Ovine artificial insemination (OAI) is not commonly performed because of specific problems related to semen application techniques, leading to highly variable results. The ideal methodology ...(frozen‐thawed semen/vaginal route) is unfeasible under field conditions due to the cervix morphology of the ewe, which prevents the process of intrauterine insemination necessary to obtain acceptable results. Currently, OAI commercial programmes use superficial cervical insemination, CAI (vaginal), with chilled semen (15°C) and intrauterine insemination, LAI (laparoscopic), with frozen‐thawed semen. The ability to improve upon these contrasting techniques may be derived from examining certain poorly studied factors such as insemination time, productive state of females and alternatives of seminal preservation, some of which we reviewed in this work. This interim solution will remain in use until AI by the vaginal route with frozen‐thawed semen is developed, but it poses new challenges in optimizing the freezing of the sperm and adapting the cervical (CAI) and/or transcervical intrauterine AI (TCAI). In this review, we address the current problems and evaluate their methodological (mechanical) and chemical (dilation) alternatives. Currently, TCAI is a methodologically complex technique with poor fertility results, so further studies are needed to improve the logistics of this procedure and the results of its application.
We have compared two methods for salvaging epididymal sperm from post-mortem samples from Iberian red deer. Of each pair of testicles (29 samples), one cauda epididymis was processed by means of cuts ...(sperm was immediately diluted with extender) and the other was detached from the corpus and flushed from the vas deferens with 1
mL of extender. Sperm was processed for cryopreservation, and analyzed just after recovery, pre-freezing and post-thawing. Total spermatozoa recovered, contamination (concentration of epididymal cells and red blood cells (RBCs)) and quality (motility by CASA, and acrosomal status, viability and mitochondrial status by flow cytometry) were used to compare both methods. The number of recovered spermatozoa was similar for both methods. Contamination was higher for the cuts method, but when considering the final dilution before freezing, only RBCs concentration was significantly higher. Motility was similar just after extraction, but higher for both pre-frozen and post-thawed flushed sperm. Pre-freezing acrosomal status (
P
<
0.05) and viability (
P
<
0.1) were better for flushing; however post-thawing results were similar for the two methods. A clustering analysis using CASA data showed that the subpopulation pattern of motile sperm was different depending on the method, being better for flushing. With regard to yield, lower contamination (especially RBCs) and, in general, better quality results, flushing seems to be a more recommendable method for post-mortem sperm recovery. The cuts method may be more practical on certain occasions, but care must be taken in order to achieve rapid extension of the sample and to avoid contamination in order to improve sample condition.
We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship ...between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menadione and DMNQ induced damage to sperm membranes after 1 h (P < 0.001; P < 0.05) and after 3 h of incubation (P < 0.001, P < 0.05). Pre-incubation with cysteine ameliorated both the menadione- and DMNQ-induced increase in 4-hydroxynonenal (P < 0.001). As cysteine is a precursor of GSH, we hypothesized that stallion spermatozoa are able to synthesize this tripeptide using exogenous cysteine. To test this hypothesis, we investigated the presence of two enzymes required to synthesize GSH (GSH and GCLC) and using western blotting and immunocytochemistry we detected both enzymes in stallion spermatozoa. The inhibition of GCLC reduced the recovery of GSH by addition of cysteine after depletion, suggesting that stallion spermatozoa may use exogenous cysteine to regulate GSH. Other findings supporting this hypothesis were changes in sperm functionality after BSO treatment and changes in GSH and GSSG validated using HPLC-MS, showing that BSO prevented the increase in GSH in the presence of cysteine, although important stallion to stallion variability occurred and suggested differences in expression of glutamate cysteine ligase. Mean concentration of GSH in stallion spermatozoa was 8.2 ± 2.1 µM/109 spermatozoa, well above the nanomolar ranges per billion spermatozoa reported for other mammals. Summary Sentence Thiols regulate stallion sperm functionality.
Global aquaculture growth will most probably face specific conditions derived from climate change. In fact, the most severe impacts of these changes will be suffered by aquatic populations in ...restrictive circumstances, such as current aquaculture locations, which represent a perfect model to study global warming effects. Although the impact of temperature on fish reproduction has been characterized in many aspects, this study was focused on recreating more realistic models of global warming, particularly considering heatwave phenomena, in order to decipher its effects on male gametes (spermatozoa). For this purpose, thermal stress via a heatwave simulation (mimicking a natural occurring heatwave, from 24 to 30 °C) was induced in adult tench (
) males and compared with a control group (55.02 ± 16.44 g of average body wet weight). The impact of the thermal stress induced by this climate change event was assessed using cellular and molecular approaches. After the heatwave recreation, a multiparametric analysis of sperm quality, including some traditional parameters (such as sperm motility) and new ones (focus on redox balance and sperm quality biomarkers), was performed. Although sperm concentration and the volume produced were not affected, the results showed a significant deleterious effect on motility parameters (e.g., reduced progressive motility and total motility during the first minute post-activation). Furthermore, the sperm produced under the thermal stress induced by this heatwave simulation exhibited an increased ROS content in spermatic cells, confirming the negative effect that this thermal stress model (heatwave recreation) might have had on sperm quality. More importantly, the expression of some known sperm quality and fertilization markers was decreased in males exposed to thermal stress. This present study not only unveils the potential effects of climate change in contemporary and future fish farming populations (and their underlying mechanisms) but also provides insights on how to mitigate and/or avoid thermal stress due to heatwave events.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
To improve artificial insemination protocols in ovine species it is crucial to optimize sperm quality evaluation after preservation technologies. Emerging technologies based on novel biomolecules and ...related to redox balance and proteins involved in sperm motility such as ProAKAP4 could be successfully applied in ram sperm evaluation. In this work, a multiparametric analysis of fresh, cooled, and cryopreserved ram sperm was performed at different complexity levels. Samples were evaluated in terms of motility (total motility, progressive motility, and curvilinear velocity), viability, apoptosis, content of reactive oxygen species, oxidation‒reduction potential, and ProAKAP4 expression and concentration. As expected, cryopreserved samples showed a significant decrease of sperm quality (
< 0.05), evidencing different freezability classes among samples that were detected by ProAKAP4 analyses. However, in cooled sperm no differences were found concerning motility, viability, apoptosis, ROS content, and redox balance compared to fresh sperm that could explain the reported decrease in fertility rates. However, although the proportion of sperm ProAKAP4 positive-cells remained unaltered in cooled sperm compared to fresh control, the concentration of this protein significantly decreased (
< 0.05) in cooled samples. This altered protein level could contribute to the decrease in fertility rates of cooled samples detected by some authors. More importantly, ProAKAP4 can be established as a promising diagnostic parameter of sperm quality allowing us to optimize sperm conservation protocols and finally improve artificial insemination in ovine species.
Computer-assisted sperm analysis (CASA) allows assessing the motility of individual spermatozoa, generating huge datasets. These datasets can be analyzed using data mining techniques such as cluster ...analysis, to group the spermatozoa in subpopulations with biological meaning. This review considers the use of statistical techniques for clustering CASA data, their challenges and possibilities. There are many clustering approaches potentially useful for grouping sperm motility data, but some options may be more appropriate than others. Future development should focus not only in improvements of subpopulation analysis, but also in finding consistent biological meanings for these subpopulations.
The optimization of sperm cryopreservation protocols in ram is a feasible tool to reinforce artificial insemination technologies considering the desirable application of sperm by vaginal/cervical or ...transcervical deposition. Cryopreservation provokes different types of damage on spermatozoa and many of these detrimental effects are triggered by redox deregulation. For this reason, the antioxidant supplementation in sperm cryopreservation protocols to decrease reactive oxygen species (ROS) levels and to equilibrate redox status has been widely employed in different species. Despite this, more fertility trials are necessary to provide the definitive tool to ensure the antioxidant effectiveness on sperm quality. For this reason, in this work, we performed a multiparametric analysis of some previously tested antioxidants (crocin, GSH and Trolox) on ram sperm cryopreservation from field trials to sperm quality analyses focused on new strategies to measure redox balance. Attending to fertility trial, Trolox supplementation registered an improvement concerning to fertility (when we considered high fertility males) and multiple lambing frequency and other complementary and descriptive data related to lambing performance such as prolificacy and fecundity. This positive effect was more evident in multiple lambing frequency when we considered low fertility males than in global male analysis. In vitro analyses of sperm quality confirmed in vivo trials registering a positive effect on sperm viability and redox balance. In this study, we provided the definitive evidence that the role of trolox on redox balance maintenance has a direct effect on fertility parameters, such as prolificacy. The effectiveness of antioxidant treatments was tested, for the first time in ovine species, using an integrative and multiparametric approach combining in vivo and in vitro analyses and novel approaches, such as RedoxSYS. These types of strategies should be applied to improve sperm conservation methods and optimize AI technologies upgrading the correlation between in vitro and in vivo analyses.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Over the years, testicular volume has been used to evaluate the reproductive capacity of rams and the effects of different factors related to reproductive performance. The aim of this study was to ...determine the most suitable tool and formula to calculate testicular volume under field conditions to guarantee a more accurate determination of sperm production. First, testicles from 25 rams (n = 50) were measured in vivo and postmortem using calipers and ultrasonography during the breeding season (BS). The accurate testicular volume (ATV) was calculated through water displacement. In addition, the sexual status of donor rams was evaluated during a period of four years in a reproduction center, and the three most crucial groups in terms of genetic value and seminal collections were studied in the second part of this experiment: ER-NBS (Elite rams during the non-breeding season), ER-BS-S (Elite rams with a standard frequency of seminal collection), and ER-BS-O (Elite rams with a high frequency of seminal collection). The total testicular volume (TTV), testosterone (T), and total spermatozoa obtained from two consecutive ejaculates in the same day (SPERM) were measured, and the relationship between SPERM and TTV and T was analyzed to predict SPERM. Although all published formulas revealed statistically significant differences (p ≤ 0.05) from the ATV, our proposed formula (ItraULE) (Testicular volume = L × W × D × 0.61) did not show significant differences. In the second part of the study, in the ER as a model donor ram for its high genetic value and high demand from farmers, TTV and T showed strong positive correlations with SPERM (r = 0.587, p = 0.007 NBS; r = 0.684, p = 0.001 BS-S; r = 0.773, p < 0.0001 BS-O). Moreover, formulas were established to predict SPERM in these practical scenarios. In conclusion, the use of ultrasonography and a new formula adapted to rams could improve the prediction of SPERM considering crucial factors such as season and semen collection frequency.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The improvement of frozen-thawed sperm quality has been mostly approached from the view of cryopreservation protocol optimization in terms of cryoprotectant solutions, freezing-thawing rates and ...antioxidant supplementation, while the impact of sperm collection frequency remains unknown in rams. In this work, a multiparametric study was carried out in cooled and frozen-thawed semen to evaluate sperm quality after different semen collection frequencies during a month: zero sperm collection (0 CW), four sperm collections per week (4 CW), and ten sperm collections per week (10 CW). Traditional analyses have been applied, in combination with novel technologies related to redox balance. Frozen-thawed semen quality showed a significant decrease (p < 0.05) in 0 CW and 10 CW in comparison to 4 CW, concerning motility and kinetics parameters. However, apoptosis showed a significant increase (p < 0.05) in 10 CW in comparison to 0 CW and 4 CW. The employment methods related to redox balance provided us with the definitive probe to ensure the influence of collection frequency on balance redox after thawing. Specifically, glutathione peroxidase (GPX) and superoxide dismutase (SOD) activity showed a significant decrease (p < 0.05) in 10 CW compared to 0 CW and 4 CW. The characterization of alternative strategies to sperm cryopreservation based on consideration of male sexual regimes, could improve the quality of frozen-thawed sperm.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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