membrane integrity, did not differ (P>0.05) between CT and EP sperm. Nevertheless, no difference (P>0.05) in in vitro fertilizing capacity between spermatozoa collected by means of the two different methods was found. In conclusion, semen collection by means of urethral catheterization after medetomidine administration yields fertilization results similar to epididymal slicing, despite the fact that several sperm variables were different. Since this novel catheterization technique is repeatable, is easy to perform and facilitates semen preparation protocols, it may be preferable for routine IVF experiments with fresh spermatozoa.

The objectives were to determine the: 1) extent of epithelial and red blood cell contamination in epididymal cat sperm samples recovered by the cutting method; 2) efficacy of simple washing, single-layer centrifugation (SLC), and swim-up for selecting epididymal cat sperm; and 3) effects of freezing and thawing on cat sperm selected by various techniques. Ten unit samples were studied; each contained sperm from the cauda epididymides of four cats (total, approximately 200 x 10(6) sperm) and was equally allocated into four treatments: 1) simple washing, 2) single-layer centrifugation through colloid prior to cryopreservation (SLC-PC), 3) single-layer centrifugation through colloid after cryopreservation (SLC-AC), and 4) swim-up. Centrifugation (300 x g for 20 min) was done for all methods. The SLC-PC had a better recovery rate than the SLC-AC and swim-up methods (mean+/-SD of 16.4+/-8.7, 10.7+/-8.9, and 2.3+/-1.7%, respectively; P<0.05). The SLC-PC, SLC-AC and swim-up samples contained less red blood cell contamination than simple washed samples (0.02+/-0.01, 0.02+/-0.04, 0.03+/-0.04, and 0.44+/-0.22 x 10(6) cells/mL, respectively; P<0.05). Although the proportion of sperm with head abnormalities did not differ among selection methods (P>0.05), SLC-PC yielded the highest percentage of sperm with normal midpieces and tails (P<0.05), due to the lowest proportion of coiled tails (P<0.05). Furthermore, the SLC-PC was as effective as swim-up in removing sperm with proximal droplets, and selecting motile sperm, as well as those with intact membranes and DNA (P>0.05). In conclusion, both SLC-PC and swim-up improved the quality of epididymal cat sperm, including better morphology, membrane and DNA integrity, and removal of cellular contamination. However, SLC had a better sperm recovery rate than swim-up., (2010 Elsevier Inc. All rights reserved.)

In Bos taurus, at ejaculation, epididymal sperm acquire a number of proteins secreted in the seminal plasma that increase their ability to interact with the female reproductive tract. Sperm-oviduct interaction comprises a transient sperm adhesion to the isthmus, the lower portion of the oviduct, followed by sperm release around ovulation. Oviductal fluid molecules, such as sulfated glycoconjugates and disulfide-reductants, are able to release bovine ejaculated sperm bound to the oviductal epithelium in vitro through the reduction of sperm surface protein disulfides to sulfhydryls. To understand whether the sperm molecules sensitive to releasing signals are already exposed on the surface of epididymal sperm, we studied the ability of cauda epididymal sperm to adhere to the oviductal epithelium and to be released by sulfated glycoconjugates and the disulfide-reductant penicillamine. Surface protein sulfhydryls in cauda epididymal sperm were analyzed in the initial suspension, in sperm bound to the in vitro-cultured oviductal epithelium, and in released sperm. Results showed that epididymal sperm are able to bind the oviductal epithelium in vitro, although at a lower extent than frozen-thawed ejaculated sperm; the interaction is mediated by oviductal cell microvilli that closely bind to the plasma membrane of the sperm head rostral region, as previously shown for ejaculated sperm. The sulfated glycoconjugates heparin, fucoidan, and dextran sulfate, as well as the disulfide-reductant penicillamine, are all powerful inducers of sperm release. The level of sulfhydryls in sperm surface proteins was (1) high in the initial sperm suspension; (2) low in bound sperm; (3) markedly increased in sperm released by heparin or by penicillamine. In conclusion, epididymal sperm are already able to bind the oviductal epithelium and to respond to the inducers of release through the reduction of sperm surface protein disulfides to sulfhydryls., ((c) 2010 Elsevier Inc. All rights reserved.)

Interspecies hybridization of bovids occurs between domestic cattle and at least three other species; American bison (Bison bison), yak (Bos grunniens) and banteng (Bos banteng). Birth of a cattlexbuffalo (Bubalus bubalis) hybrid has reportedly occurred in Russia and in China, but these reports were not authenticated. Such hybrids could be important in improving livestock production and management of diseases that impede production in tropical Africa. This study investigated hybridization between cattle and its closest African wild bovid relative, the African buffalo (Syncerus caffer caffer). In an attempt to produce cattlexbuffalo hybrid embryos in vitro, matured cattle oocytes were subjected to a standard in vitro fertilization (IVF) procedure with either homologous cattle (n=1166 oocytes) or heterologous African buffalo (n=1202 oocytes) frozen-thawed epididymal sperm. After IVF, 67.2% of the oocytes inseminated with the homologous cattle sperm cleaved. In contrast, fertilization with buffalo sperm resulted in only a 4.6% cleavage rate. The cleavage intervals were also slower in hybrid embryos than in the IVF-derived cattle embryos. Of the cleaved homologous cattle embryos 52.2% progressed to the morula stage compared with 12.7% for the buffalo hybrid embryos. No hybrid embryos developed beyond the early morula stage, while 40.1% of the cleaved cattlexcattle embryos developed to the blastocyst stage. Transfer of buffalo hybrid IVF embryos to domestic cattle surrogates resulted in no pregnancies at 60 days post-transfer. This study indicates that interspecies fertilization of cattle oocytes with African buffalo epididymal sperm can occur in vitro, and that a barrier to hybridization occurs in the early stages of embryonic development. Chromosomal disparity is likely the cause of the fertilization abnormalities, abnormal development and subsequent arrest impairing the formation of hybrid embryos beyond the early morula stage. Transfer of the buffalo hybrid embryos did not rescue the embryos from development arrest.

This study was carried out to investigate the cryoprotective efficacy of Equex STM Paste on the quality of canine post-thaw epididymal spermatozoa. Following castration, spermatozoa were flushed from the cauda epididymides. Epididymal spermatozoa from 13 of 16 dogs with a sperm motility of >70% were frozen in an egg yolk-Tris extender, supplemented with Equex STM Paste (0.5%, v/v); the extender free of Equex STM Paste served as a control cryoprotective diluent. The quality of spermatozoa, judged by its motility, plasma membrane integrity and acrosome integrity, was evaluated on four occasions, immediately after collection, after equilibration and at 0 and 2h post-thaw. Reducing the temperature to 4 degrees C for 2h prior to freezing decreased sperm motility (P=0.001), but had no effects on membrane integrity or acrosome integrity. Immediately after thawing, the percentage of acrosome-intact spermatozoa significantly decreased in samples frozen without Equex STM Paste compared to freshly collected or Equex-treated samples. After incubation at 37 degrees C for 2h post-thaw, a greater percentage of motile spermatozoa (P=0.018) and spermatozoa with intact acrosomes (P=0.001) were observed in Equex-treated samples compared with the control. The percentage of membrane-intact spermatozoa did not differ significantly between Equex-treated and control samples at any time. Supplementation with Equex STM Paste in the semen extender was effective for freezing canine epididymal spermatozoa because it protected acrosome integrity against damage induced by cryopreservation and it prolonged post-thaw sperm motility during in vitro incubation at 37 degrees C.

A proteomic approach was used in this study to follow the protein expression of epididymal cells during the different phases of a cell culture protocol which was able to obtain an epididymal cell monolayer. The secretory activity of intact proximal and middle caput epididymal fragments and caput, corpus and cauda epithelial cell monolayers was examined on different days of culture. Transcriptomic activity was also followed by RT-PCR for the mRNA of several previously identified major proteins. During the establishment of epididymal cell cultures, a progressive shift was found in the pattern of protein secretion. The normal epididymal protein profile, specific for each epididymal region, was progressively replaced by a less specific profile with the secretion of new proteins. A correlation between protein secretion and the presence of the mRNA of the marker proteins was observed only in the first phase of culture. Most of the new proteins which appeared were characteristic of the secretion of cell monolayers cultivated over several weeks. Despite the significant modifications of the epididymal cell secretome, the presence of new proteins secreted only by cell cultures originating from a specific epididymal region shows the presence of remaining endogenous differentiation.

Although dog prostatic fluid decreases the longevity of ejaculated dog spermatozoa, it also increases their rate of motility and their fertility after vaginal insemination, as well as the fertility of epididymal spermatozoa after uterine insemination. These findings indicate a need to further characterize the effects of prostatic fluid on dog spermatozoa. This study was done to determine the effects (P<0.05) of homologous prostatic fluid added prior to cooling, after thawing, or at both times to epididymal spermatozoa from 21 dogs. The effects of two extenders were also determined. The one extender was Biladyl(*) with Equex STM paste(**) (BilEq) and the other Andromed(*) (Minitüb, Tiefenbach, Germany (*); Nova Chemical Sales, Scituate, MA, USA (**)). The response variables were percentage progressively motile spermatozoa (Prog) and morphology after thawing. Prog was measured at various times until 8h after extension (unfrozen spermatozoa) or until 2h after thawing. Prog after thawing was higher with BilEq than Andromed, when no prostatic fluid was added prior to cooling, and when prostatic fluid was added after thawing. BilEq resulted in a higher mean percentage of spermatozoa with bent principle pieces than Andromed and the addition of prostatic fluid prior to cooling resulted in lower mean percentages of cytoplasmic droplets and bent principle pieces than when none was added. The optimal combination was BilEq with prostatic fluid added prior to cooling (in order to inhibit the development of bent principle pieces) and after thawing (to achieve higher motility until 1h after thawing). This study shows that BilEq is more suitable for the freezing of epididymal spermatozoa than Andromed and that prostatic fluid improves the freezability and post-thaw longevity of epididymal spermatozoa frozen in BilEq.

Sperm recovery from the cauda epididymis can be very advantageous, for example, in case of the unexpected death of a genetically highly valuable animal, for preserving endangered species, or when the collection of sperm by other means becomes impossible. Studies indicate that epididymides stored at cooler temperatures result in better quality sperm. One of the factors that could negatively affect sperm viability during storage is lipid peroxidation, in which the sperm membrane's ability to resist attacks by reactive oxygen species (ROS) plays an important role. Another factor is the presence of cytoplasmic droplets, which appear in high numbers in epididymal sperm, and are known to influence oxidative stress. The objectives of this study were: to determine whether the post-slaughter storage temperature of the epididymis would effect the sperm membrane's resistance to lipid peroxidation and/or the sperm cell's fertilizing capacity in vitro and to elucidate the role played by the cytoplasmic droplets. Forty-eight testicles with epididymides (24 bulls) were collected following slaughter, and divided into two groups. One testicle from each pair was stored at 4 degrees C, and the other at 34 degrees C, for 2h, after which sperm was collected from the caudae epididymides. Sperm concentration was measured, and an aliquot containing 10(8)sperm was subjected to induced lipid peroxidation with ferrous sulphate and ascorbate (37 degrees C, 2h). Subsequently, thiobarbituric acid reactive substances (TBARS), as an index of lipid peroxidation, were measured. A second aliquot of the same sample was used in a routine in vitro fertilization performed in duplicate. Sperm from caudae epididymides stored at 34 degrees C resulted in lower rates of total blastocyst formation and had a higher percentage of distal droplets, when compared to sperm from epididymides stored at 4 degrees C (21.2+/-2.42 and 71.8+/-4.7% versus 33.5+/-1.8 and 23.7+/-4.7%, respectively, P<0.05). Storage temperature had no effect on TBARS levels. For samples stored at 4 degrees C, TBARS were negatively correlated with distal droplets (r=-0.63, P<0.05) and positively correlated with proximal droplets (r=0.42, P<0.05). In conclusion, our results show that short-term storage of epididymides at 4 degrees C provided sperm of higher quality and in vitro fertilizing capacity than storage at 34 degrees C. Although resistance to oxidative stress could not be shown to directly influence these results, distal sperm droplets that appeared in high numbers in the cooled epididymal sperm samples, may have exerted an antioxidant effect. We hypothesize that this protection against ROS is one of the functions of distal sperm droplets in the epididymis.

Two studies were performed to determine annual reproductive patterns in stray male dogs in the tropics. In Study 1, four dogs housed individually outdoors were monitored once monthly for 12 months, including collection and assessment of semen, measurements of scrotal width, and determination of serum testosterone and prolactin concentrations. In Study 2 (conducted concurrently), a single blood sample (for serum testosterone concentration) was collected from 220 clinically healthy dogs, and after euthanasia, scrotal width and morphology of epididymal sperm were determined. The year was divided into three seasons: warm-dry (March to June); warm-humid (July to October) and fresh-humid (November to February). In Study 1, scrotal width, ejaculate volume, sperm count and motility were significantly lower during the fresh-humid season and sperm midpiece abnormalities were significantly more common during the warm-humid and fresh-humid seasons. Serum testosterone concentrations remained constant during the year. Prolactin concentrations did not differ significantly among seasons, but had a well-defined increase from the beginning of March to the end of August. In Study 2, sperm morphology was similar to in Study 1 and serum testosterone concentrations varied nonsignificantly during the year. Environmental factors, e.g. daylength may have influenced circannual changes in prolactin secretion. Seasonal variations in some reproductive tract and seminal traits were significant but of small magnitude and the percentage of morphologically normal sperm did not vary significantly among seasons. In conclusion, healthy male dogs constantly produced sperm and were apparently fertile throughout the year.

The European Union has introduced transmissible spongiform encephalopathy (TSE) resistance breeding programmes for several sheep breeds to cope with the genetic susceptibility to Scrapie infections. Due to the different allele frequencies among breeds, strong selection for ARR alleles is associated with a loss of genetic diversity in small populations and in larger populations with unfavourable ARR allele frequencies. To ensure maintenance of genetic diversity, an adhoc cryopreservation programme was initiated employing epididymal sperm from 109 rams representing 16 different breeds within one breeding season. Epididymal semen was chosen for this adhoc programme because time consuming training of rams for ejaculated semen collection via an artificial vagina was not possible. Prior to freezing, average sperm motility was 79.7% and acrosome integrity was 93.7%. After freezing, these levels were decreased to 60.5 and 72.8%, respectively. An insemination trial using frozen-thawed epididymal semen resulted in a lambing rate of 87.5%. Results show that this semen preservation method is robust and efficient and associated with high fertility. It may also be useful for other animal species.

Abnormal chromatin condensation is not detected using classical techniques for sperm analysis. SCSA has demonstrated its usefulness in sperm chromatin analysis in several species (human, bull, stallion and boar). In this work, we studied sperm samples from red deer, ram and dog to analyze the differentiation of chromatin structure applying SCSA in epididymal and ejaculated spermatozoa. Epididymal samples were obtained from the caput, corpus and cauda by means of cuts, and ejaculated ones were obtained by electroejaculation (deer), artificial vagina (ram) and digital manipulation (dog). SCSA results suggested different critical points in sperm maturation (spermatozoa with loose chromatin to more condensed chromatin) among species: from corpus to cauda in ram and from caput to corpus in deer and dog. Moreover, we also detected differences in ruminants and dog, reflected in the appearance of SCSA plots. Indeed, ram and deer samples rendered two peaks within the sperm main population (sperm with condensed chromatin), whereas only one was detected in dog. Although some differences were observed between cauda and ejaculated samples, SCSA parameters indicated good chromatin condensation, making these samples suitable for germplasm banking. Some species-dependent modifications in the analysis of the results may be necessary to take full advantage of its analytical power.

Two experiments were conducted to determine the effects of egg yolk (EY), glycerol, and cooling rate on the cryosurvival of red deer epididymal spermatozoa. The aim of Experiment 1 was to examine the effects of two EY types (clarified EY, CE, prepared by centrifugation, and whole EY, WE), and four EY concentrations (0, 5, 10 and 20%) on cryosurvival of red deer epididymal spermatozoa. Sperm samples were diluted to a final sperm concentration of approximately 200 x 10(6)spermatozoa/ml with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility, viability and of plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Cryopreservation of red deer epididymal spermatozoa frozen in a clarified EY extender, and with a 20% EY resulted in more vigorous post-thaw and post-incubation motilities (P<0.0001). Moreover, our results showed that regardless of the egg yolk concentration tested, the best sperm quality was obtained with the use of CE. Therefore, the objective of Experiment 2 was to explore the post-thaw effects of four clarified egg yolk concentrations (0, 5, 10 and 20%), two final glycerol concentrations (3 and 6%), and two cooling rates from 22 to 5 degrees C (slow: 0.23 degrees C/min; rapid: 4.2 degrees C/min) on red deer epididymal spermatozoa. At thawing, the effects of CE and glycerol concentrations, and cooling rate, all independently affected post-thaw sperm quality, while there were no effects of interactions on post-thawing sperm quality. Therefore, we studied each variable separately. Differences (P<0.05) for most of the semen parameters evaluated were found between the two final glycerol concentrations tested, with the high values after thawing found with the use of 6% glycerol (58.8+/-1.4 versus 46.2+/-1.4, for sperm motility). Moreover, the cooling rate did not have an effect on the semen characteristics, except for NAR (P<0.05), with the high values after thawing found with the use of the rapid protocol (64.5+/-1.4 versus 59.9+/-1.4). In conclusion, the use of 20% CE and 6% glycerol in combination with a rapid cooling rate, significantly improved red deer epididymal spermatozoa freezability.

We tested the protective action of seminal plasma on epididymal spermatozoa from Iberian red deer, especially considering cryopreservation, as a means for germplasm banking improvement. We obtained seminal plasma by centrifuging electroejaculated semen, and part of it was thermically inactivated (denatured plasma; 55 degrees C 30 min). Epididymal samples (always at 5 degrees C) were obtained from genitalia harvested after regulated hunting, and pooled for each assay (five in total). We tested three seminal plasma treatments (mixing seminal plasma with samples 2:1): no plasma, untreated plasma and denatured plasma; and four incubation treatments: 32 degrees C 15 min, 5 degrees C 15 min, 5 degrees C 2h and 5 degrees C 6h. After each incubation, samples were diluted 1:1 with extender: Tes-Tris-Fructose, 10% egg yolk, 4% glycerol; equilibrated for 2h at 5 degrees C, extended down to 10(8) spz./mL and frozen. Sperm quality was evaluated before 1:1 dilution, before freezing and after thawing the samples, assessing motility (CASA) and viability (percentage of viable and acrosome-intact spermatozoa; PI/PNA-FITC and fluorescent microscopy). Plasma treatment, both untreated and denatured, rendered higher viability before freezing and higher results for most parameters after thawing. The improvement was irrespective of incubation treatment, except for viability, which rendered slightly different results for untreated and denatured plasma. This may be due to the presence of thermolabile components. We still have to determine the underlying mechanisms involved in this protection. These results might help to improve the design of cryopreservation extenders for red deer epididymal sperm.

The optimization of cryopreservation extenders is a fundamental issue for adequately performing germplasm banking on wild species. We have tested two glycerol concentrations (4 and 8%), and three extender osmolalities (320, 380 and 430 mOsm/kg; before adding cryoprotectants), for cryopreservation of epididymal and ejaculated sperm samples from Iberian red deer. All the extenders were based on Tes-Tris and fructose (for osmolality adjustment), and complemented with 20% egg yolk. Epididymal and ejaculated sperm samples were obtained from the cauda epididymis (post-mortem) and using electroejaculation, respectively. Samples were diluted 1:1 with each extender and equilibrated for 2 h at 5 degrees C. Then, they were diluted down to 100x10(6) sperm/mL and frozen at -20 degrees C/min. Post-thawed samples were assessed for motility (CASA), HOS test, proportion of swollen (osmotically challenged) cells in the untreated sample, viability and acrosomal status. For epididymal samples, 8% glycerol rendered a slightly higher proportion of intact acrosomes on viable spermatozoa than 4%; regarding extender osmolality, 380 and 430 mOsm/kg rendered higher motility results, and the 430 mOsm/kg yielded the lowest proportion of swollen spermatozoa. For ejaculated samples, 4% glycerol yielded more viable spermatozoa than 8%; for extender osmolality, 320 mOsm/kg rendered the highest percentages of progressively motile and viable spermatozoa, although 380 mOsm/kg extender was not significantly different. These results show that sample source influences extender suitability, and that extenders should be isoosmotic or rather slightly hyperosmotic. Future studies should test multiple glycerol concentrations and extender osmolalities in order to adjust them to these kinds of sample.