Seminal plasma removal for medium-term preservation of ram sperm at 5 °C.

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.

The Adaptation Time to the Extender as a Crucial Step for an Accurate Evaluation of Ram Sperm Quality during the Liquid Storage.

Accurate assessment of ram sperm quality is crucial to optimizing assisted reproductive technologies in sheep. However, semen preservation can induce sperm due to osmotic, biochemical, and thermal stress. Stabilizing sperm with a suitable cooling rate and adaptation period to the extender could mitigate these effects for a more reliable evaluation.

The characterization of CellROX™ probes could be a crucial factor in ram sperm quality assessment.

Several authors have demonstrated that low levels of reactive oxygen species (ROS) are necessary for the physiological functions of sperm, such as capacitation, hyperactivation, acrosomal reaction and fertilization. However, high levels of ROS are associated with oxidative stress and detrimental effects on fertility. Consequently, deep characterization of ROS presence using different fluorescent probes could be crucial.

Does Size Matter? Testicular Volume and Its Predictive Ability of Sperm Production in Rams.

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 ( = 50) were measured in vivo and postmortem using calipers and ultrasonography during the breeding season (BS).

Ovine fertility by artificial insemination in the breeding season could be affected by intraseasonal variations in ram sperm proteomic profile.

It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21 to July 20) and at the end (November 20 to December 21) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6-19 rams at two very distant points in the mating season (July as Early Breeding Season -EBS- and November as Late Breeding Season -LBS-).

Application of ultrasound technique to evaluate the testicular function and its correlation to the sperm quality after different collection frequency in rams.

The frequency of semen collection is a crucial factor to consider in the rams performance inside breeding centers workout. To evaluate this factor, ram Breeding Soundness Evaluation could include sperm quality evaluation and new predictive and non-invasive tools such as ultrasound technique. In this work, an advanced ultrasonography technology, analyzing the testicular volume, echotexture, and vascular function, was used in three different frequencies of semen collection (abstinence frequency, AF; standard frequency, SF; and intensive frequency, IF).

An optimized centrifugation protocol for ram sperm ensuring high sample yield, quality and fertility.

The optimization and implementation of artificial insemination (AI) in sheep is necessary to increase the livestock productivity through enhanced control of reproductive function. Sperm centrifugation is a common procedure in the ejaculate handling in AI and other assisted reproductive technologies (ART), as part of new methods of sperm analysis, selection or preservation. However, our research group previously established that this simple procedure might cause a large sperm loss and induce deleterious effects on the sperm function of the ovine species when high centrifugation forces are employed.

Frequency of Semen Collection Affects Ram Sperm Cryoresistance.

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.

Centrifugal force assessment in ram sperm: identifying species-specific impact.

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.

Comparing the Effect of Different Antibiotics in Frozen-Thawed Ram Sperm: Is It Possible to Avoid Their Addition?

It is crucial to perform a deep study about the most extensively used antibiotics in sperm extenders. Most of the protocols and concentrations used in ram are direct extrapolations from other species. It is important to establish species-specific antibiotic treatments to optimize their use and if it is possible to reduce the quantity.

Multiparametric Study of Antioxidant Effect on Ram Sperm Cryopreservation-From Field Trials to Research Bench.

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.

ProAKAP4 as Novel Molecular Marker of Sperm Quality in Ram: An Integrative Study in Fresh, Cooled and Cryopreserved Sperm.

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.

Current challenges in sheep artificial insemination: A particular insight.

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.

Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: a possible role for GSH regulation in spermatozoa†.

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).

Extender osmolality, glycerol and egg yolk on the cryopreservation of epididymal spermatozoa for gamete banking of the Cantabric Chamois (Rupicapra pyrenaica parva).

Germplasm banking is a key technology enabling the ex-situ conservation of wild species. However, cryopreservation protocols must be tested to assure the applicability of the banked material. The objective of this study was defining a range of parameters for the composition of a semen extender for Cantabrian chamois epididymal spermatozoa (post-mortem collection).

Progesterone stimulates the long-distance migration of capacitated ram spermatozoa through viscous media under geotactic condition.

Forward progressive motility of spermatozoa is an essential prerequisite for reproductive success, and sperm navigation is assisted by guidance mechanisms that may depend on micro-environmental factors. In the present study, we performed an integrated analysis of long-distance ram sperm migration in vitro that combined two environmental factors (10 μM progesterone and a geotactic effect) and the physiological status of the cells (capacitation treatment). A penetration assay was used in which spermatozoa had to travel 20 mm in a viscous medium (two media of differing viscosity: acrylamide and hyaluronic acid) through a tube device.

Influence of insemination time on the fertility of sex sorted frozen-thawed Y-sperm in red deer.

The aim of this study was to assess the effect of insemination timing on pregnancy rates in red deer (Cervus elaphus) when using sex-sorted sperm samples. Semen was collected by electroejaculation from 8 mature stags and processed to obtain: Conventional samples, following standard freezing procedures for commercial purposes; Control sorted samples, diluted and handled as per sorted samples but without being submitted to the sorter passage; and Y Sex Sorted (YSS) samples. Hinds were synchronized via intravaginal CIDR (Controlled Internal Drug Release) placement and given eCG (Folligon PMSG Serum Gonadotrophin) on day 12, upon CIDR removal.

Pulse Doppler ultrasound as a tool for the diagnosis of chronic testicular dysfunction in stallions.

Testicular function is particularly susceptible to vascular insult, resulting in a negative impact on sperm production and quality of the ejaculate. A prompt diagnosis of testicular dysfunction enables implementation of appropriate treatment, hence improving fertility forecasts for stallions. The present research aims to: (1) assess if Doppler ultrasonography is a good tool to diagnose stallions with testicular dysfunction; (2) to study the relationship between Doppler parameters of the testicular artery and those of sperm quality assessed by flow cytometry and (3) to establish cut off values to differentiate fertile stallions from those with pathologies causing testicular dysfunction.

Optimization of protocols for Iberian red deer (Cervus elaphus hispanicus) sperm handling before sex sorting by flow cytometry.

Currently, sperm reproductive biotechnologies such as sex sorting and cryopreservation are undoubtedly valuable tools for improving the economic and biological efficiency of red deer production systems. In this context, and because of the particular characteristics of this species (extensive exploitation typically far from laboratory facilities), a key goal is to optimize the design of an adequate handling protocol of sperm samples before samples are subjected to sex sorting and cryopreservation procedures to obtain better outputs from the application of these technologies. The main aim of this paper was to design an adequate protocol for Iberian red deer sperm handling before sex sorting by flow cytometry to obtain optimal yields when sex sorting is used in this species.

Caspase 3 Activity and Lipoperoxidative Status in Raw Semen Predict the Outcome of Cryopreservation of Stallion Spermatozoa.

Stallion-to-stallion variability in the quality of cryopreserved ejaculates postthaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry.

Head morphology of ram spermatozoa is associated with their ability to migrate in vitro and correlates with fertility.

Fertility is a highly complex biological function that depends on several properties of spermatozoa that are necessary for them to overcome various barriers in the female reproductive tract to reach the fertilisation site. This ability has been evaluated in vitro using cervical mucus migration tests. Head morphology has been widely studied, and various studies have reported correlations between head morphology and motility, fertility and DNA fragmentation.

Free-radical production after post-thaw incubation of ram spermatozoa is related to decreased in vivo fertility.

The aim of the present study was to evaluate the effect of sperm reactive oxygen species (ROS) production and DNA changes on male fertility. For that purpose, six rams with significantly different pregnancy rates were used; these were classified as having high fertility, i.e.

Ram spermatozoa migrating through artificial mucus in vitro have reduced mitochondrial membrane potential but retain their viability.

Sperm motility in vitro is one of the most common predictors of fertility in male screening. We propose that a mucus-penetration assay can isolate a cellular subpopulation critical to reproductive success. To this end, a device was designed with three modules (sample, test and collection) and its conditions of use evaluated (length of mucus, incubation time, mucus medium, sperm concentration and position in relation to the horizontal).

Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm(®) gradient between freeze-thaw cycles.

The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze-thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed.