Semen Cryopreservation

Background and Aim: The Javan leopard (Panthera pardus melas Cuvier, 1809) is a subspecies of Panthera pardus spp., spread across the African and Asian regions. Information on reproductive aspects is crucial for wild animals, including the Javan leopard. In this study, we aimed to develop electroejaculator (EE) techniques and evaluate cryopreservation success in Javan leopard semen.

Materials and Methods: The semen of four adult Javan leopards was collected once a week using EE. Placement of the EE probe in the rectum was performed after ultrasound imaging (ultrasonography) to determine the prostate body location. The semen obtained was then evaluated macroscopically and microscopically. Three Javan leopards were used for cryopreservation. The ejaculate was divided into two parts [i.e., one part diluted with AndroMed® (Minitüb, Tiefenbach, Germany) and the other part with Steridyl® (Minitüb)] at a 1:1 ratio immediately after collection and evaluation. The semen was then packed in a 0.25 mL MiniStraw® (Minitüb) then equilibrated at 4°C for 2 h. After equilibration, the straw was then frozen in liquid nitrogen vapor. Frozen semen was then stored in containers until further evaluation.

Results: The results showed that ejaculation response occurred at all levels of stimulation, while erections did not always occur. The fastest ejaculation and erection occurred at the fourth voltage. The macroscopic evaluation showed that the semen volume was 0.80±0.26 mL, cloudy white, pH 7.44±0.14, and with watery semen consistency. The microscopic evaluation showed that the sperm motility was 66.98±0.39%, with sperm viability of 75.6±1.79%. Sperm concentration was 62.17±46.95×106 mL–1 with a total concentration of 42.14±23.51×106 cells. Normal sperm morphology is only 40.72±6.26%.

Conclusion: This study concluded that the development of a semen collection technique using an EE preceded by imaging of the EE probe location using ultrasound was effective for the ejaculation of Javan leopards. The characteristics of the semen of the Javan leopard showed moderate semen volume, sperm motility, and viability. Javan leopard showed low sperm concentration and normal sperm morphology.

Objective: To test the effects of extenders and packaging methods on morphological and functional characteristics of frozen thawed Ossimi ram semen. Methods: Ram semen was pooled, diluted in 3 different extenders: Tris-egg yolk (TEY), Tris-soybean lecithin (TSBL), and Tris-butylatedhydroxytoluene (TBHT), equilibrated at 5 C for 4 h, and packaged in straws or pellets for freezing. Semen was evaluated for sperm progressive motility, viability, abnormality, and membrane integrity after dilution, equilibration and thawing. The percentages of viable, early apoptotic, apoptotic, and necrotic spermatozoa as well as comet assay parameters were determined in post-thawed semen. Total antioxidants capacity, malondialdehyde and lactic dehydrogenase were assayed in thawed seminal plasma. Results: After equilibration, only sperm membrane integrity was significantly higher (P<0.05) in TEY and TSBL than in TBHT. After thawing, TEY or TSBL in straws significantly improved sperm progressive motility and vitality (P<0.05). In thawed seminal plasma, TBHT or TSBL in straws and TBHT in pellets significantly reduced malondialdehyde, and TBHT in pellets significantly increased lactic dehydrogenase (P<0.05). TEY in straws increased viable sperm, while significantly decreased early apoptotic and apoptotic sperm (P<0.05). DNA damage was significantly decreased (P<0.05) in straws with TEY and TSBL, and tail moment decreased in straws with all extenders (P<0.05). Conclusions: Despite the disadvantages of dilution of cryopreserved semen with egg yolk, ram semen cryopreserved with TEY gives the best physical, morphological and functional characteristics in straws compared with pellets, followed by semen diluted with TSBL. However, semen diluted with TBHT or TSBL, regardless of packaging method, showed the highest impact on antioxidant status of cyopreserved ram semen.

Simple Summary: This is a comprehensive study to compare between the effects of different supplements (vitamins C and E, trace elements Na 2 SeO 3 and ZnSO 4 , and nanoparticles of zinc oxide and selenium) to the semen extender of camel epididymal spermatozoa during cooling and freezing/thawing cryopreservation. Supplementation of the semen SHOTOR extender with zinc oxide and selenium nanoparticles lead to improved progressive motility, vitality, and anti-oxidative defense, and reduced the ultrastructural abnormalities in camel epididymal spermatozoa. Abstract: There are several obstacles in camel semen cryopreservation; such as increasing semen viscosity and the reduction in motile spermatozoa after ejaculation. Epididymal spermatozoa offer an efficient alternative to overcome these problems and are well-suited for artificial insemination in camels. In the current study, we compared the effects of supplementation with vitamin C, E, inorganic trace elements of selenium (Na 2 SeO 3) and zinc (ZnSO 4), and zinc and selenium nanoparticles (ZnONPs and SeNPs, respectively) on the cryopreservation of dromedary camel epididymal spermatozoa. When the SHOTOR extender was supplemented with ZnONPs and SeNPs; the sperm showed increased progressive motility; vitality; and membrane integrity after cooling at 5 • C for 2 h; when compared to the control and vitamin-supplemented groups. Moreover, the ZnONPs and SeNPs supplementation improved the progressive motility, vitality, sperm membrane integrity, ultrastructural morphology, and decreased apoptosis when frozen and thawed. SeNPs significantly increased reduced glutathione (GSH), superoxide dismutase (SOD), and decreased lipid peroxide malondialdehyde (MDA) levels. The advantageous effects of the trace elements were potentiated by reduction into a nano-sized particle, which could increase bioavailability and reduce the undesired liberation of toxic concentrations. We recommend the inclusion of SeNPs or ZnONPs to SHOTOR extenders to improve the cryotolerance of camel epididymal spermatozoa.

Background and Aim: The Javan leopard (Panthera pardus melas Cuvier, 1809) is a subspecies of Panthera pardus spp., spread across the African and Asian regions. Information on reproductive aspects is crucial for wild animals, including the Javan leopard. In this study, we aimed to develop electroejaculator (EE) techniques and evaluate cryopreservation success in Javan leopard semen. Materials and Methods: The semen of four adult Javan leopards was collected once a week using EE. Placement of the EE probe in the rectum was performed after ultrasound imaging (ultrasonography) to determine the prostate body location. The semen obtained was then evaluated macroscopically and microscopically. Three Javan leopards were used for cryopreservation. The ejaculate was divided into two parts [i.e., one part diluted with AndroMed® (Minitüb, Tiefenbach, Germany) and the other part with Steridyl® (Minitüb)] at a 1:1 ratio immediately after collection and evaluation. The semen was then packed in a ...

There are various challenges during semen cryopreservation used for artificial insemination, oxidative stress is considered as the major factor. Usage of nano-particle materials is an efficient tool to overcome these challenges. In the present article, effects of adding green synthesized gold nanoparticles (GSGNPs, 5; 10ppm/mL) to Tris-extender on sperm characteristics in post-equilibrated and post-thawed semen. Apoptosis and sperm ultrastructure post-thawed were also examined as well as enzyme activity and antioxidant status in post-thawed extender of goat semen were evaluated. Supplementation of GSGNPs (10 ppm) increased (P<0.05) motility, livability, and membrane integrity post-equilibration, and also in addition to acrosome integrity post-thawing. Also, viable sperm increased (P<0.05), early apoptotic, apoptotic, and necrotic sperm decreased (P<0.05) by 10 ppm GSGPNs. In the extender of thawed semen, AST and ALT activities were not affected, total antioxidant capacity and catalase activity were higher (P<0.05), while malondialdehyde decreased (P<0.05) by 10 ppm GSGNPs. Frequency distribution of sperm cells with intact plasma or acrosomal membranes increased (P<0.05), while of those with swollen plasma membranes or typical acrosome reaction decreased (P<0.05). However, frequency distribution of sperm cells with slightly swollen or lost plasma membranes as well as atypical acrosome reaction and lost acrosomal membrane was not affected by GSGNPs. Transmission electron microscopy examination revealed no entry of GSGNPs with sperm cells.The present article may suggest considerable addition of GSGNPs at a level of 10 ppm/mL to Tris-extender of cryopreserved goat semen during cryopreservation.

This study was aimed to explore the effect of adding pentoxifylline (PTX) and nitric oxide (NO) to Tris extender on some post-cryopreserved semen attributes of Holstein bulls for different preservation periods (cooling at 5•C, 48 hrs., 1, 2 and 3 months post cryopreservation, PC). Seven Holstein bulls of 2.5-3 years old were used in the current study during the period from 20 th November, 2017 to 20 th August, 2018. Pooled semen was equally divided into three groups within one experiment. PTX (0.192 g / 100 ml extender) and NO (0.018 g / 100 ml extender) were added to Tris extender and comparisons in response were made with the control group (Tris extender, C). The PTX group exhibited greater (P≤ 0.01) sperm's cell individual motility percentage as compared with the C group at cooling (5ºc) and 48 hr PC periods, while, PTX and NO groups were superior in these percentages at the remaining PC periods than C group. Excluding data of 2 months PC, greater (P≤ 0.01) live sperm percentage was observed in PTX and NO groups in comparison with the C group at all preservation periods. Lesser (P≤ 0.01) abnormal sperm percentage were noticed for PTX and NO groups as compared with the C group at all preservation time periods. The PTX and NO groups exhibited greater (P≤ 0.01) acrosome and plasma membrane integrity percentages in comparison with the C group at all preservation time periods. In conclusion, adding PTX and NO to Tris extender enhanced some of PC semen characteristics of Holstein bulls at different preservation periods. 1 1 ‫االصطناعي،دائرة‬ ‫التلقيح‬ ‫قسم‬ ‫اقية.‬ ‫العر‬ ‫اعة‬ ‫الزر‬ ‫ارة‬ ‫انية،وز‬ ‫الحيو‬ ‫الثروة‬ 2 ‫بغداد.‬ ‫اعية،جامعة‬ ‫الزر‬ ‫الهندسة‬ ‫علوم‬ ‫كلية‬ ‫المستخلص‬ ‫مخفف‬ ‫الى‬ ‫النتريك‬ ‫اوكسيد‬ ‫و‬ ‫البنتوكسفايلين‬ ‫اضافة‬ ‫تأثير‬ ‫بيان‬ ‫بهدف‬ ‫اسة‬ ‫الدر‬ ‫هذة‬ ‫أجريت‬ Tris ‫بعد‬ ‫الهولشتاين‬ ‫ان‬ ‫لثير‬ ‫المنوي‬ ‫السائل‬ ‫صفات‬ ‫بعض‬ ‫في‬ ‫(التبري‬ ‫مختلفة‬ ‫زمنية‬ ‫لمدد‬ ‫الحفظ‬ ‫ارة‬ ‫حر‬ ‫بدرجة‬ ‫د‬ 5 º ‫لمدة‬ ‫التجميد‬ ‫و‬ ‫م‬ 48 ‫ان‬ ‫ثير‬ ‫سبعة‬ ‫استخدام‬ ‫تم‬ ‫بالتجميد).‬ ‫الحفظ‬ ‫من‬ ‫اشهر‬ ‫وثالثة‬ ‫وشهرين‬ ‫وشهر‬ ‫ساعة‬ ‫بعمر‬ ‫هولشتاين‬ 2.5-3 ‫من‬ ‫للمدة‬ ‫ات‬ ‫سنو‬ 20 ‫الثاني/‬ ‫تشرين‬ 2017 ‫اب/‬ ‫ولغاية‬ 2018 ‫ثالث‬ ‫الى‬ ‫تقسيمه‬ ‫ثم‬ ‫ومن‬ ‫وتجميعه‬ ‫المنوي‬ ‫السائل‬ ‫جمع‬ ‫تم‬. ‫مخفف‬ ‫باستخدام‬ ‫اء‬ ‫اجز‬ Tris. (‫البنتوكسفايلين‬ ‫مركب‬ ‫من‬ ‫كل‬ ‫اضيف‬ 0.192 ‫غم/‬ 100 (‫النتريك‬ ‫اوكسيد‬ ‫و‬ ‫مخفف)‬ ‫مل‬ 0.018 ‫غم/‬ 100 ‫الى‬ ‫مخفف)‬ ‫مل‬ ‫مخفف‬ Tris ‫(مخفف‬ ‫السيطرة‬ ‫مجموعة‬ ‫مع‬ ‫ومقارنتها‬ Tris ‫البنتوكسفايلين‬ ‫مجموعة‬ ‫اظهرت‬ ‫لوحده).‬ ‫معنوي‬ ‫ارتفاع‬ (P≤ 0.01) ‫ال‬ ‫في‬ ‫نسبة‬ ‫ال‬ ‫مئوية‬

This study was conducted to investigate the effect of adding aqueous extracts of Olea europaea (AEOEL), Rosmarinus officinalis leaves (AEROL), calcium chloride (CaCl 2) and their combinations to Tris extender on post-cryopreserved sperm's cell individual motility and live sperm percentage for low-semen quality of Holstein bulls for different preservation periods (cooling at 5 0 C, 48 hrs., 1, 2 and 3 months post cryopreservation, PC). The experiment was carried out using five Holstein bulls of 2.5-3 years old. Pooled semen was equally divided into six groups within one experiment. AEROL (0.04 mg / 25 ml; T 2), AEOEL (0.01 mg / 25 ml; T 3), CaCl 2 (0.02 M; T 4), AEROL (0.04 mg / 25 ml) + CaCl 2 (T 5) and AEOEL (0.01 mg / 25 ml) + CaCl 2 (T 6) were added to Tris extender and comparisons in response were made with the control group (Tris extender, T 1). The productive revenue percentage of the aqueous extracts of Olea europaea leaves was 6.6%, the total phenolic compounds of the extract was 28.9 mg GAE / g extract and the total flavonoid compounds was 16.4 mg Rutin / g extract. On the other hand, the productive revenue percentage of the aqueous extracts of Rosmarinus officinalis leaves was 7.2%, the total phenolic compounds of the extract was 73.2 mg GAE / g extract and the total flavonoid compounds was 39.5 mg Rutin / g extract. The differences in sperm's head, tail's midpiece, principal, terminal and total abnormalities (except for S 4 , 3 months PC) percentages lacked significance. Moreover, non-significant differences among PC periods were observed for most sperm's abnormality types, but significantly different with their cooling periods. In conclusion, adding the AEOEL and AEROL as alone or in combinations with CaCl 2 to Soybean-Lecithin extender did not significantly loweredthe sperms abnormality percentages for low-semen quality Holstein bulls. Abdulkareem and Zaidi, 2018a). That the increase in the quantity of low-quality semen in artificial insemination centers is significant and the economic loss is not low, which is reflected negatively in the low rates of fertilization of cattle herds and the financial yields of cattle breeders (Alam et al, 1993; Anann and Hammerstedt, 2002). There was obvious relationship between a low quality semen and the low concentration of calcium in sperm plasma of men (Slivkova et al, 2009; Li et al, 2012), due to the large role of calcium in the hyper activation of semen, on the other hand. The continuous release of reactive oxygen species by abnormal sperms and immature sperms, as well as the result of freezing and sagging of semen, often accompanied by a decrease in antioxidant concentrations in the seminal plasma as well as in semen extenders,

The Splinter bean, Entada abyssinica, is widely used in folk medicine. In the current work, we profiled the secondary metabolites from E. abyssinica bark extract using LC-MS and investigated its effect on cryopreserved ram semen. Twenty-eight compounds, including tannins and gallic acid derivatives that prevailed in the extract, were tentatively identified. Results showed that the quality of the post-thawed semen showed a significant improvement when the extract was added to the extender at a concentration of 375 µg/mL. The progressive motility and plasma membrane integrity of sperm cells were significantly increased in the post-thawed semen; however, the total antioxidant capacity (TAC) was insignificantly increased. A significant decrease in the concentration of hydrogen peroxide was detected as well. No significant changes were observed in activities of lactate dehydrogenase (LDH), alanine aminotransaminase (ALT), and aspartate transaminase (AST) within the treated samples. Intact sperm percentage was significantly increased, while apoptotic and necrotic sperm percentages were reduced significantly. Molecular docking of some individual components from the extract revealed their potential to interfere with the apoptosis cascade in which Bcl-2 is involved. In conclusion, Entada abyssinica appears to be useful for cryopreservation presumably owing to its polyphenol content that has potent antioxidant capacity scavenging reactive oxygen species (ROS), enhancing the endogenous antioxidant system and inhibiting lipid peroxidation.

There are various challenges during semen cryopreservation used for artificial insemination, oxidative stress is considered as the major factor. Usage of nano-particle materials is an efficient tool to overcome these challenges. In the present article, effects of adding green synthesized gold nanoparticles (GSGNPs, 5; 10ppm/mL) to Tris-extender on sperm characteristics in post-equilibrated and post-thawed semen. Apoptosis and sperm ultrastructure post-thawed were also examined as well as enzyme activity and antioxidant status in post-thawed extender of goat semen were evaluated. Supplementation of GSGNPs (10 ppm) increased (P<0.05) motility, livability, and membrane integrity post-equilibration, and also in addition to acrosome integrity post-thawing. Also, viable sperm increased (P<0.05), early apoptotic, apoptotic, and necrotic sperm decreased (P<0.05) by 10 ppm GSGPNs. In the extender of thawed semen, AST and ALT activities were not affected, total antioxidant capacity and catalase activity were higher (P<0.05), while malondialdehyde decreased (P<0.05) by 10 ppm GSGNPs. Frequency distribution of sperm cells with intact plasma or acrosomal membranes increased (P<0.05), while of those with swollen plasma membranes or typical acrosome reaction decreased (P<0.05). However, frequency distribution of sperm cells with slightly swollen or lost plasma membranes as well as atypical acrosome reaction and lost acrosomal membrane was not affected by GSGNPs. Transmission electron microscopy examination revealed no entry of GSGNPs with sperm cells.The present article may suggest considerable addition of GSGNPs at a level of 10 ppm/mL to Tris-extender of cryopreserved goat semen during cryopreservation.