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Abstract This study aimed to investigate if washing ram sperm from seminal plasma (SP) could be an effective tool to extend sperm lifespan in medium-term preservation in liquid form to optimize ovine artificial insemination protocols. To this end, in Experiment 1 SP was added to a sperm model without previous contact with this substance (ram epididymal sperm) at the beginning or the end of a 48-hour preservation protocol at 5 °C (n = 13). Sperm motility and kinetic parameters and sperm functionality in terms of sperm viability, apoptosis, mitochondrial activity and reacted acrosomes were assessed after 6 h of storage at 15 °C (standard liquid preservation method) and 24 and 48 h at 5 °C. Extended sperm showed better results after 48 h when stored in the absence than in the presence of SP in most sperm quality parameters. Moreover, the final SP supplementation of this experimental group resulted in the highest sperm motility and kinetic parameters, viability and mitochondrial activity. These results suggested that initial SP deprivation could be beneficial in a medium-term ram sperm preservation protocol in liquid form, as well as a final supplementation. Therefore, we conducted Experiment 2 to evaluate the effect of SP removal from freshly ejaculated ram semen under the same storage conditions as in Experiment 1 (n = 12). Surprisingly, SP withdrawal impaired sperm functionality, leading to increased apoptosis and decreased mitochondrial activity after 24 and 48 h at 5 °C. Conversely, SP supplementation at the end of the preservation protocol of the ejaculate processed as usual had a positive effect on sperm quality and fertility. To summarize, SP absence was beneficial for a medium-term preservation protocol (up to 48 h at 5 °C) of ram epididymal sperm, but the same preservation protocol for ram ejaculated sperm revealed a possible failure of the SP removal method in avoiding the sperm-SP interaction effect. Meanwhile, SP supplementation of ram semen at the end of the preservation protocol increased in vitro sperm quality and fertility after artificial insemination.

This study aimed to investigate if washing ram sperm from seminal plasma (SP) could be an effective tool to extend sperm lifespan in medium-term preservation in liquid form to optimize ovine artificial insemination protocols. To this end, in Experiment 1 SP was added to a sperm model without previous contact with this substance (ram epididymal sperm) at the beginning or the end of a 48-hour preservation protocol at 5 °C (n = 13). Sperm motility and kinetic parameters and sperm functionality in terms of sperm viability, apoptosis, mitochondrial activity and reacted acrosomes were assessed after 6 h of storage at 15 °C (standard liquid preservation method) and 24 and 48 h at 5 °C. Extended sperm showed better results after 48 h when stored in the absence than in the presence of SP in most sperm quality parameters. Moreover, the final SP supplementation of this experimental group resulted in the highest sperm motility and kinetic parameters, viability and mitochondrial activity. These results suggested that initial SP deprivation could be beneficial in a medium-term ram sperm preservation protocol in liquid form, as well as a final supplementation. Therefore, we conducted Experiment 2 to evaluate the effect of SP removal from freshly ejaculated ram semen under the same storage conditions as in Experiment 1 (n = 12). Surprisingly, SP withdrawal impaired sperm functionality, leading to increased apoptosis and decreased mitochondrial activity after 24 and 48 h at 5 °C. Conversely, SP supplementation at the end of the preservation protocol of the ejaculate processed as usual had a positive effect on sperm quality and fertility. To summarize, SP absence was beneficial for a medium-term preservation protocol (up to 48 h at 5 °C) of ram epididymal sperm, but the same preservation protocol for ram ejaculated sperm revealed a possible failure of the SP removal method in avoiding the sperm-SP interaction effect. Meanwhile, SP supplementation of ram semen at the end of the preservation protocol increased in vitro sperm quality and fertility after artificial insemination. [ABSTRACT FROM AUTHOR]

Simple Summary: The choice of the optimal sperm preservation method is an important consideration in animal breeding. Stored semen can be used for reproductive purposes to introduce new genotypes and prevent inbreeding, which poses a considerable problem in cervid farms. The aim of this study was to evaluate and compare the effect of storage time and storage method (liquid state/epididymides) on the motility, morphology, and antioxidant status of European red deer sperm stored at 5 °C for up to six days (D0-D6). Sperm samples were assessed for motility, viability, morphology, activity of antioxidant enzymes (superoxide dismutase, SOD; glutathione peroxidase, GPx; catalase, CAT), and lipid peroxidation (based on malondialdehyde, MDA, content). Significant differences between storage variants were noted on D2 in sperm morphology; on D4 in the percentage of progressively motile sperm, MDA content, and SOD and GPx activity; and on D6 in the percentage of motile and viable spermatozoa. Sperm motility, viability, and antioxidant status are more effectively preserved during liquid storage than epididymal storage. Morphological and functional abnormalities of sperm were observed earlier during epididymal storage, which suggests that spermatozoa can be stored for shorter periods of time in the epididymides than in a liquid state. The aim of this study was to evaluate the motility, morphology, and antioxidant status of European red deer sperm stored in a liquid state (variant I) and in the epididymides (variant II). Spermatozoa were harvested post-mortem from the cauda epididymis. Sperm samples in both variants were stored for up to six days (D6) at 5 °C. Spermatozoa were assessed for motility, viability, morphology, activity of antioxidant enzymes (superoxide dismutase, SOD; glutathione peroxidase, GPx; catalase, CAT), and lipid peroxidation (malondialdehyde, MDA, content). Sperm samples were analyzed on storage days 0, 2, 4, and 6 (D0-D6). Storage time and storage method significantly (p ≤ 0.05) influenced the examined variables. On D2, a decrease in motility and acrosomal integrity was observed in both storage variants, whereas a decrease in viability and an increase in MDA content were noted in spermatozoa stored in the epididymides. On D4, higher values of SOD and GPx activity and MDA content were noted in variant I than in variant II. Catalase activity was very low. GPx is the key enzyme that participates in the reduction of hydrogen peroxide in sperm cells. Spermatozoa stored in a liquid state were characterized by higher motility and viability, improved morphology and antioxidant status than those stored in the epididymides; therefore, liquid storage is more recommended for short-term preservation of epididymal spermatozoa. [ABSTRACT FROM AUTHOR]

Background: Epididymal sperm preservation is a simple conservation approach that can help prevent the loss of high genetic quality of farm animals. The chance of loss increases, especially during disease outbreaks or other interruptions to normal reproduction function. Aim: This study looked into the ability of preserved ram epididymal sperm to fertilize oocytes. Due to motility becoming an issue following sperm storage for fertilization, the sperm micro injection of known as intracytoplasmic sperm injection (ICSI) approach was employed. Methods: The study was divided into two parts. First, involved preservation of epididymal sperm at 5°C for 12 days. During preservation, sperm quality parameters namely motility, viability, intact membrane, acrosome, and DNA are evaluated every three days. For fertility test in the second experiment, matured oocytes were injected with immotile sperm discovered in the last days of preservation. The presence of pronucleus development following in vitro culture is used as an indicator of sperm ability to activate and fertilize oocytes. Results: All sperm quality parameters significantly (p [Open Vet J 2024; 14(2.000): 707-715]

Background: Epididymal sperm preservation is a simple conservation approach that can help prevent the loss of high genetic quality of farm animals. The chance of loss increases, especially during disease outbreaks or other interruptions to normal reproduction function. Aim: This study looked into the ability of preserved ram epididymal sperm to fertilize oocytes. Due to motility becoming an issue following sperm storage for fertilization, the sperm microinjection known as intracytoplasmic sperm injection approach was employed. Methods: The study was divided into two parts. First, involved the preservation of epididymal sperm at 5°C for 12 days. During preservation, sperm quality parameters namely motility, viability, intact membrane, acrosome, and Deoxyribonucleic acid (DNA) are evaluated every three days. For the fertility test in the second experiment, matured oocytes were injected with immotile sperm discovered in the last days of preservation. The presence of pronucleus development following in vitro culture is used as an indicator of sperm's ability to activate and fertilize oocytes. Results: All sperm quality parameters significantly (p < 0.05) declined during preservation time. On day 12, motility was discovered to be 0%, but viable sperm, sperm with intact membrane, acrosome, and DNA remained at 41.86% ± 9.30%, 31.18% ± 5.15%, 21.88% ± 1.93%, and 33.35% ± 8.74%, respectively. On the fertility test, we inject immotile sperm from day 12 of preservation, which has the lowest motility found, into matured oocytes. Those sperms are able to activate (52.05% ± 7.15%) and fertilize (31.37% ± 1.75%) the injected oocytes, but their fertilizing ability is significantly lower (p < 0.05) when compared to the sperm derived from the ejaculate. Conclusion: In this study, simple preservation of epididymal sperm reduces all sperm quality criteria, particularly motility. Using the microinjection approach preserved sperm which had no motility, still demonstrated its ability to activate and fertilize the oocytes. According to that, this study provides potential approaches and tools for using genetically superior animals that have lost their ability to execute regular fertilization, and also prolong reproduction function. [ABSTRACT FROM AUTHOR]

The goals of the present study were to develop a simple method for obtain highly purified pig sperm hyaluronidase (pHyase) and to assess its activity, function, and safety. In mammals, sperm-specific glycophosphatidylinositol (GPI)-anchored Hyase assists sperm penetration through the cumulus mass surrounding the egg and aids in the dispersal of the cumulus–oocyte complex. Recently, Purified bovine sperm hyaluronidase (bHyase) has been shown to enhance therapeutic drug transport by breaking down the hyaluronan barrier to the lymphatic and capillary vessels, thereby facilitating tissue absorption. Commercially available Hyase is typically isolated from bovine or ovine; which have several disadvantages, including the risk of bovine spongiform encephalopathy, low homology with human Hyase, and the requirement for relatively complex isolation procedures. This study successfully isolated highly purified pHyase in only two steps, using ammonium sulfate precipitation and fast protein liquid chromatography. The isolated Hyase had activity equal to that of commercial bHyase, facilitated in vitro fertilization, and effectively dissolved high molecule hyaluronic acid. This simple, effective isolation method could improve the availability of pHyase for research and clinical applications.

In laboratory mice, sperm quality is usually assessed in spermatozoa collected from the cauda epididymidis of freshly sacrificed males. Percutaneous epididymal sperm aspiration (PESA) is a non-terminal alternative that would allow repeated sperm collection for sperm quality assessment in living males. To test whether PESA is a suitable method to assess sperm quality, we compared sperm traits between samples collected by PESA vs the commonly applied terminal cauda epididymidis dissection. The collected sperm samples were analyzed using computer-assisted sperm analysis and various parameters, including sperm motility, swimming velocity and morphology, were determined. We were able to retrieve motile sperm from all mice using PESA and the terminal cauda epididymidis dissection. Based on computer-assisted sperm analysis, however, sperm motility and swimming velocity were significantly lower after PESA compared to samples obtained by cauda epididymidis dissection. In addition, we found significantly more morphological abnormalities in PESA samples, probably induced as a side effect of the sampling technique. Although sperm samples collected by PESA are successfully used for in vitro fertilization, we cannot recommend PESA as a suitable method to assess sperm quality in mice, since the procedure seems to impair various sperm traits.

The aim of this study was to evaluate the motility, morphology, and antioxidant status of European red deer sperm stored in a liquid state (variant I) and in the epididymides (variant II). Spermatozoa were harvested post-mortem from the cauda epididymis. Sperm samples in both variants were stored for up to six days (D6) at 5 °C. Spermatozoa were assessed for motility, viability, morphology, activity of antioxidant enzymes (superoxide dismutase, SOD; glutathione peroxidase, GPx; catalase, CAT), and lipid peroxidation (malondialdehyde, MDA, content). Sperm samples were analyzed on storage days 0, 2, 4, and 6 (D0-D6). Storage time and storage method significantly (p ≤ 0.05) influenced the examined variables. On D2, a decrease in motility and acrosomal integrity was observed in both storage variants, whereas a decrease in viability and an increase in MDA content were noted in spermatozoa stored in the epididymides. On D4, higher values of SOD and GPx activity and MDA content were noted in variant I than in variant II. Catalase activity was very low. GPx is the key enzyme that participates in the reduction of hydrogen peroxide in sperm cells. Spermatozoa stored in a liquid state were characterized by higher motility and viability, improved morphology and antioxidant status than those stored in the epididymides; therefore, liquid storage is more recommended for short-term preservation of epididymal spermatozoa.

Objective To investigate the efficacy, safety and the influence on pregnancy outcome of L-carnitine before intracytoplasmic sperm injection (ICSI) from different sources of sperm in patients with obstructive azoospermia (OA). Methods A total of 141 patients with OA were treated with L-carnitine for three months, and sperms were obtained by testicular sperm aspiration (TESA) and percutaneous epididymal sperm aspiration (PESA) respectively. According to the source of sperm, they were divided into the two groups: the TESA group (n=78) and the PESA group (n=63). The general clinical data, sperm quality, embryonic development and clinical outcome of the two groups were compared. Results In the TESA/PESA group, sperm DFI and sperm spontaneous acrosome reaction rate were significantly lower than those before treatment, and sperm acrosome integrity rate was significantly higher than that before treatment (P＜0.05). There were no significant differences in sperm DFI, sperm acrosome integrity rate and sperm spontaneous acrosome reaction rate between the two groups(P＞0.05). There were no significant differences in the fertilization rate, 2PN fertilization rate, cleavage rate, excellent embryo rate, implantation rate, clinical pregnancy rate, live birth rate, premature birth rate, abortion rate and neonatal malformation rate between the two groups (P＞0.05). A total of 103 fresh transplant cycles, 989 MII oocytes, 773 zygotes, 49 clinical pregnancies and 39 live births were obtained (including 17 in the TESA group and 22 in the PESA group). During a 3-month follow-up after birth, it was found that one newborn had cardiac abnormalities in the TESA group, while the other newborns had no abnormalities. Conclusion In OA patients, L-carnitine before TESA-ICSI and PESAICSI can improve the sperm quality, optimize clinical outcome, and the medication is safe. [ABSTRACT FROM AUTHOR]

Simple Summary: Flow cytometry (FC) is the recommended technique for assessing sperm quality. In comparison with fluorescence microscopy (FM), FC supports analyses of much larger sperm populations and generates more reliable results. However, FC is not always accessible, and sperm populations are often evaluated with the use of FM. In the present study, FC and FM were used to assess the functionality of various organelles in European red deer epididymal spermatozoa stored in liquid nitrogen. Spermatozoa were collected from the epididymides of hunter-harvested European red deer stags. The epididymides were stored in a refrigerator (2–4 °C) for 12 h before analysis. The study demonstrated that the refrigerated storage of the epididymides for 12 h had no significant effect on the sperm quality before cryopreservation, but it significantly influenced the percentage of early necrotic sperm after thawing. The results of FM and FC assays differed significantly, excluding in the assessment of the plasma membrane integrity. However, the results of both assays revealed significant correlations between the examined variables, except for mitochondrial activity. The study demonstrated that the spermatozoa from epididymides chill-stored for 12 h can be used for cryopreservation. Fluorescence microscopy and FC are equally reliable techniques, but FM was more useful for evaluating mitochondrial activity. Thawed spermatozoa, sampled post mortem from the fresh epididymides of European red deer and epididymides stored for up to 12 h at 2–4 °C, were evaluated by fluorescence microscopy (FM) and flow cytometry (FC). The sperm samples were extended and cryopreserved. The sperm motility (CASA), sperm viability (SYBR+/PI-), acrosome integrity, mitochondrial activity, apoptotic changes, and chromatin stability were assessed. Sperm were analyzed by FM before cryopreservation, and by FM and FC after thawing. Epididymal storage time (for 12 h) had no significant effect (p > 0.05) on the examined variables before cryopreservation. After thawing, the storage variants differed (p ˂ 0.05) in the percentage of apoptotic sperm (FM and FC) and DNA integrity (FC). The results of FM and FC differed (p ˂ 0.05) in all the analyzed parameters, excluding SYBR+/PI. Significant correlations (p ˂ 0.01) were observed between the sperm viability, acrosome integrity, and the percentage of non-apoptotic spermatozoa, regardless of the applied technique. In FM, the above parameters were also significantly correlated with mitochondrial activity. The study demonstrated that European red deer spermatozoa stored in the epididymides at 2–4 °C for 12 h can be used for cryopreservation. Both techniques were equally reliable, but FM was better suited for evaluating mitochondrial activity whereas FC was more useful in the evaluation of DNA fragmentation. [ABSTRACT FROM AUTHOR]

Intracytoplasmic Sperm Injection (ICSI) using frozen/thawed sperm is a common procedure to obtain embryos from fertile or subfertile mares and stallions. Stallion-associated factors that impact the efficiency of ICSI have been studied less than those associated with the mare. Three experiments were conducted: Experiment 1: the effect of freezing extender composition and cryoprotectant; Experiment 2: the effect of sperm exposure to seminal plasma prior to freezing (ejaculated vs. epididymal sperm; two-freeze/thaw cycles each); and Experiment 3: the effect of sperm morphologic feature used for fertilization (normal vs. cytoplasmic droplet vs. bent tail); on the blastocyst rate after ICSI. In Experiment 1, stallion sperm was cryopreserved using commercially available extenders containing: a) 2% egg-yolk + milk + 4% glycerol (MFR5); b) 2% egg-yolk + milk + 2% glycerol + 3% methyl formamide (CMMFR5); c) 20% egg-yolk + 4.75% glycerol (LE); or d) 20% egg-yolk + 2% glycerol + 3% methyl formamide (CMLE). Sperm from each of the treatment groups were used for Piezo-driven ICSI on in vitro -matured equine oocytes (n = 321). Extender CMLE resulted in a lower cleavage rate (35%) than the other treatment groups (MFR5: 74%, CMMFR5: 62%, LE: 68%; P < 0.05). Extender MFR5 yielded a higher blastocyst rate per injected oocyte (21/82 [26%]) than the Groups LE (8/77 [10%]), CMLE (4/80 [5%]) or CMMFR5 (4/82 [5%]; P < 0.05). Extender MFR5 also yielded a higher blastocyst rate per cleaved oocyte (34%) than Groups LE, CMLE or CMMFR5 (15%, 14%, 8%; respectively P < 0.05). In Experiment 2, ejaculated (EJ) and epididymal (EPD) sperm from a fertile stallion which was initially cryopreserved in the CMLE extender, was thawed and re-cryopreserved in MFR5 extender for use in ICSI. Sperm from both groups (EJ vs. EPD) were used for ICSI on in vitro matured oocytes (n = 127). Differences were not detected for cleavage rate (EJ: 36/63 [57%] vs. EPD: 49/64 [77%]), blastocyst rate per injected oocyte (EJ: 11/63 [17%] vs. EPD: 11/64 [17%]), or blastocyst rate per cleaved oocyte (EJ: 31% vs. EPD: 22%) between treatment groups (P > 0.05). In Experiment 3, morphologically normal sperm (N), or sperm with proximal droplets (PD) or bent tails (BT), were obtained from a single fertile stallion and were used for ICSI on in vitro matured oocytes (n = 75). No differences were detected among treatment groups for cleavage rate (N: 19/25 [77%] vs. PD: 20/25 [88%] vs. BT: 18/25 [72%]), blastocyst rate per injected oocyte (N: 6/25 [24%] vs. PD: 5/25 [20%] vs. BT: 2/25 [8%]), and blastocyst rate per cleaved oocyte (N: 32% vs. PD: 23% vs. BT: 11%; P > 0.05). In conclusion, the current study indicates that freezing extender composition used for stallion sperm cryopreservation has an impact on the developmental competence of in vitro -matured equine oocytes after ICSI and in vitro culture. Furthermore, we were unable to detect differences on cleavage and blastocyst rates when performing ICSI when using: 1) ejaculated or epididymal sperm; or 2) sperm with different morphologic features. The results from the current study provide additional insight regarding stallion-related factors that should be considered when performing ICSI in horses. • Composition of semen freezing extender affects the efficiency of ICSI in horses. • A freezing semen extender with low egg yolk, milk proteins, and glycerol yielded the highest rates of blastocysts after ICSI. • Presence of high levels of egg yolk (i.e., 20%), or amides negatively impact the production of equine blastocysts by ICSI. • Frozen/thawed epididymal and ejaculated sperm resulted in similar blastocyst rates after ICSI. • Sperm morphology features (i.e., proximal droplets or bent tails) can be used to produce equine blastocysts by ICSI. [ABSTRACT FROM AUTHOR]

Lyophilisation is an alternative method for sperm preservation. The aim of this study was to evaluate the effects of freeze-thawing (F/T) and freeze-drying (F/D) on the quality of epididymal goat sperm. Sperm from each region of the epididymis (caput, corpus and cauda) were collected and evaluated for the expression of phospholipase C zeta (PLC-ζ), protamine 1 (PRM1), transition protein 1 (TNP1) and 2 (TNP2). The effects of F/T and F/D on sperm quality in terms of PLC-ζ expression, chromatin stability (Chromomycin A3; CMA3) and DNA integrity were examined. The fertilising ability after intracytoplasmic sperm injection (ICSI) was also tested. Fresh sperm existed PLC-ζ, PRM1, TNP1 and TNP2, irrespective of the regions of the epididymis. However, different patterns of PLC-ζ expression were found. Although PRM1, TNP1, TNP2 were still expressed after F/T or F/D, only F/T could preserve the presence of PLC-ζ. For fresh sperm, caput epididymal sperm had the lowest evidence of chromatin stability when compared to sperm harvested from other regions of the epididymis. The F/T and F/D further increased the numbers of CMA3-positive sperm (P < 0.001). In all cases, no CMA3 staining was observed in caudal epididymal sperm. The caudal epididymal sperm had significantly greater proportions of sperm with intact DNA compared with caput and corpus epididymal sperm, especially when F/T and F/D were performed. The fertilisation rates of F/D sperm tended to decrease when compared with F/T sperm (4.2 ± 3.2 vs. 13.6 ± 9.0, P = 0.08). It is concluded that the sperm recovered from the caudal epididymis is suitable for freezing and lyophilisation. However, poor fertilisation rates of F/D sperm were coincidently observed, with a deficit demonstration of PLC-ζ. • Cauda epididymal spermatozoa are suitable for freeze-drying compared with sperm from other regions of epididymis. • Freeze-drying disrupts PLC-ζ expression, sperm quality and fertilising ability of epididymal sperm. • Freeze-dry cauda epididymal spermatozoa could fertilise MII goat oocytes following ICSI and sperm activation. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effect of different glycerol concentrations and different sperm concentrations on stallion epididymal sperm motility indicators after thawing. For statistical analysis, 25 stallions were used. Collection of the epidydimal spermatozoa was performed as a retrograde flush of cauda epididymis and ductus deferens within 12 h post castration. After evaluation, the resuspended spermatozoa were centrifuged, the supernatant removed and the spermatozoa resuspended in Gent semen extender to get three different groups with different concentrations of sperm (250 x 106 in ml, 500 x 106 in ml, 1000 x 106 in ml) and different final glycerol concentrations (2%, 4%, and 6%). Therefore, 9 different samples were finally obtained from each stallion. The spermatozoa were packed and placed in a fridge (4 °C) for 2 h, then placed in liquid nitrogen vapour (-80 to -100 °C) and after 25 min plunged into the liquid nitrogen and stored at -196 °C for at least 5 days. The selected straws were individually thawed in a 38 °C water bath for 30 s prior to post-freezing analysis. Motility indicators were assessed at 0, 60, 120, and 180 min after thawing. Parametric test was used for analysis; the measured indicators were total motility, progressive motility, curvilinear velocity, straightness, and average-path velocity. In this study, the best results were reached in samples diluted into a concentration of 1,000 x 106 in ml, regardless of the concentrations of glycerol. [ABSTRACT FROM AUTHOR]

In this study, cauda epididymal sperm were collected from Amur leopard cats with various causes of death as well as Tsushima leopard cats that underwent castration surgery, and sperm quality was compared with that in domestic cats. A sufficient number of sperm similar to those in domestic cats could be collected from the cauda epididymis of Amur leopard cats. However, in old leopard cats, no or very few cauda epididymal sperm were recovered, although there were no differences in sperm motility and sperm abnormality. There were no significant differences in sperm quality immediately after collection and after freezing-thawing of cauda epididymal sperm compared with corresponding estimates in domestic cats. [ABSTRACT FROM AUTHOR]

Epididymal sperm cryopreservation provides a potential method for preserving genetic material from males of endangered species. This pilot study was conducted to develop a freezing method for tiger epididymal sperm. We evaluated post-thaw sperm condition using testes with intact epididymides obtained from a Siberian tiger (Panthera tigris altaica) after castration. The epididymis was chopped in Tyrode's albumin-lactate-pyruvate 1x and incubated at 5% CO2, 95% air for 10 min. The Percoll separation density gradient method was used for selective recovery of motile spermatozoa after sperm collection using a cell strainer. The spermatozoa were diluted with modified Norwegian extender supplemented with 20 mM trehalose (extender 1) and subsequent extender 2 (extender 1 with 10% glycerol) and frozen using LN2 vapor. After thawing at 37℃ for 25 s, Isolate® solution was used for more effective recovery of live sperm. Sperm motility (computerized assisted sperm analysis, CASA), viability (SYBR-14 and Propidium Iodide) and acrosome integrity (Pisum sativum agglutinin with FITC) were evaluated. The motility of tiger epididymal spermatozoa was 40.1 ± 2.0%, and progressively motile sperm comprised 32.7 ± 2.3%. Viability was 56.3 ± 1.6% and acrosome integrity was 62.3 ± 4.4%. Cryopreservation of tiger epididymal sperm using a modified Norwegian extender and density gradient method could be effective to obtain functional spermatozoa for future assisted reproductive practices in endangered species.

The presented study covers testicular tissue and epididymal spermatozoa cryopreservation processes in bulls and aims to investigate the effects of these applications on spermatological parameters, cell viability in testicular tissue, and the expression of the PARP-1 gene, a DNA repair enzyme. Testes of 20 bulls over 2 years old, slaughtered in a slaughterhouse, were used in the study. After spermatological evaluations, the semen obtained from the cauda epididymis was frozen in liquid nitrogen vapor according to the straw method and stored in liquid nitrogen (−196 °C). Testicular tissue pieces obtained from the testicles were frozen by the slow freezing method in cryotubes in diluents containing Dimethylsulfoxide (DMSO) and Ethylene Glycol (EG) cryoprotectants and stored in liquid nitrogen (−196 °C). The total motility (TM) (85.89 ± 12.83 %), progressive motility (PM) (54.02 ± 15.77 %), and kinematic parameter values of fresh sperm were significantly higher compared to the TM (57.62 ± 13.13 %), PM (29.60 ± 10.76 %), and kinematic parameter values after thawing (P < 0.05). Significant decreases in plasma membrane integrity (PMI) and viability and an increase in chromatin condensation and morphological disorders in the head, middle part, and tail regions were observed in post-thaw semen samples (P < 0.05). When the effects of DMSO and EG on cell viability after thaw in frozen testicular tissue were evaluated, it was observed that the cell viability values of testicular tissues frozen with EG (45.70 ± 10.00) were statistically significantly lower than those frozen with DMSO (51.20 ± 7.70) (P < 0.05). When the effects of both cryoprotectants on gene expression in tissue and semen samples were examined, it was determined that gene expression increased on average 0.19 ± 0.27 times in the tissue samples in the DMSO group compared to fresh tissue samples and 0.17 ± 0.19 times in the tissue samples in the EG group. It was determined that gene expression levels increased by an average of 1.20 ± 1.08 times in post-thaw epididymal spermatozoa samples compared to fresh semen samples. The results show that cryopreservation can activate cellular repair mechanisms by stimulating PARP-1 gene expression and affect gene expression by activating specific pathways in tissues and cells. • Cryopreservation triggers biological processes like DNA repair by increasing PARP-1 gene expression. • DMSO provides higher cell viability in testicular tissue than EG during cryopreservation. • Cryopreservation significantly reduces spermatozoa progressive motility, and kinematic parameters. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the quality and fertilizing potential of red deer epididymal spermatozoa stored in a liquid state for up to 11 days (D11). In Experiment 1, sperm quality was determined. In Experiment 2, the efficiency of in vitro fertilization (IVF) and artificial insemination (AI) of stored sperm were evaluated. An analysis of sperm quality on D5 of storage revealed a decrease (p < 0.05) in motility and morphology, and a higher proportion of apoptotic spermatozoa. On D1, D7 and D10, the total motility of sperm for IVF and AI was determined to be 82.6%, 71.0% and 64.8%, respectively. The results of IVF and AI demonstrated that the fertilizing potential of spermatozoa differs between days of storage. The percentage of blastocysts was higher when oocytes were fertilized on D1 (17.4 %) compared to D7 (8.5%) and D10 sperm (10.5%). Differences were noted in the pregnancy rates of inseminated hinds. The insemination with D1, D7 and D10 sperm led to live births (33% from D7 and D10). The results indicate that the quality of red deer epididymal spermatozoa remains satisfactory during ten days of storage in a liquid state, and that these spermatozoa maintain their fertility potential. [ABSTRACT FROM AUTHOR]

Simple Summary: The use of commercial extenders for storing cervid epididymal spermatozoa in a liquid state can contribute to popularizing this method of semen preservation in cervid farms. The aim of this study was to assess the effect of various extenders (Bovidyl®, BoviFree®, and BioXcell®) on the quality of European red deer epididymal spermatozoa stored at 5 °C. The analyses demonstrated that the tested extenders and storage time significantly affected sperm motility and functionality of different sperm structures. The Bovidyl® extender containing chicken egg yolk exerted the most beneficial influence on the quality of stored sperm. Spermatozoa stored in the Bovidyl® extender were characterized by satisfactory motility (above 60% on storage day 9) and the functionality over a long period of time. In turn, extenders containing plant-based ingredients and glycerol (BioXcell® and BoviFree®) significantly decreased membrane integrity and mitochondrial activity of spermatozoa already on the first day of storage. These results indicate that European red deer epididymal spermatozoa can be stored in commercial extenders, but their viability and functionality are significantly influenced by the composition of the applied extenders. The extender containing chicken egg yolk without glycerol produced the most satisfactory results. The aim of this study was to evaluate the effect of different extenders on the quality of European red deer epididymal sperm stored at 5 °C. Epididymal spermatozoa were collected post mortem from 10 stags and diluted with three extenders (Bovidyl®, BoviFree®, and BioXcell®) and stored at 5 °C. Sperm motility (TMOT), motility parameters (system CASA), plasma membrane integrity (SYBR-14+/PI−), acrosomal membrane integrity (FITC-PNA−/PI−), mitochondrial activity (JC-1/PI), viability, and apoptotic-like changes (YOPRO/PI) were evaluated. The analyses were conducted on the first and successive days of storage (D1–D7). The applied extender, storage time, and the interactions between these factors significantly (p < 0.001) affected most of the analyzed parameters whose values were highest in sperm samples stored in Bovidyl®, regardless of storage time. In Bovidyl®, BoviFree®, and BioXcell® extenders, TMOT values were estimated at 83%, 63%, and 59%, respectively, on D3. The extenders significantly influenced DNA integrity on D7. The percentage of dead sperm increased from D3. The quality of stored sperm cells was significantly influenced by the extenders' biochemical composition. BoviFree® and BioXcell® contain glycerol which could contribute to deteriorating the quality of spermatozoa stored at 5 °C. Sperm cells stored in the egg yolk-based extender (Bovidyl®) were characterized by the highest viability and functionality. [ABSTRACT FROM AUTHOR]

The objective of the current experiment was to study the effect of different avian egg yolks (EYs) on rams epididymal sperms stored at 4 ºC. Sixteen pairs of testes (n=32) were mature Awassi rams of about 1-4 years, slaughtered at Al-Sadoon abattoir, Mosul city were collected. The spermatozoa were collected from the tail of the epididymis by cutting and squeezing; after that, a total of 0.5 ml of spermatozoa volume was diluted by using sodium citrate 2.9% and the volume was completed to 1 ml, then the sample was divided into four aliquots (0.25 ml for each one). The extender consisted of egg yolk (EY) 10% of four avian types (chicken, quail, duck, turkey), sodium citrate 2.9%, fructose 2.4gm, penicillin, and streptomycin (100.000 IU and 100 mg, respectively) in 100 distal water. Sperms individual motility, live sperm percentage, sperms abnormalities were checked at 0, 72, 144 hours of storage. Results of the present study revealed that quail egg yolk improved sperms, individual motility, live sperm percentage with longer life span and reduce sperms abnormalities, followed by Turkey, chicken and duck EYs, respectively. Individual motility of the spermatozoa after 144 h of storage in quail EY extender were (36.00%± 1.20), which higher than turkey EY (32.30%± 1.30). However, these results were higher and significant (P˂ 0.05) when compared with chicken EY extender (24.60%± 2.30) and duck EY extender (19.00%± 1.50). Similar results of quail and turkey EYs were manifested in the percentage of live spermatozoa, which were (38.00%± 1.20), (33.30%± 1.30), (24.00%± 2.30), and (19.90%± 1.50), respectively. The duck EY was significantly lower (P˂ 0.05) when comparing the four egg yolks types after storage for 144 h. In conclusion: we advise potentially using a quail or turkey EYs extender for the chilled storage for ram epididymal sperms.

We studied the sperm membrane functionality through the epididymal transit by comparing different hypoosmotic solutions and verifying possible associations among osmotic response and functional parameters of sperm in red‐rumped agouti (Dasyprocta leporina). For this purpose, epididymal sperm from six sexually mature male agoutis were collected via flotation. Then, analyses of sperm parameters and hypoosmotic swelling test using different hypoosmotic solutions (0, 50 and 200 mOsm/L) in different regions of the epididymis (caput, corpus and cauda) were performed. There was an increase (p <.05) in the values for sperm concentration, the total number of sperm recovered, total and progressive motility, average path velocity, straight‐line velocity, curvilinear velocity, and rapid and medium subpopulations following the caput‐corpus‐cauda direction. Regardless of the hypoosmotic solution, the agouti sperm membrane presented similar functional integrity in all the epididymal regions. Moreover, the highest (p <.05) osmotic responses were reached with the use of 50 mOsm/L solution in comparison to 0 and 200 mOsm/L for all the regions. Significant correlations among osmotic response and some sperm kinetic parameters were observed, especially in epididymal caput, while no correlations were found in the region of the cauda. In summary, red‐rumped agouti sperm present similar membrane functionality during epididymal transit, but there are evident correlations among such functionality and sperm kinetic parameters, especially in the caput region. Moreover, we indicate the use of a 50 mOsm/L hypoosmotic solution for the analysis of this parameter through the hypoosmotic swelling test. [ABSTRACT FROM AUTHOR]

A variety of surgical options exists for sperm retrieval in the setting of obstructive azoospermia (OA). With appropriate preparation, the majority of these techniques can safely be performed in the office with local anesthesia and with or without monitored anesthesia care (MAC). The available techniques include percutaneous options such as percutaneous epididymal sperm aspiration (PESA) and testicular sperm aspiration (TESA), as well as open techniques that include testicular sperm extraction (TESE) and microsurgical epididymal sperm aspiration (MESA). In addition to providing a step-by-step description of each available approach, we introduce and describe a new technique for sperm retrieval for OA called minimally invasive epididymal sperm aspiration (MIESA). The MIESA utilizes a tiny keyhole incision, and the epididymis is exposed without testicular delivery. Epididymal aspiration is performed in the style of MESA, except using loupe magnification rather than an operating microscope. MIESA is a safe, office-based procedure in which millions of motile sperm can be retrieved for cryopreservation. While we prefer the MIESA technique for OA, there remain distinct advantages of each open and percutaneous approach. In the current era of assisted reproductive technology, sperm retrieval rates for OA should approach 100% regardless of the technique. This reference provides a roadmap for both advanced and novice male reproductive surgeons to guide them through every stage of sperm retrieval for OA, including preoperative evaluation, patient selection, procedural techniques, and complications. With the incredible advances in in vitro fertilization (IVF), combined with innovative surgical treatment for male factor infertility in recent years, OA is no longer a barrier for men to become biologic fathers.

As an alternative to ejaculated semen, epididymal spermatozoa (ES) can be recovered and cryopreserved for use in artificial insemination and other assisted reproductive technologies. However, variabilities in the sperm response to freeze-thaw procedures challenge its inherent value. Therefore, the present study aims to clarify the freezability phenomena in the swamp buffalo ES, where pieces of literature do not abound. Here, we isolated ES from swamp buffaloes using abattoir-derived, post-mortem caudal epididymis by slicing-flushing technique. Following cryopreservation by slow-freezing, ES samples were classified into high (HF) and low freezability (LF) based on their post-thaw total and progressive motilities. Conventional sperm parameters and proteins of interest, such as glutathione S-transferase Mu 3 (GSTM3) and ATP synthase beta subunit (ATP1B1), were assessed and compared between HF and LF. Computer-assisted sperm analysis revealed that nearly all motion and kinematic parameters significantly differed among freezability groups except for wobble, linearity, and straightness. Moreover, intracellular reactive oxygen species production was evident in both HF and LF after fluorescence staining, with the latter having considerably greater malondialdehyde levels than the former. Immunohistochemical labeling demonstrated that both GSTM3 and ATP1B1 proteins were present in the ES and the epididymal tubular epithelium. Although the GSTM3 relative amounts, as analyzed through Western blot, were significantly higher in LF than HF in association with lipid peroxidation, no significant differences were observed in the case of ATP1B1. Such variations in motility, motion and kinematics, oxidative stress status, and specific sperm proteins suggest their potential utility in distinguishing freezability phenotypes in swamp buffalo ES. [Display omitted] • Freezability differences exist even in the epididymal spermatozoa of swamp buffalo. • Sperm motion, kinematics and lipid peroxidation vary among freezability phenotypes. • GST and ATP1B1 occur in the epididymal sperm and epithelium. • Sperm proteins of swamp buffalo can further be explored as freezability biomarkers. [ABSTRACT FROM AUTHOR]

Abstract Background The industrial revolution has resulted in increased synthesis and the introduction of a variety of compounds into the environment and their potentially hazardous effects have been observed in the biota. The present study was aimed to evaluate the potential endocrine-disrupting effects of chronic exposure to the low concentrations of bisphenol S (BPS) in male rats. Methods Weaning male Sprague-Dawley rats (22 days old) were either exposed to water containing 0.1% ethanol for control or different concentrations of BPS (0.5, 5, and 50 μg/L) in drinking water for 48 weeks in the chronic exposure study. After completion of the experimental period, animals were dissected and different parameters (hormone concentrations, histology of testis and epididymis, oxidative stress and level of antioxidant enzymes in the testis, daily sperm production (DSP), and sperm parameters) were determined. Results Results of the present study showed a significant alteration in the gonadosomatic index (GSI) and relative reproductive organ weights. Oxidative stress in the testis was significantly elevated while sperm motility, daily sperm production, and the number of sperm in epididymis were reduced. Plasma testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) concentrations were reduced and estradiol levels were high in the 50 μg/L-exposed group. Histological observations involved a significant reduction in the epithelial height of the testis along with disrupted spermatogenesis, an empty lumen of the seminiferous tubules, and the caput region of the epididymis. Conclusion These results suggest that exposure to 5 and 50 μg/L of BPS for the chronic duration started from an early age can induce structural changes in testicular tissue architecture and endocrine alterations in the male reproductive system which may lead to infertility in males.

The current study was conducted to investigate the role of alpha lipoic acid (ALA) against testicular toxicity- induced by lead acetate (PbAc) in rats. Four groups of adult Wistar albino rats (8 for each) were intubated daily for 56 days as follows; control (C)received dislled water; lead acetate at dose of 5mg/kg b.w (T1); ALA at dose of 60mg/kg b.w (T2) and and group T3 received both of PbAc + ALA at the same doses of above. Blood samples were collected at 0, 28 and 56 day of the experiment, then the sera were collected for determination of testosterone(T) and follicular stimulating hormone (FSH). At the end of the experiment, body weight, testes weight and epididymal sperm parameters was studied. Furthermore, histo-morphometric and histopathological study changes were examined. The results revealed a significant decrease in testes weight to body weight ratio, serum testosterone, sperm concentration and motility, diameters of seminiferous tubules, height of seminiferous epithelia and number of Leydig cells, moreover the results showed a significant increase in serum FSH, dead sperm and abnormal sperm morphology in group T1 when compared with the other groups. Comparing to lead acetate treated rats, group T3 showed an improvement at the level of the studied parameters, accompanied with mild congestion in the interstitial tissue with a marked developing proliferation of spermatogenic cells, as well as presence of mature spermatozoa in the seminiferous tubules. In conclusion, subchronic exposure of rats to lead acetate showed an amelioration of all reproductive parameters near to normal values due to the antioxidant effects of ALA and the histological changes of the testes confirmed such change in serum parameters and the beneficial role of ALA.

Objective: To determine the in-vitro effect of the total alkaloid extract of Peganum (P.) harmala seeds on ram epididymal sperm. Methods: Semen was divided into six groups according to the following concentrations of the P. harmala total alkaloids: 1, 5, 10, 50, and 100 μg/mL, and the control group. The samples were incubated at ambient temperature (21 °C-24 °C) for 24 h, and analyzed in terms of motility, membrane integrity, and oxidative status. Results: The sperm kinematic parameters, i.e. straight-line velocity, curvilinear velocity, average path velocity, were significantly higher when treated with P. harmala at concentrations ranging from 1 to 10 μg/mL compared to the control group (P0.05). Conclusions: Low concentrations (1-10 μg/mL) of P. harmala extracts stimulate sperm motility, preserve membrane integrity and protect ram spermatozoa from lipid peroxidation.

Thawed spermatozoa, sampled post mortem from the fresh epididymides of European red deer and epididymides stored for up to 12 h at 2–4 °C, were evaluated by fluorescence microscopy (FM) and flow cytometry (FC). The sperm samples were extended and cryopreserved. The sperm motility (CASA), sperm viability (SYBR+/PI-), acrosome integrity, mitochondrial activity, apoptotic changes, and chromatin stability were assessed. Sperm were analyzed by FM before cryopreservation, and by FM and FC after thawing. Epididymal storage time (for 12 h) had no significant effect (p > 0.05) on the examined variables before cryopreservation. After thawing, the storage variants differed (p ˂ 0.05) in the percentage of apoptotic sperm (FM and FC) and DNA integrity (FC). The results of FM and FC differed (p ˂ 0.05) in all the analyzed parameters, excluding SYBR+/PI. Significant correlations (p ˂ 0.01) were observed between the sperm viability, acrosome integrity, and the percentage of non-apoptotic spermatozoa, regardless of the applied technique. In FM, the above parameters were also significantly correlated with mitochondrial activity. The study demonstrated that European red deer spermatozoa stored in the epididymides at 2–4 °C for 12 h can be used for cryopreservation. Both techniques were equally reliable, but FM was better suited for evaluating mitochondrial activity whereas FC was more useful in the evaluation of DNA fragmentation.

The objective of the current experiment was to study the effect of different avian egg yolks (EYs) on rams epididymal sperms stored at 4 °C. Sixteen pairs of testes (n=32) were mature Awassi rams of about 1-4 years, slaughtered at Al-Sadoon abattoir, Mosul city were collected. The spermatozoa were collected from the tail of the epididymis by cutting and squeezing; after that, a total of 0.5 ml of spermatozoa volume was diluted by using sodium citrate 2.9% and the volume was completed to 1 ml, then the sample was divided into four aliquots (0.25 ml for each one). The extender consisted of egg yolk (EY) 10% of four avian types (chicken, quail, duck, turkey), sodium citrate 2.9%, fructose 2.4gm, penicillin, and streptomycin (100.000 IU and 100 mg, respectively) in 100 distal water. Sperms individual motility, live sperm percentage, sperms abnormalities were checked at 0, 72, 144 hours of storage. Results of the present study revealed that quail egg yolk improved sperms, individual motility, live sperm percentage with longer life span and reduce sperms abnormalities, followed by Turkey, chicken and duck EYs, respectively. Individual motility of the spermatozoa after 144 h of storage in quail EY extender were (36.00%± 1.20), which higher than turkey EY (32.30%± 1.30). However, these results were higher and significant (P=0.05) when compared with chicken EY extender (24.60%± 2.30) and duck EY extender (19.00%± 1.50). Similar results of quail and turkey EYs were manifested in the percentage of live spermatozoa, which were (38.00%± 1.20), (33.30%± 1.30), (24.00%± 2.30), and (19.90%± 1.50), respectively. The duck EY was significantly lower (P=0.05) when comparing the four egg yolks types after storage for 144 h. In conclusion: we advise potentially using a quail or turkey EYs extender for the chilled storage for ram epididymal sperms. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effect of different extenders on the quality of European red deer epididymal sperm stored at 5 °C. Epididymal spermatozoa were collected post mortem from 10 stags and diluted with three extenders (Bovidyl®, BoviFree®, and BioXcell®) and stored at 5 °C. Sperm motility (TMOT), motility parameters (system CASA), plasma membrane integrity (SYBR-14+/PI−), acrosomal membrane integrity (FITC-PNA−/PI−), mitochondrial activity (JC-1/PI), viability, and apoptotic-like changes (YOPRO/PI) were evaluated. The analyses were conducted on the first and successive days of storage (D1–D7). The applied extender, storage time, and the interactions between these factors significantly (p < 0.001) affected most of the analyzed parameters whose values were highest in sperm samples stored in Bovidyl®, regardless of storage time. In Bovidyl®, BoviFree®, and BioXcell® extenders, TMOT values were estimated at 83%, 63%, and 59%, respectively, on D3. The extenders significantly influenced DNA integrity on D7. The percentage of dead sperm increased from D3. The quality of stored sperm cells was significantly influenced by the extenders’ biochemical composition. BoviFree® and BioXcell® contain glycerol which could contribute to deteriorating the quality of spermatozoa stored at 5 °C. Sperm cells stored in the egg yolk-based extender (Bovidyl®) were characterized by the highest viability and functionality.

The article presents data of research on the long-term exposure (two or three months) effect of mobile phone EMF (1745 MHz) in the immediate (1st day) and remote (30th day) period on the number of leukocytes and leukocyte blood elements and indices of the reproductive system of male rats. It was established that on the 1st day after EMF exposure from mobile phones (1745 MHz) notes some deviations hematological parameters of control, particularly important for the number of granulocytes after 3 months of exposure, observed disturbance spermatogenesis, as well as reducing the number of epididymal spermatozoa and drop their viability. By the 30th day revealed changes in the parameters studied mostly normalized, with the exception of significant fall viability of mature germ cells at 60-day and the number of granulocytes in the 90-day exposure.

Techniques for the cryopreservation of epididymal sperm was are widely used in clinical practice. However, given the unique characteristics of sperm from patients with obstructive azoospermia, epididymal sperm cryopreservation is more difficult because of low count and weak motility; therefore, conventional methods of sperm cryopreservation may not result in the best outcomes. We used the micro-straw method to store small quantities of sperm obtained from patients with severe oligozoospermia or azoospermia and achieved successful deliveries in the previous study. This retrospective study of ICSI cycles included the first ICSI cycles of fresh or frozen/thawed epididymal sperm that were performed in patients suffering from obstructive azoospermia who were admitted to the CITIC-Xiangya Hospital of Reproduction and Genetics of China from June 1, 2015 to June 31, 2019. A total of 2441 patients with obstructive azoospermia were divided according to the use of fresh (n = 2342) or frozen/thawed (n = 99) epididymal sperm. The results showed that the fertilisation rate was higher with fresh epididymal sperm than that with frozen/thawed epididymal sperm (85.14% vs. 79.26%, respectively; p = 0.000). However, the rates of embryo cleavage, high-quality embryos, clinical pregnancy, miscarriage, singletons and birth defect were similar between fresh and frozen/thawed epididymal sperm (98.28% vs. 99.13%, 60.34% vs. 57.29%, 67.90% vs. 70.51%, 8.12% vs. 10.91%, 57.76% vs. 49.09%, 1.59% vs. 1.45%respectively; p = 0.088, 0.109, 0.628, 0.462,0.203 and 0.686). In addition, the short-term cryostorage of small quantities of epididymal sperm did not affect clinical outcomes. The results indicated that in cases of obstructive azoospermia, cryostorage of small quantities epididymal sperm is a reliable option. [ABSTRACT FROM AUTHOR]

Background: The industrial revolution has resulted in increased synthesis and the introduction of a variety of compounds into the environment and their potentially hazardous effects have been observed in the biota. The present study was aimed to evaluate the potential endocrine-disrupting effects of chronic exposure to the low concentrations of bisphenol S (BPS) in male rats. Methods: Weaning male Sprague-Dawley rats (22 days old) were either exposed to water containing 0.1% ethanol for control or different concentrations of BPS (0.5, 5, and 50 μg/L) in drinking water for 48 weeks in the chronic exposure study. After completion of the experimental period, animals were dissected and different parameters (hormone concentrations, histology of testis and epididymis, oxidative stress and level of antioxidant enzymes in the testis, daily sperm production (DSP), and sperm parameters) were determined. Results: Results of the present study showed a significant alteration in the gonadosomatic index (GSI) and relative reproductive organ weights. Oxidative stress in the testis was significantly elevated while sperm motility, daily sperm production, and the number of sperm in epididymis were reduced. Plasma testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) concentrations were reduced and estradiol levels were high in the 50 μg/L-exposed group. Histological observations involved a significant reduction in the epithelial height of the testis along with disrupted spermatogenesis, an empty lumen of the seminiferous tubules, and the caput region of the epididymis. Conclusion: These results suggest that exposure to 5 and 50 μg/L of BPS for the chronic duration started from an early age can induce structural changes in testicular tissue architecture and endocrine alterations in the male reproductive system which may lead to infertility in males. [ABSTRACT FROM AUTHOR]

Despite encountering new challenges in using epididymal sperm recovered from cauda epididymides, this accessible and, in some species, worthwhile sample makes inevitable the further development of a suitable cryopreservation protocol. In this study, sperm was recovered from the epididymis of 4°C overnight stored slaughtered bulls' testes and the effects of cryopreservation on the bovine epididymal sperm motility (with CASA) and gene expression patterns (with quantitative Real time‐PCR) were evaluated. Moreover the fertilizing potential of cryopreserved epididymal sperm was used in in vitro fertilization (IVF). After freezing and thawing of epididymal sperm, total and slow progressive sperm motility, VCL, VAP, MAD, ALH and BCF were significantly decreased (p <.05), while in the parameters of fast progressive motility, VSL, LIN, WOB and STR there were not any significant variations in the frozen sperm compared to fresh (non‐frozen) counterpart. The assessment of abundance of transcripts encoding motility (TSSK6) and fertility (PRM1 and PRM2)‐related genes in epididymal sperm, showed that these transcripts were affected by freezing especially in slow progressive motility status (p <.01). Furthermore, cleavage and blastocyst rate did not present any significant differences between bovine embryos produced in vitro by fresh or frozen‐thawed epididymal sperm. It can be concluded that epididymal sperm has enough freezability after overnight testes storage, and cryopreservation could not affect the percentage of in vitro produced embryos in spite of the changes of relative abundance of some transcripts and direction progressive motility pattern of sperm. [ABSTRACT FROM AUTHOR]

Creating germplasm banks of wild species, such as the Iberian red Deer (Cervus elaphus hispanicus) can be challenging. One of the main difficulties is the obtention and cryopreservation of good-quality reproductive cells when the spermatozoa are obtained from epididymides after death. To avoid a loss of seminal quality during transport, developing alternative methods for cooling and freezing sperm samples under field conditions is necessary. The objective of this study was to evaluate the effects of different durations of equilibrium and different techniques of cooling and freezing on Iberian red deer epididymal sperm quality after thawing to optimize the processing conditions in this species. Three experiments were carried out: (I) evaluation of refrigeration in straws or tubes of 15 mL; (II) study of equilibration period (0, 30, 60, or 120 min); and (III) comparison of four freezing techniques (liquid nitrogen vapor in a tank (C), liquid nitrogen vapor in a polystyrene box (B), dry ice (DY), and placing straws on a solid metallic plate floating on the surface of liquid nitrogen (MP)). For all experiments, sperm motility and kinematic parameters, acrosomal integrity, sperm viability, mitochondrial membrane potential, and DNA integrity were evaluated after thawing. All statistical analyses were performed by GLM-ANOVA analysis. Samples refrigerated in straws showed higher values (p ≤ 0.05) for mitochondrial activity and lower values (p ≤ 0.05) for apoptotic cells. Moreover, the acrosome integrity showed significant differences (p ≤ 0.05) between 0 and 120 min, but not between 30 and 60 min, of equilibration. Finally, no significant differences were found between freezing in liquid nitrogen vapors in a tank or in a box, although there was a low quality after thawing when the samples were cryopreserved in dry ice or by placing straws on a solid metallic plate floating on the surface of liquid nitrogen. In conclusion, under field conditions, it would be possible to refrigerate the sperm samples by storing them in straws with a 120 min equilibration period and freezing them in liquid nitrogen vapors in a tank or box.

The biological properties of European red deer (Cervus elaphus elaphus) spermatozoa stored in the epididymides and in a liquid state were compared. Spermatozoa were collected from the epididymides harvested post-mortem. In the first variant, spermatozoa were diluted in two extenders (Bovidyl® and Salomon's), and were stored at 5 °C for up to 144 h. In the second variant, spermatozoa were stored in the epididymides at 5 °C for up to 144 h, and then diluted in the same extenders. Biological properties were evaluated after 0, 48, 96, and 144 h of storage. Sperm motility parameters were determined in the CASA system. Plasma and acrosomal membrane integrity, mitochondrial activity, apoptotic changes, and DNA integrity were assessed by the fluorescence method. Most variables were significantly influenced by the storage method and time. At 144 h, differences (P ≤ 0.05) in sperm parameters were observed between storage variants. Total motility (TMOT), plasma membrane integrity, and mitochondrial activity decreased below 50% of baseline values in the spermatozoa stored in the epididymides, but remained above 70% of baseline values in the spermatozoa stored in a liquid state. The compared storage variants did not differ in TMOT, mitochondrial activity, or the percentage of viable spermatozoa without apoptotic-like changes up to 96 h of storage, regardless of the applied extender. The earliest significant changes were noted in sperm motility parameters. In conclusion, European red deer spermatozoa can be stored in the epididymides at 5 °C for up to 96 h, but their biological parameters are more effectively preserved during liquid storage. ● Sperm can be stored in the epididymides in the scrotum at 5 °C for up to 96 h. ● Motility parameters decline earliest during epididymal storage. ● Liquid storage is recommended for short-term preservation of epididymal sperm. ● Bovidyl® has a more positive effect on the progressive movement of sperm than Salomon's. [ABSTRACT FROM AUTHOR]

Follicular fluid is the liquid fraction that surrounds the oocyte during its growth and maturation in the ovary. The components of the follicular fluid can favor the functionality of the sperm. In the present research, the binding to the zona pellucida of epididymal spermatozoa of alpaca incubated with follicular fluid was studied. In the experiment a control group and a treatment group were used. The sperm from the control group and the treatment were incubated with zona pellucidae. The incubation of the treatment group was performed in the presence of follicular fluid at a concentration of 30%; while the incubation of the control group occurred in the absence of follicular fluid. It was observed that a greater number of spermatozoa joined the zona pellucida in the group incubated in the presence of follicular fluid. It was found that there were statistical differences between treatment and control. It is concluded that alpaca follicular fluid enhances the in vitro binding of spermatozoa to the zona pellucida of this species.

Context: Ionidium suffruticosum (L.) Ging (Violaceae) is an important medicinal plant widely used as a herbal traditional medicine in Ayurveda for the treatment of infertility. Currently, little pharmacological information is available on its male fertility properties following prolonged use. Objective: To investigate I. suffruticosum leaf extracts for male fertility parameters. Materials and methods: The ethanol lyophilized fraction was administered orally on carbendazim-induced sub-fertility rats (250 mg/kg body weight for 28 days). The effects of fractions on rat’s fertility parameters i.e., body and testes weight, sperm motility, sperm vitality, epididymal sperm counts, its morphology, enzyme and antioxidant stress and histopathology were studied and compared with clomiphene citrate. Results: The sub-fertile male rats treated with I. suffruticosum leaf extract increased the body weight of 7 g, testis weight of 97 mg, increased cauda epididymal sperm counts of 34.2 × 106 sperm/mL, motility of sperm 46% and vitality 28% also increased and normal sperm morphology also improved up to 32%. The carbendazim-treated group showed loss in body weight of 33 g, testis weight of 851 mg, decreased epididymal sperm counts of 15 × 106 sperm/mL, with sluggish motility and a highly significant fall in the live sperms of about 57%. Discussion and conclusion: The leaf fraction of I. suffructicosum increased the testicular weight, spermatogenesis, sperm counts, lessened sperm agglutination, and increased testicular oxidative biomarkers, SOD, and CAT. This study therefore supports the usage of I. suffructicosum in traditional medicine for infertility.

This study compared three cryopreservation protocols on sperm functions, IVF outcomes, and embryo development. Epididymal spermatozoa cryopreserved using slow-cooling (18% w/v raffinose, RS-C) were compared with spermatozoa vitrified using 0.25 M sucrose (SV) or 18% w/v raffinose (RV). The motility, vitality, and DNA damage (TUNEL assay) of fresh control (FC) spermatozoa were compared with post-thawed or warmed RS-C, RV, and SV samples. Mouse oocytes (n = 267) were randomly assigned into three groups for insemination: RV (n = 102), RS-C (n = 86), and FC (n = 79). The number and the proportion of two-cell embryos and blastocysts from each treatment were assessed. Sperm motility (P < 0.01) and vitality (P < 0.05) were significantly reduced after vitrification compared with slow-cooled spermatozoa. However, DNA fragmentation was significantly reduced in spermatozoa vitrified using sucrose (15 ± 1.8% [SV] vs 26 ± 2.8% [RV] and 27 ± 1.2% [RS-C]; P < 0.01). Although the number of two-cell embryos produced by RS-C, RV, and FC spermatozoa was not significantly different, the number of blastocysts produced from two-cell embryos using RV spermatozoa was significantly higher than FC spermatozoa (P = 0.0053). This simple, small volume vitrification protocol and standard insemination method allows successful embryo production from small numbers of epididymal spermatozoa and may be applied clinically to circumvent the need for ICSI, which has the disadvantage of bypassing sperm selection.

The objective of the study was to investigate the effect of porcine circovirus type 2 (PCV2) vaccination in piglets on sperm quality of young boars. A total of 136 sows were divided in four groups of 34 animals each (17 vaccinated with Circovac®, Merial and 17 unvaccinated in each group). A total of 1 200 piglets were selected, half of which were vaccinated against PCV2 on the 21st day (Porcilis® PCV, MSD) and the other half was left unvaccinated. Four groups of 300 pigs each were formed as follows: PS group (vaccinated sows + piglets), S group (vaccinated sows + unvaccinated piglets), P group (unvaccinated sows + vaccinated piglets), C group (unvaccinated sows + piglets). Furthermore, 80 boars (20 piglets per group) were selected and slaughtered at the age of 5.5 months and weight of 95 ± 5.5 kg and their epididymal sperm was collected and evaluated for motility and kinetics, concentration and morphology. Additionally, 10 pigs from each group were used for blood sampling and serological testing for PCV2 IgM and IgG antibodies at the age of 21, 70, 110 and 150 days. The IgG and IgM patterns suggested that the piglets were coming into contact with PCV2 early in life. The S group demonstrated significantly lower curvilinear velocity (VCL, µm/s), amplitude of lateral head displacement (ALH, µm) and significantly higher head abnormalities (%) compared to other groups (P < 0.05). In conclusion, vaccinated young boars showed some improved epididymal sperm kinetic indices and head morphology. [ABSTRACT FROM AUTHOR]

Abstract This study compares the effectiveness of the ultra-rapid and conventional freezing of sperm from captive bovids, giraffids, cervids, ursids, a cercopithecid, a delphinid and a phascolarctid. The relationship between sperm head dimensions and cryosurvival was also examined. Compared to conventional freezing, the ultra-rapid freezing of epididymal sperm from the dama gazelle, giraffe and brown bear returned higher cryoresistance ratios (CR, the ratio, in percentage, between the value of the variable after thawing/value before thawing) for sperm viability and motility. In the remaining species, the conventional freezing of epididymal sperm returned better CR values. The conventional freezing method also returned better CR values for ejaculated samples from all species. The head dimensions of both fresh epididymal and ejaculated sperm differed widely among species: for epididymal sperm, dolphin sperm heads were the smallest (7.189 ± 0.049 μm2) and dama gazelle sperm heads the largest (43.746 ± 0.291 μm2), while for ejaculated sperm, giant panda sperm heads were the smallest (15.926 ± 0.150 μm2) and mouflon sperm heads the largest (38.258 ± 0.104 μm2). However, no significant correlations were detected between the CR for motility, viability, membrane functional integrity or acrosome integrity and the sperm head area, either for epididymal or ejaculated sperm. In conclusion, ultra-rapid freezing is especially recommended for the cryopreservation of dama gazelle, giraffe and brown bear epididymal sperm. Sperm head dimensions appear not to be useful predictors of how well sperm might survive freezing. Highlights • The study compares the effectiveness of the ultra-rapid and conventional sperm freezing. • The sperm head dimensions differed widely among species. • Ultra-rapid freezing is recommended in dama gazelle, giraffe, brown bear epididymal sperm. • Sperm head dimensions appear not to be useful as predictors of cryosurvival. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study was to evaluate some reproduction performances in Ouled-Djellal rams. Materials and Methods: This study involved genital organs removed after slaughter from 54 rams at the municipal slaughterhouse of Batna (East Algeria). Results: The measurements of survival and mobility of epididymal sperm followed at 0, 24, 48 and 72 h after collection, revealed significant (p0.001) according to time. Thus, concerning the sperm motility the values were 91.00±2.40%, 89.20±2.40%, 77.00±6.20% and 62.60±1.20% at 0, 24, 48 and 72 h, respectively. Indeed, in live sperm, the viability rates were 82.15±1.48%, 77.67±1.74%, 66.56±1.95% and 52.30±1.46 at 0, 24, 48 and 72 h, respectively. Conclusion: This study revealed that epididymal spermatozoa stored at 04°C for 72 h kept their mobility and vitality at nearly a half of their the original parameters.

The goals of the present study were to develop a simple method for obtain highly purified pig sperm hyaluronidase (pHyase) and to assess its activity, function, and safety. In mammals, sperm-specific glycophosphatidylinositol (GPI)-anchored Hyase assists sperm penetration through the cumulus mass surrounding the egg and aids in the dispersal of the cumulus-oocyte complex. Recently, Purified bovine sperm hyaluronidase (bHyase) has been shown to enhance therapeutic drug transport by breaking down the hyaluronan barrier to the lymphatic and capillary vessels, thereby facilitating tissue absorption. Commercially available Hyase is typically isolated from bovine or ovine; which have several disadvantages, including the risk of bovine spongiform encephalopathy, low homology with human Hyase, and the requirement for relatively complex isolation procedures. This study successfully isolated highly purified pHyase in only two steps, using ammonium sulfate precipitation and fast protein liquid chromatography. The isolated Hyase had activity equal to that of commercial bHyase, facilitated in vitro fertilization, and effectively dissolved high molecule hyaluronic acid. This simple, effective isolation method could improve the availability of pHyase for research and clinical applications., Competing Interests: No potential conflict of interest relevant to this article was reported., (© Copyright 2023 Korean Society of Animal Science and Technology.)

Abstract Genome Resource Banks are keystones in the ex-situ conservation of wild species. Post-mortem (PM) collection of epididymal spermatozoa is an opportunistic and valuable source of germplasm, the time from the death of the animal limits its use. Seeking to improve germplasm preservation strategies for the chamois (Rupicapra sp.), the effect of PM time on epididymal sperm quality and freezability was studied using the Cantabrian chamois. Samples were classified according to PM collection time, up to 216 h (refrigerated), and cryopreserved (Tris-citric acid-fructose, 430 mOsm/kg, 15% egg yolk, 8% glycerol; freezing at −20 °C/min). Sperm quality was assessed after recovery and post-thawing (motility by CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). The sperm mass pH and osmolality showed a positive correlation with time. Total sperm motility dropped after 2 days PM, with progressivity and sperm velocities remained similar up to 3 days PM. Sperm freezability was acceptable, with the post-thawing HOST, motility, progressivity, VAP, VCL, VSL and BCF negatively correlating with PM time. Overall, chamois epidydimal samples were not adequate for preservation after 6 days PM. Freezability capacity could make these spermatozoa suitable for specific ART even if kept refrigerated for several days PM. [ABSTRACT FROM AUTHOR]

Contents: The aim of the study was to compare the characteristics of chinchilla epididymal sperm: fresh, stored at liquid state and cryopreserved. Epididymal spermatozoa obtained from 11 males were assessed for subjective motility, concentration, motility parameters measured by CASA, viability, morphology, membrane integrity, acrosome integrity, mitochondrial potential, lipid peroxidation, chromatin structure, apoptotic changes and capacitation. Then half of the spermatozoa were stored at 5°C for 30 hr, and the second half was cryopreserved. After storage and thawing the same parameters as in fresh semen were assessed. Fresh semen showed good quality, with low levels of lipid peroxidation, chromatin fragmentation and capacitation. CASA evaluation showed significantly lower values for MOT, PMOT, RAPID, VCL, VAP and VSL after both storage at liquid state and cryopreservation (p < 0.05). Cold storage did not induce membrane and acrosome damage (p > 0.05), conversely to cryopreservation (p < 0.05). After storage, there was a drop in high mitochondrial potential in live cells (p < 0.05) and an increase in the percentage of non‐apoptotic, capacitated cells (p < 0.05). These changes were not seen after cryopreservation (p > 0.05). Lipid peroxidation in live cells and chromatin structure remained unchanged both after storage and cryopreservation (p > 0.05). The study showed that examined methods of semen preservation exerted different patterns of changes in spermatozoa and that sperm quality after both of them allowed for further use of preserved spermatozoa in artificial reproductive techniques. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the present study was to improve the penetration during in vitro fertilization (IVF) of a frozen lot of epididymal sperm with a notoriously low fertilization ability of a Ban boar which is a native Vietnamese breed by optimizing different parameters of the IVF system. In Experiment 1, we determined that Pig‐fertilization medium was superior medium to Tyrode's albumin lactate pyruvate‐polyvinyl alcohol medium for IVF and defined the optimum the sperm concentration (1 × 106 sperm/ml). In Experiment 2, we clarified that partial removal of cumulus cells from cumulus‐oocyte complexes by hyaluronidase treatment before IVF enhances sperm penetration, whereas complete cumulus removal reduces penetration. Finally, in Experiment 3 the elevation of concentration of caffeine in Pig‐fertilization medium from 2 to 5 mmol/L and the prolongation of the co‐culture of gametes from 3 to 5 hr significantly increased the total penetration rate from 15.2% to over 50%. In conclusion, the combination of partial oocyte denudation, an elevated caffeine concentration in Pig‐fertilization medium and an extended interval of IVF with using an optimized sperm concentration was a potent way to improve the fertilization results for a frozen epididymal Ban sperm lot with low fertility. [ABSTRACT FROM AUTHOR]

Purpose: The aims of this study were (1) to evaluate clinical outcomes after ICSI cycles using surgically recovered sperm and (2) to assess the influence of maternal age on those outcomes.Methods: A retrospective cohort study of 24,763 IVF cycles of fresh autologous oocytes and ICSI using surgically recovered sperm reported to the SART CORS database from 2004 to 2015.Results and conclusions: Older women had significantly longer stimulation (p < 0.001), a lower number of oocytes retrieved (p < 0.001), a lower number of 2PN zygotes (p < 0.001), a lower chance of having a blastocyst transferred (p < 0.001), and a higher number of fresh embryos transferred (p < 0.001). There was no significant association between the number of 2PNs per oocyte retrieved and maternal age (p = 0.214). Both clinical pregnancy rates and live birth rates (LBR) decreased with advanced maternal age (p < 0.001). LBR ranged from 50.4% in women < 30 to 7.2% in women > 42 years, and for cleavage-stage transfers, the LBR ranged from 47.3% in women< 30 to 6.3% in women > 42 years. There were no differences in gestational age at delivery, proportion of term deliveries, preterm deliveries, neonatal birth weight < 2500 g, neonatal birth weight > 4000 g and average birthweight of neonates for singleton pregnancies according to age. For twin pregnancies, women < 30 years had significantly higher number of live births, term deliveries, and lower preterm deliveries than older women. There was a similar number of female (6051) and male neonates (5858; p = 0.2). Overall, pregnancy outcomes with ICSI using surgically recovered sperm are reassuring and comparable to those of ICSI with ejaculated sperm. [ABSTRACT FROM AUTHOR]

This study examines the effectiveness of two methods for cryopreserving post-mortem epididymal sperm - conventional slow freezing employing a short equilibration time with glycerol, and ultra-rapid freezing - from the wild ruminant species Ovis musimon (mouflon) and Dama dama (fallow deer). A Tris-citric acid-glucose (TCG) + 12% egg yolk-based medium was used for the conventional slow freezing of the fallow deer sperm, whereas a Tes-Tris-glucose (TEST) + 6% egg yolk-based medium was used for the mouflon sperm. Glycerol was added to a final concentration of 5% to both media. The same diluents were used for ultra-rapid freezing but replacing the glycerol with 100 mM of sucrose. Sperm variables (motility, viability, acrosome integrity, membrane integrity, and morphological abnormalities) were analyzed before and after cryopreservation. Although values were generally better after the thawing of the conventionally cryopreserved sperm, total sperm motility (38.40 ± 4.44% in mouflon and 31.25 ± 3.37% in fallow deer) and total live sperm (47.19 ± 5.18% in mouflon and 43.13 ± 2.43% in fallow deer) were acceptable for the ultra-rapidly cooled sperm. Independent of the cryopreservation method, membrane integrity, acrosome integrity and the percentages of dead sperm and sperms with a damaged acrosome were better for the cryopreserved mouflon sperm than the fallow deer sperm (P < 0.05). Despite exerting a more harmful effect on sperm variables than conventional freezing, ultra-rapid freezing may be a useful alternative for the cryopreservation of these species' epididymal sperm in the field, as this simple technique does not require sophisticated equipment and expertise. [ABSTRACT FROM AUTHOR]