163 EVALUATION OF VIABILITY OF BULL SEMEN COLLECTED BY ELECTRO-EJACULATION USING COMMERCIAL SEMEN EXTENDER AND 2 CULTURE MEDIA AT CONTROLLED ROOM TEMPERATURE

2017 ◽  
Vol 29 (1) ◽  
pp. 190
Author(s):  
A. M. Raseona ◽  
O. A. Ajao ◽  
L. D. Nethengwe ◽  
L. R. Madzhie ◽  
T. L. Nedambale ◽  
...  

Preservation of semen is an important process to ensure that semen quality is sufficient for assisted reproductive technologies. The aim of this study was to evaluate the viability of bull semen collected by electro-ejaculation using commercial semen extender and 2 modified culture media stored at controlled RT (24°C) for 72 h. Two Nguni bulls were used for semen collection; after collection, the semen was evaluated macroscopically for volume, pH, and colour, and microscopically for sperm motility, viability, and morphology. Uncontaminated semen samples with progressive motility >70% and morphological defects <20% were pooled after collection before being aliquoted into 3 extenders, namely Triladyl, modified Ham’s F10, and TCM-199 culture media, at a dilution ratio of 1:4 and then stored at controlled RT (24°C). Sperm motility rate was analysed using the computer-aided sperm analyser after 0, 24, 48, and 72 h of storage. Sperm morphology and viability was performed after staining the sperm cells with spermac and nigrosine-eosin stain, respectively. The study was replicated 4 times and data were analysed using ANOVA. Triladyl had a higher sperm viability rate (41.3%) and total motility rate (96.3%) for 72 h (P < 0.01) compared with the 2 modified culture media, Ham’s F10 (26.5 and 86.8%) and TCM-199 (25.0 and 86.7%), respectively. However, Ham’s F10 had higher progressive motility rate (37.8%) as compared with the other extenders, TCM-199 (31.7%) and Triladyl (23.4). There was no significant difference (P > 0.05), in viability rate between Ham’s F10 (26.5%) and TCM-199 (25.0%). No significant difference (P > 0.05) in straight line velocity was observed for the three extenders. Furthermore, no significant difference was observed in total sperm abnormalities, except for reacted acrosomes and absent tails (P > 0.05), between the 2 Nguni bulls. Nguni semen can be preserved in Triladyl or modified Ham’s F10 and TCM-199 culture media stored at 24°C and stay viable for 72 h. Triladyl proved to be the best suitable extender of the 3 extenders, showing higher sperm viability and total motility rate as compared with Ham’s F10 and TCM-199 modified culture media.

2020 ◽  
Vol 1 (1) ◽  
pp. 83-92
Author(s):  
Jennifer B Nagashima ◽  
Marcia de Almeida Monteiro Melo Ferraz ◽  
Sarah H Kamen ◽  
Nucharin Songsasen

The red wolf is a critically endangered canid, with ~250 and ~20 individuals in the ex situ and reintroduced wild populations, respectively. Assisted reproductive technologies such as sperm cryopreservation and in vitro fertilization therefore represent critically-needed tools to manage these populations. However, the motility of post-thaw red wolf sperm rapidly declines during in vitro incubation, hindering the ability to develop these technologies. In this study, we evaluated the influence of several culture media (a modified canine capacitation medium (mCCM), a modified North Carolina State University-23 medium (mNCSU-23), a synthetic oviductal fluid (SOF), a fertilization Tyrode’s medium base or Fert-TALP (FERT), and a TRIS-based buffer (TRIS)) on the survival and capacitation of red wolf sperm during extended (18 h) incubation at 38.5°C and 5% CO2. Red wolf sperm motility averaged (±s.e.m.) 73.8 ± 7.1% at the time of collection, and was better maintained over 4 h incubation in mCCM (55.0 ± 9.8%) and mNCSU-23 (54.7 ± 10.4), compared to mSOF (43.8 ± 8.3%), FERT (30 ± 10.5), and TRIS (16.4 ± 4.1%) solutions. Patterns of tyrosine phosphorylation signal, as assessed via immunocytochemistry, indicated induction of capacitation between 2 and 4 h in vitro culture. Tyrosine phosphorylation signal was particularly robust in mCCM and mNCSU-23 incubated sperm, although significant acrosome exocytosis was not observed in response to progesterone supplementation after 3 h incubation in any of the media. In sum, results indicate mCCM and mNCSU-23 are promising base media for the in vitro incubation and capacitation of red wolf sperm, for assisted reproduction applications. Lay summary Development of assisted reproductive technologies such as in vitro fertilization and artificial insemination is of high importance to the genetic management of critically endangered species such as the red wolf (Canis rufus). However, these technologies require the ability to maintain sperm viability and function during extended incubation, which has not been successful for the red wolf thus far. In this study, various culture media developed for sperm/egg/embryo culture in large mammalian species were evaluated for their ability to maintain red wolf sperm motility under physiological incubation conditions. Media and conditions previously utilized for domestic dog sperm were found to best support sperm incubation and capacitation (process of becoming competent to fertilize an egg) in the red wolf, representing a key step for future development of assisted reproductive technologies for the species.


2021 ◽  
Vol 33 (9) ◽  
pp. 619
Author(s):  
Lachlan Campbell ◽  
John Clulow ◽  
Belinda Howe ◽  
Rose Upton ◽  
Sean Doody ◽  
...  

Assisted reproductive technologies (ARTs) have a significant role to play in reptile conservation, yet are severely lacking. Previous attempts to cryopreserve spermatozoa in the threatened lizard Varanus panoptes achieved approximately 48% motile sperm post-thaw for samples frozen immediately after collection. However, the feasibility of extended cold storage before cryopreservation has not been tested. We held V. panoptes spermatozoa at either 25°C or 4°C for 8 days, assessing sperm motility at days 1, 2, 4 and 8. Subsamples were cryopreserved on days 1 and 4 following the previously reported protocol for this species. Percentage motility decreased rapidly at 25°C, but did not decrease significantly until 4 days after collection at 4°C, with &gt;30% motility maintained after 8 days. There was no significant difference in post-thaw motility or viability of samples cryopreserved after 1 or 4 days storage at 4°C, yielding substantial results for both parameters (mean motility 23.8% and 28.1% and mean viability 50.1% and 57.5% after 1 and 4 days respectively). We demonstrate the capacity to extend sperm viability for up to 8 days in unfrozen samples and to produce acceptable post-thaw motility in samples frozen after 4 days of storage, contributing to the development of valuable ARTs for lizards and other reptiles.


2020 ◽  
Vol 32 (2) ◽  
pp. 242
Author(s):  
S. Kamen ◽  
J. Nagashima ◽  
N. Songsasen ◽  
M. Ferraz

Development of assisted reproductive technologies for the critically endangered red wolf (Canis rufus) is crucial to the maintenance of genetic diversity to support species recovery. Towards this goal, a cryopreservation protocol has previously been developed for red wolf sperm; however, the ability of the gametes to undergo capacitation has not been assessed in this species. Previously, we have shown that oviductal extracellular vesicles (oEVs) improve cat sperm motility and fertilizing ability. The objectives were to (1) compare the effects of culture media on motility and acrosomal integrity of fresh sperm, and select the best medium that can be used in a capacitation protocol; (2) identify potential biomarkers for sperm cryo-tolerance; and (3) determine the influence of canine oEVs on sperm survival and motility post-thaw. In Study 1, sperm were collected by electro-ejaculation from adult red wolves (n=8) and immediately cryopreserved in TRIS-egg yolk buffer with 8% glycerol or incubated for 18h in 5% CO2 and 38.5°C in one of the following media: canine capacitation medium (CCM), FERT-TALP (FERT), NCSU, synthetic oviductal fluid (SOF) and TRIS. At 0, 1, 2, 3, 4, and 18h, sperm were evaluated for total motility and acrosome integrity (FITC-PNA). In Study 2, sperm with high (&gt;80%, HM, n=2 wolves) and low (&lt;15%, LM, n=2 wolves) motility post-incubation at 4°C in the cryopreservation medium for 18h were subjected to proteomic analysis. In Study 3, oviducts were collected from domestic dogs (1-9 years, n=12) after elective spaying, and oEVs from various stages of the oestrous cycle [early follicular (EF), late follicular (LF), early luteal (EL), and late luteal (LL)] were isolated using the Total Exosome Isolation kit (Invitrogen). Frozen-thawed red wolf sperm (n=4 males) were incubated with 30×106 oEVs in non-capacitating CCM, and assessed as in study 1 at 0, 0.5, 1, 2, 3, 4, 6, 8, and 10h. Data were analysed using a paired samples t-test with 95% CI (Prism8, GraphPad Inc.). Sperm incubated in CCM and NCSU had higher motility than those in FERT, SOF, and TRIS after 2h of incubation and onward (2 h: 65±6, 68±6, 42±10, 57±8, and 43±5; 3 h: 60±9, 63±8, 36±11, 46±9, and 34±6; 4 h: 60±9, 60±10, 30±10, 43±8, and 20±5; 18 h: 12±7, 15±7, 9±5, 3±2, and 0, respectively; P&lt;0.05). After 1h of incubation, samples incubated in CCM, NCSU, and SOF had a higher number of sperm with intact acrosomal membranes (P&gt;0.05) than other treatments. A total of 179 proteins were identified, of which 129, including those regulating energy metabolism and mitochondrial mediated apoptosis, were differentially expressed between HM and LM. Preliminary data from Study 3 suggested that thawing and incubating sperm in the presence of LF, EL, and LL oEVs improved sperm motility. In conclusion, CCM and NCSU sustained sperm survival after invitro incubation and could be candidates for invitro fertilization studies in the red wolf. Data generated from sperm proteomic analysis provided insights into cellular pathways regulating sperm cryo-sensitivity. Finally, we demonstrated the potential of oEVs in improving wolf sperm survival post-thawing.


2014 ◽  
Vol 26 (1) ◽  
pp. 140
Author(s):  
M. M. Seshoka ◽  
M. L. Mphaphathi ◽  
T. L. Nedambale

Cryopreservation has been reported to damage approximately 40 to 50% of viable spermatozoa in bulls. It is critical to evaluate frozen-thawed spermatozoa with computer-aided sperm analysis (CASA) and find a suitable cryoprotectant for Nguni semen. The study was conducted to compare cryo-effectiveness of glycerol (GLY), dimethyl sulfoxide (DMSO), propanediol (PND), and ethylene glycol (EG) cryoprotectants at 12% concentrations during freezing of Nguni bull semen. Semen was collected from 18 stud Nguni bulls of proven fertility with the use of an electro-ejaculator. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories. Semen samples were pooled and sperm motility rate was evaluated using CASA. Semen was then diluted (1 : 2; vol : vol) with egg-yolk citrate extender supplemented with either 12% GLY, DMSO, PND, or EG. Semen samples were equilibrated for 4 h at 5°C. After equilibration, samples were loaded into 0.25-mL straws and placed into the controlled rate programmable freezer using a customized freezing curve (from 5 to –5°C at 0.008°C min–1 and from –3 to – 130°C at 6°C min–1). Following thawing of semen, artificial insemination was conducted on 104 oestrus-synchronised Nguni cows. The IVF was also conducted on 120 oocytes to check the cleavage rate. Data were analysed using ANOVA. There was a significant difference (P < 0.05) between raw total sperm motility (94.70 ± 2.63) and frozen-thawed sperm total motility with GLY (77.80 ± 11.03), EG (20.35 ± 11.86), DMSO (15.68 ± 10.14), and PND (11.19 ± 11.27) groups. The pregnancy rate following artificial insemination was 75.9% and a total of 86.6% oocytes had cleaved after fertilization with frozen (12% GLY)/thawed semen. In conclusion, cryopreservation process reduced sperm motility and velocity rates, regardless of cryoprotectant. Egg-yolk citrate extender supplemented with 12% glycerol had recorded the highest post-thaw sperm motility rate.


2018 ◽  
Vol 30 (1) ◽  
pp. 174
Author(s):  
N. C. Negota ◽  
M. L. Mphaphathi ◽  
L. P. Nethenzheni ◽  
T. L. Rammutla ◽  
N. R. Serota ◽  
...  

Mammalian blastocysts must hatch out from the zona pellucida before implantation. In vitro embryo culture and grouping of mice blastocysts are conducive options of assisted reproductive technologies (ART) to speed up the hatching rate of mice embryos. The number of embryos per unit volume has the greatest impact on hatching rates due to autocrine signalling. The study aimed to determine the effect of two in vitro culture (IVC) media (TCM-199 and Ham’s F10) and embryo groupings (1, 2, 3, and 4 embryos per 50-µL droplet) after 24 h of culture on hatching rate. Breeds of C57BL/6 (n = 10) and BALB/c (n = 10) were raised until they reached maturity and bred naturally to produce the first filial generation. The photoperiod was 14 h of light followed by 10 h of darkness in the breeding house, and feed and water were provided ad libitum. Female mice were superovulated using eCG and hCG. The first filial generations from 2 breeds were used for the collection of 160 blastocysts and randomly allocated into 2 IVC media (80 embryos for TCM-199 and 80 embryos for Ham’s F10) and again subjected to 4 embryo groupings (1, 2, 3, and 4 embryos per droplet) treatments. Four replicates were done per treatment group. The general linear model of Minitab version 17 (Minitab Inc., State College, PA, USA) was used to analyse the data. The hatching rate of blastocyst stage was significantly higher for TCM-199 (56.9 ± 27.2) compared with Ham’s F10 (50.0 ± 35.1%). The comparison of all embryo groupings, 1 (20.0 ± 40.5), 2 (28.8 ± 29.7), 3 (59.1 ± 38.8), and 4 (43.8 ± 32.4%) per 50-µL droplet showed significant differences, irrespective of IVC medium and breed. In TCM-199, groupings of 1 (20.0 ± 41.0), 2 (30.0 ± 29.9), 3 (63.3 ± 40.3), and 4 (42.5 ± 33.5%) had a significant difference on blastocyst hatching percent. In Ham’s F10, groupings of 1 (20.0 ± 41.0), 2 (27.5 ± 30.2), 3 (55.0 ± 37.9), and 4 (45.0 ± 32.0%) were significantly different on blastocyst hatching rate. However, an increase in hatching rate was observed for the interaction of media and embryo groupings and especially when embryos were increased per droplet in all breeds. In conclusion, the use of TCM-199 and grouping of 3 embryos per 50-µL droplet during culture had the highest hatching rate compared with the use of Ham’s F10.


2016 ◽  
Vol 28 (2) ◽  
pp. 149
Author(s):  
M. M. Seshoka ◽  
M. L. Mphaphathi ◽  
K. S. Mafolo ◽  
M. Nkadimeng ◽  
Z. C. Raphalalani ◽  
...  

Traditionally, commercial hen egg yolk has been used in extenders or freezing media because of its easy availability. However, the use of quail egg yolk has not been used for preserving Nguni bull semen. The aim of the study was to compare the suitability of different quail egg yolk concentrations (5, 10, 15, and 20%) for cryopreserving Nguni bull semen. Semen was collected from 14 stud Nguni bulls with the aid of electro ejaculator. Collected semen samples were kept in a thermos-flask containing warm water at a temperature of 37°C and transported to the laboratory for further analyses. The sperm motility traits were evaluated using computer-aided sperm analysis prior extension. Semen samples were then randomly allocated into 5, 10, 15, and 20% of quail egg yolk and 20% concentration of chicken egg yolk (control) in citrate extender. The extender was supplemented with 12% of glycerol (Seshoka et al. 2012) as a cryoprotectant, and semen samples were diluted (1 : 2) and equilibrated for a period of 4 h at 5°C. After equilibration, semen samples were loaded into 0.25-mL straws, placed into a controlled rate programmable freezer, and stored in a LN tank (–196°C) until thawing. Frozen semen straws were thawed in a water bath at 37°C for 1 min. Thawed semen was evaluated for sperm motility traits using a computer-aided sperm analysis system. Data were analysed with ANOVA. A significant difference was recorded between fresh total sperm motility rate (99%) and frozen-thawed semen samples with either 5% (87.3%) quail or 20% (87.6%) chicken egg yolk citrate extender compared with 10% (92.6%), 15% (91.2%), or 20% (89.9%) quail egg yolk citrate extender. Moreover, fresh semen also resulted in a significantly higher progressive sperm motility rate (39.3%) as compared with frozen-thawed with 5% (26.2%) or 20% quail (28.5%) or 20% chicken (22.7%) egg yolk citrate extender. The results also demonstrated that the use of 10, 15, and 20% quail egg yolk in citrate extender yielded comparable results on total sperm motility with fresh semen as compared with 5% quail and 20% chicken egg yolk. In conclusion, quail egg yolk extender provided sufficient cryo-effectiveness to cryopreservation of Nguni bull semen.


2021 ◽  
Vol 63 (1) ◽  
Author(s):  
Marta Neila-Montero ◽  
Marta F. Riesco ◽  
Mercedes Alvarez ◽  
Rafael Montes-Garrido ◽  
Juan Carlos Boixo ◽  
...  

Abstract Background Centrifugation is routinely employed in handling the ejaculates of some species, but it is not part of the commonly used protocols in ram. However, the development and implementation of new assisted reproductive technologies, alternative preservation models based on washing sperm from a cellular ageing-accelerating substance such as the seminal plasma, and basic studies in spermatology is associated with the use of centrifugation. This requires a specific evaluation of the centrifugation protocols considering the species-specific relationship with the potential damage produced by this procedure. No previous studies have determined the effect of different centrifugation forces on ram sperm. Therefore, we aimed to assess the performance of three centrifugal forces (600×g, 3000×g, and 6000×g for 10 min at room temperature) and their effects on ram sperm motility and functionality. Results Sperm motility and functionality parameters were assessed at 0 h and after 2 h of incubation at 37 °C. As expected, a higher cell packaging degree was obtained at high centrifugation forces (P ≤ 0.0001). Cell packaging was unstable at all centrifugal forces. Thus, there was a high cell resuspension rate after less than 2 min. Regarding sperm quality, there was a change in movement pattern of 3000×g and 6000×g centrifuged sperm after 2 h of incubation at 37 °C, characterized by an increase in rapid progressive motility, linearity, straightness, and beat frequency, and a decrease in medium progressive motility, curvilinear velocity, path velocity, and head lateral amplitude. Non-significant differences were obtained among the different treatments concerning the total viability. However, we observed a significant increase (P ≤ 0.05) in the percentage of viable apoptotic sperm in the samples centrifuged at 6000×g at 0 h. Conclusions Centrifugal forces equal to or greater than 3000×g induced some deleterious effects in ram sperm quality, and lower forces did not provide a successful cell packaging degree.


2020 ◽  
Vol 147 ◽  
pp. 01015
Author(s):  
Dimas Fendy Pradana ◽  
Ignatius Hardaningsih ◽  
Dini Wahyu Kartika Sari

The objectives of this study were to evaluate the sperm viability of Najawa carp (Cyprinus carpio L.) in cryopreservation pre-conditions at 4°C. The design used in this study was Complete Randomized Design with 4 treatments, BSS as a control, 10% DMSO, 0,2 M Sucrose, and 5% DMSO + 0,1 M Sucrose; each consist of three replications. The parameters observed were progressive motility of fresh sperm, diluted sperm before low temperature storage, and 2 hours; 3 hours; 4 hours; 5 hours; one day; one week; a month after 4°C storage. The data were analyzed by ANOVA. The data showed that there was no significant difference between treatment (P>0.05). The best viability was 40.56% of sperm motility which survive for one week, it was achieved by 5% DMSO + 0,1 M Sucrose.


Author(s):  
I. S. Esua ◽  
U. U. Uno ◽  
U. B. Ekaluo

Background and Aim: Tramadol is a potent analgesic effective in the treatment of mild to severe pains. However, the use of the drug can pose a threat to other organs and systems. Therefore, this study evaluated the effect of graded doses of tramadol on sperm profile of male albino rats. Materials and Methods: Eighteen male rats were divided into three groups (A, B and C) using completely randomized design (CRD) with six rats in each group. Rats in group A served as the control group and were given just food and water while groups B and C were given tramadol at 50 and 100 mg/kg body weight (BW) respectively, daily for the period of 65 days. The treatment was administered via oral gavage and at the end of the treatments, the rats were sacrificed. Immediately after sacrifice, a puncture was made in the epididymis with a sterile pin and examined for semen pH. The epididymes were processed for epididymal sperm motility, viability, count and sperm head abnormality. Results: There was no significant difference in the weight of testes and semen pH. Sperm viability, sperm motility, sperm count and weight of epididymes significantly reduced (p<0.05) in tramadol treated animals when compared with the control. Results also indicated statistically significant (p<0.05) increase in sperm head abnormalities in rats treated with tramadol when compared with the control. Conclusion: The results obtained from this study reveal that tramadol has negative effects on weight of epididymes, sperm count, sperm viability, sperm motility and sperm head abnormalities in male albino rat as mammalian models in a dose dependent manner.


2020 ◽  
Author(s):  
Evelynne Paris-Oller ◽  
Sergio Navarro-Serna ◽  
Cristina Soriano-Úbeda ◽  
Jordana Sena Lopes ◽  
Carmen Matas ◽  
...  

Abstract Background: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits.Results: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group.Conclusions: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.


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