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7 Pág. Departamento de Reproducción Animal​, In both captive wildlife and production animals is important to develop strategies for population control. Immunization against GnRH is an easy and inexpensive immunocastration method that reduces the concentration of testosterone and decreases sperm quality. However, its effectiveness depends on the species and repetition of the treatment. This study aimed to compare the effectiveness of a single treatment (initial immunization plus a booster with Improvac) vs repeated treatment (six doses of Improvac) to inhibit testicular function and maintain the contraceptive status during long periods in bucks. Three Dwarf bucks (Capra hircus) received two doses of Improvac, the first on Week 0, and the booster 4 weeks later (single immunization, group SI) while three Dwarf bucks received one dose of Improvac every 6 months during 3 consecutive years (repeated immunization, group RI). The other three Dwarf bucks remained untreated (control bucks, group CON). Bucks from RI had a greater decrease in scrotal circumference, testosterone concentration, male odor intensity, and sperm quality than SI bucks. However, there were no differences between SI and CON bucks in any of the variables studied. Overall, repeated treatment of Improvac decreased the testicular function of Dwarf bucks, although did not produce complete infertility. However, the repetition of the treatment produced more intensive negative effects, indicating that the strength of the effects of Improvac is rapidly lost in bucks., This study was funded by the Fundación Parques Reunidos—INIA agreement CC19‐096. The authors thank Matías Villagrán forproviding the photograph of the graphical abstract and Analía deLeón for reviewing the English of the manuscript

7 Pág., Transrectal ultrasonic-guided massage of the accessory sex glands (TUMASG) is a technique that allows collecting semen requiring few electrical stimuli or even no pulse. A long-acting analogue of oxytocin (carbetocin, 0.1 mg) was i.v. administered before TUMASG in 10 conscious bucks (Experiment 1) and 10 anaesthetized Iberian ibexes (Experiment 2) to shorten the time of semen collection, decrease the number of electrical stimuli and/or improve the semen quality. The ejaculated volume, concentration, quality parameters and kinetics variables of the sperm were determined in fresh semen. The time length of the procedures and the number of electric pulses applied were recorded. Furthermore, stress response indicators (number of vocalizations in Experiment 1; heart and respiratory rates, rectal temperature, cortisol levels, totals proteins and neutrophil-to-lymphocyte ratio in Experiment 2) were documented. In bucks, the administration of carbetocin tended to shorten the time needed for semen collection but no-showed differences in the fresh seminal quality. In the Iberian ibexes, there were no significant differences between groups in the time length of procedures or in the number of animals that ejaculated. Carbetocin administration only reduced the respiratory rate, did it modify fresh semen characteristics in ibexes. In conclusion, the administration of carbetocin did not appear as a useful tool to improve welfare during semen collection with TUMASG or semen quality in conscious bucks and anaesthetized ibexes, having only slight advantages related to the procedure.

Aim: This work examined the influence of induced changes in prolactin (PRL) secretion on sperm cryoresistance of ibex and the mouflon. Materials and Methods: PRL secretion was modified in a first experiment by the use of bromocriptine (BCR, dopamine agonist) during the non-breeding season, and in a second experiment by the use of sulpiride (SLP, dopamine D2-receptor antagonist) during the rutting season. Slow and ultra-rapid freezing protocols were used to cryopreserve sperm samples. Results: BCR decreased blood plasma PRL concentrations, whereas SLP increased them. Cryoresistance ratios (CRs) for curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP) in BCR-treated mouflons were lower than in controls using slow-freezing (p

Neurons, from the time of their birth until their death, are always accompanied by neuroglial cells, maintaining very close morphological and functional relationships among themselves. Classically three main neuroglial families have been considered within the central nervous system (CNS): astroglia, oligodendroglia, and microglia. However, using morphological and immuno-cytochemical criteria, several different types/subtypes of neuroglial cells have been identified, each exerting specific physiological and pathological effects on the neurons. The neuron–neuroglial relationships are quite complex (glio-glial, glio-neuronal, and neuro-glial cell signaling, including glio-transmission), and all these routes of cell communication are essential for supporting brain function. Subtle morpho-functional changes resulting from glial cell plasticity occur in parallel to the plastic neuronal changes that serve to optimize the functionality of neurons and neuronal circuits. Moreover, all the different types/subtypes of neuroglia may adopt a reactive status, referred to as “gliosis”, in which novel actions are performed. These latter morpho-functional changes are difficult to understand as they involve both neuroprotection/neuro-repair and neurodegeneration. Diverse and profound regional and local neuroglial changes occur in all the involutive processes [physiological and pathological aging or neurodegenerative disorders such as Alzheimer´s disease (AD)], although the exact implications of these spatiotemporal modifications for the different neuroglial types are not sufficiently well understood. Owing to all these features, neuroglial cells become elements of paramount importance when attempting to elucidate any physiological or pathological processes occurring in the CNS. Furthermore, in recent years, potential therapeutic interventions capable of positively modifying the responses of neuroglial cells have been proposed to prevent and/or treat degenerative CNS conditions. In the present review, the different neuroglial cells and their responses are discussed in order to understand their possible role in the pathogenesis, diagnosis and treatment (preventive or palliative) of involutive CNS processes. In addition, the existence of differentiated and/or concurrent pathogenic and neuro-protective/neuro-restorative astroglial and microglial responses is addressed.

This work identified the presence of AQPs in frozen-thawed sperm of wild ruminants and assessed the influence of the interaction between photoperiod and thyroxine on AQP expression, and on testosterone secretion. Thyroxine and melatonin were administered to ibexes. In a second experiment, performed in mouflons, circulating thyroxine was reduced via treatment with propylthiouracil (PTU), and an artificial long day (LD) photoperiod established. In the ibexes, the melatonin treatment increased the blood plasma testosterone concentration, reduced the cryoresistance ratio (CR) for sperm viability and the presence of an intact acrosome, and increased the percentage of sperm with AQP7 in the acrosome and of AQP3 and AQP10 in the midpiece. In the mouflons, neither the PTU treatment, the LD, nor the combination of both affected the CR of any sperm variable. The percentage of sperm with AQP3 increased in the post-acrosome region but decreased in the tail in the LD+PTU group. The percentage of sperm with AQP10 in the principal piece and endpiece was lower in the PTU+LD group than in the control and LD groups. The influence of photoperiod/melatonin on AQP expression might be indirectly exerted through changes in the testosterone concentration, and thus ultimately affect sperm cryoresistance. Funding Agencies|MCINN/AEI/FEDER; EUEuropean Commission [AGL2017-85753-R, PID2020-113288RB-I00]; Spanish Ministry of Science, Innovation and UniversitiesSpanish Government [PRX19/00149]; Swedish Research Council FORMAS, Stockholm, Sweden [2017-00946, 2019-00288]

7 Pág. Departamento de Reproducción Animal., The expression of aquaglyceroporin 3 (AQP-3) has been demonstrated in the spermatozoa of several mammalian species and its role has been associated with cryotolerance. Post-thaw sperm quality from individual dromedary males with different response to freezing-thawing process was evaluated through sperm head morphometry. In order to understand the cellular mechanisms affected by cryoinjury we have explored the presence and distribution of sperm AQP-3 using western blotting and immunocytochemistry. WB showed different intensity of the specific signal bands at 28 kDa. Immunofluorescence assessments allowed us to identify five different and clear AQP-3 distribution patterns of labelling in the sperm plasma membrane; acrosome, post-acrosome, mid-piece, and principal and final tail. Although expression of AQP-3 varied among male ejaculates, the individual sperm response to freeze-thawing was not associated with AQP-3 expression. Thus, AQP3 expressions do not seem like a reliable predictor of sperm response to freeze-thawing process in this species. This work is the first to describe the morphometric characteristics of the heads of dromedary spermatozoa. No correlation was found between sperm head dimensions and sperm quality variables after freeze-thawing suggesting that dromedary camel sperm head morphometry is also not a reliable predictor of cryosurvival., This work was supported by Zoitechlab S.L. (Arquimea Group) - INIA contract CON18-141, MCINN/AEI/FEDER and EU grant AGL2017-85753-R and PID2020-113288RB-100/AEI/10.13039/501100011033 grant.

Since the original description of Alzheimer´s disease (AD), research into this condition has mainly focused on assessing the alterations to neurons associated with dementia, and those to the circuits in which they are involved. In most of the studies on human brains and in many models of AD, the glial cells accompanying these neurons undergo concomitant alterations that aggravate the course of neurodegeneration. As a result, these changes to neuroglial cells are now included in all the "pathogenic cascades" described in AD. Accordingly, astrogliosis and microgliosis, the main components of neuroinflammation, have been integrated into all the pathogenic theories of this disease, as discussed in this part of the two-part monograph that follows an accompanying article on gliopathogenesis and glioprotection. This initial reflection verified the implication of alterations to the neuroglia in AD, suggesting that these cells may also represent therapeutic targets to prevent neurodegeneration. In this second part of the monograph, we will analyze the possibilities of acting on glial cells to prevent or treat the neurodegeneration that is the hallmark of AD and other pathologies. Evidence of the potential of different pharmacological, non-pharmacological, cell and gene therapies (widely treated) to prevent or treat this disease is now forthcoming, in most cases as adjuncts to other therapies. A comprehensive AD multimodal therapy is proposed in which neuronal and neuroglial pharmacological treatments are jointly considered, as well as the use of new cell and gene therapies and non-pharmacological therapies that tend to slow down the progress of dementia.

9 Pàg., 1. Birchen and Blue Leonesa are two endangered chicken breeds mainly raised in Curueño Valley in North Spain. The establishment of a germplasm bank to guarantee the preservation of these breeds is needed. However, cockerels from different breeder flocks can show variance in semen cryoresistance.2. The following work focused on the sperm characterisation and cryopreservation of Birchen and Blue Leonesa cockerels from four different breeders. A total of 30 semen pools were analysed. Besides conventional sperm analysis, including motility by computer-aided sperm analysis (CASA) and DNA fragmentation by TUNEL, the present study tested a double staining method (MitoTrackerTM Green FM/propidium iodide). This gave simultaneous assessment of plasma and acrosomal and mitochondrial membranes, which were previously validated by SYBR-14/PI, CASA, aniline blue and TUNEL.3. No significant differences were found among fresh semen variables between breeds and breeders. For post-thawed variables, significant differences (P, r, This research is part of a project that received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement Nº 677353 IMAGE. Part of this research was funded by the project [RTA2017-00034-00-00].

9 Pág., The domestication process has resulted in profound changes in the reproductive physiology of the animals that might have affected the sperm characteristics and thus their sensitivity to handling and cryopreservation procedures. This work assesses the response of the sperm of domestic and wild ungulates to a cooling storage at 15°C for 20 h followed by incubation at 38.5°C, 5% CO2, for 2 h. In addition, this paper examines the most representative sperm traits to assess their responsiveness to these stress conditions. Sperm samples were collected from domestic and their wild ancestor species: ram, mouflon, buck, Iberian ibex, domestic boar, and wild boar. Sperm motility, viability, mitochondrial membrane status, DNA fragmentation, and reactive oxygen species production were evaluated at the beginning of the experiment, after 20 h of refrigeration at 15°C, and, finally, at 2 h of incubation at 38.5°C. Sperm from all domestic species (ram, buck, and domestic boar) suffered more stress than their wild relatives (mouflon, Iberian Ibex, and wild boar). In pigs, the percentage of intact mitochondria was lower in the domestic species compared to wild boar. In sheep, we found a higher reactive oxygen species production in rams, while in goats, the curvilinear velocity was lower in the domestic species. The PCA (principal components analysis) showed that the motility and their kinetic variables were the most represented variables in the principal components of all species, indicating that they are essential biomarkers for evaluating the stress response. Sperm viability was highlighted as a representative variable for evaluating the stress response in domestic boar, mouflon, ram, and ibex., This research was funded by Zoitechlab S.L.-INIA contract CON18-141.

In this review, the most important neuropathological changes found in the cerebella of sheep affected by classical natural scrapie are discussed. This disease is the oldest known of a group of unconventional “infections” caused by toxic prions of different origins. Scrapie is currently considered a “transmissible spongiform encephalopathy” (due to its neuropathological characteristics and its transmission), which is the paradigm of prion pathologies as well as many encephalopathies (prion-like) that present aberrant deposits of insoluble protein with neurotoxic effects due to errors in their catabolization (“misfolding protein diseases”). The study of this disease is, therefore, of great relevance. Our work data from the authors’ previous publications as well as other research in the field. The four most important types of neuropathological changes are neuron abnormalities and loss, neurogliosis, tissue vacuolization (spongiosis) and pathological or abnormal prion protein (PrP) deposits/deposition. These findings were analyzed and compared to other neuropathologies. Various aspects related to the presentation and progression of the disease, the involution of different neuronal types, the neuroglial responses and the appearance of abnormal PrP deposits are discussed. The most important points of controversy in scrapie neuropathology are presented., No special funding is obtained for this review. Different support for research projets has been obtained during years (MM and AT).

10 Pág. Centro de Investigación en Sanidad Animal (CISA) / Departamento de Reproducción animal, The roe deer is a monoestrous species with a very short rutting season. The present work reports the most suitable period for collecting epididymal sperm and describes the effect of two cooling rates on the post-thaw quality of sperm. Testes were collected 24–48 h after death. Samples of sperm flushed from the epididymis were subjected to either (1) dilution in a Tris-citric acid-glucose-egg yolk-based medium with glycerol, and slow freezing in straws, or (2) dilution in the same extender but replacing the glycerol with 100 mM of sucrose, and ultra-rapid freezing in pellets. Sperm motility, acrosome and membrane integrity, morphometry and morphological abnormalities were analysed before and after cryopreservation. Spermatogenic activity was investigated via histological examination of testis sections. Several testes collected between April, May and September showed no spermatogenic activity. All those collected in June–August showed spermatogenic activity. No significant difference was detected in the cryoresistance ratios associated with the conventional slow freezing, between sperm collected during the pre-rutting (April–May) and rutting (June–August) periods. No significant differences were seen between the slow-frozen-thawed and the ultra-rapid-frozen-thawed sperm in terms of percentage of viable sperm or the percentage of sperm with morphological abnormalities. Slow freezing returned significantly better (P, This research was funded by MINECO/AEI/FEDER and EU grant AGL2017-85753-R. P. Bóveda was the recipient of a grant for pre-doctoral researchers from MINECO (AEI/FSE, UE). Octavio Mejía was the recipient of a research fellowship from the PASPA-DGAPA-UNAM (México). V.N. Flores-Gil was funded by FONDECYT-CONCYTEC (grant contract number 000245-2015-FONDECYT).

Aim of study: To examine the influence of administering cyproterone acetate (CPA), at the beginning of the mating season, on the testosterone concentration and morphometric and functional characteristics of ram and buck semen.Area of study: Madrid, SpainMaterial and methods: Five rams and five bucks were intramuscularly administered 200 mg of CPA in 2 mL of olive oil twice per week - from July 1st to 31st in the rams, and from August 1st to 31st in the bucks. Five control animals of each species were administered 2 mL of olive oil. Blood samples and ejaculates analysed from the start of treatment until eight weeks after the last day of treatment.Main results: GLM-ANOVA showed the interaction species × CPA treatment to have effect (p

12 Pág. Departamento de Reproducción animal, Recent studies have noted that the circulating testosterone concentration may affect the ability of spermatozoa to survive cryopreservation. However, few attempts to confirm such a relationship have been made. Wild ruminant species have very marked seasonal changes in their reproductive function and strong annual changes in their plasma testosterone concentration., This study was funded by MICINN/AEI/FEDER and EU grant AGL2017-85753R. Paula Bóveda was the recipient of a grant for pre-doctoral researchers from MINECO (AEI/FSE, UE). Octavio Mejía was supported by a research fellowship from the PASPA-DGAPA-UNAM (Mexico).

The domestication process has resulted in profound changes in the reproductive physiology of the animals that might have affected the sperm characteristics and thus their sensitivity to handling and cryopreservation procedures. This work assesses the response of the sperm of domestic and wild ungulates to a cooling storage at 15°C for 20 h followed by incubation at 38.5°C, 5% CO2, for 2 h. In addition, this paper examines the most representative sperm traits to assess their responsiveness to these stress conditions. Sperm samples were collected from domestic and their wild ancestor species: ram, mouflon, buck, Iberian ibex, domestic boar, and wild boar. Sperm motility, viability, mitochondrial membrane status, DNA fragmentation, and reactive oxygen species production were evaluated at the beginning of the experiment, after 20 h of refrigeration at 15°C, and, finally, at 2 h of incubation at 38.5°C. Sperm from all domestic species (ram, buck, and domestic boar) suffered more stress than their wild relatives (mouflon, Iberian Ibex, and wild boar). In pigs, the percentage of intact mitochondria was lower in the domestic species compared to wild boar. In sheep, we found a higher reactive oxygen species production in rams, while in goats, the curvilinear velocity was lower in the domestic species. The PCA (principal components analysis) showed that the motility and their kinetic variables were the most represented variables in the principal components of all species, indicating that they are essential biomarkers for evaluating the stress response. Sperm viability was highlighted as a representative variable for evaluating the stress response in domestic boar, mouflon, ram, and ibex.

The American flamingo is a useful model for the development of successful semen cryopreservation procedures to be applied to threatened related species from the family Phoenicopteridae, and to permit genetic material banking. Current study sought to develop effective sperm cryopreservation protocols through examining the influences of two permeating cryoprotectants and the seminal plasma removal. During two consecutive years (April), semen samples were collected and frozen from American flamingos. In the first year, the effect of two permeating cryoprotectants, DMA (dimethylacetamide) (6%) or Me2SO (dimethylsulphoxide) (8%), on frozen&ndash, thawed sperm variables were compared in 21 males. No differences were seen between DMA and Me2SO for sperm motility, sperm viability, and DNA fragmentation after thawing. In the second year, the role of seminal plasma on sperm cryoresistance was investigated in 31 flamingos. Sperm samples were cryopreserved with and without seminal plasma, using Me2SO (8%) as a cryoprotectant. The results showed that samples with seminal plasma had higher values than samples without seminal plasma for the following sperm variables: Straight line velocity (22.40 &micro, m/s vs. 16.64 &micro, m/s), wobble (75.83% vs. 69.40%), (p &lt, 0.05), linearity (62.73% vs. 52.01%) and straightness (82.38% vs. 73.79%) (p &lt, 0.01), but acrosome integrity was lower (55.56% vs. 66.88%) (p &lt, 0.05). The cryoresistance ratio (CR) was greater in samples frozen with seminal plasma than without seminal plasma for CR-progressive motility (138.72 vs. 54.59), CR-curvilinear velocity (105.98 vs. 89.32), CR-straight line velocity (152.77 vs. 112.58), CR-average path velocity (122.48 vs. 98.12), CR-wobble (111.75 vs. 102.04) (p &lt, 0.05), CR-linearity (139.41 vs. 113.18), and CR-straightness (124.02 vs. 109.97) (p &lt, 0.01). This research demonstrated that there were not differences between Me2SO and DMA to successful freezing sperm of flamingos, seminal plasma removal did not provide a benefit for sperm cryopreservation.

Departamento de Reproducción Animal​ (INIA), The domestic ferret (Mustela putorius furo) provides a good model for developing new reproductive technologies for use with threatened related species. Such technologies could also be used in the reproductive management of this pet species. The present work reports an improved freezing protocol for ferret sperm. Semen was collected by electroejaculation plus rectal massage (in an attempt to reduce the electrical stimulation necessary) from five adult male ferrets, and then subjected to one of two freezing protocols: (a) from 5 to -35°C at 40°C/min, then from -35 to -65°C at 17°C/min, and finally from -65 to -85°C at 3°C/min-a decelerating freezing rate; and (b) from 5 to - 10°C at 5°C/min, and then from -10 to -130°C at 60°C/min-an accelerating freezing rate. After thawing, the viability and acrosomal integrity of the sperm frozen via the two-step accelerating method were better than those frozen via the three-step decelerating method (43.3 ± 3.5% and 71.2 ± 3.4% compared with 29.7 ± 3.7% and 58.8 ± 3.4% respectively; p 90%) remained static with both freezing methods. In conclusion, although the method with accelerating freezing rate was associated with better post-thaw sperm viability and acrosome integrity values, neither of the two freezing methods tested provided adequate motility results after thawing. Combining rectal massage with electrical stimuli seemed to reduce the number of the latter required for successful sperm collection., This work was funded by the Fundación Biodiversidad - Ministerio para la Transición Ecológica y el Reto Demográfico, Spain (grant DOC2017458). Special thanks are owed to the INIA animal husbandry team for help in the handling and care of the experimental animals., 8 Pág

International audience; Recent reports showed a positive correlation between frozen-thawed rooster sperm DNA integrity and the concentrations of valine in seminal plasma. The present study evaluated the effect of supplementing valine to semen extender for freezing sperm of 2 endangered local Spanish chicken breeds with different sperm cryoresistance: Red Villafranquina (VF) showing low sperm DNA integrity after cryopreservation and Quail Castellana that shows higher DNA integrity. One pool of semen per breed was obtained twice a week for 10 wk (n = 40, 20 per breed). Each pool was divided into 2 fractions. One of these fractions was frozen in presence of valine as additive in the extender (concentration 10 mmol), whereas the other was used as control. The evaluation of the samples before and after freezing-thawing included motility (CASA-Mot system), viability (propidium iodide and SYBR-14), DNA integrity (terminal deoxynucleotidyl transferase dUTP nick end labeling), and fertility rate (percentage of eggs with blastoderm development after artificial insemination). Supplementation of valine increased several motility variables of fresh semen. In VF breed, valine increased percentage of progressive motile sperm (P = 0.025), curvilinear velocity (P = 0.033), straight-line velocity (P 5 0.040), and average path velocity (P = 0.033), whereas progressive motile sperm (P = 0.019), curvilinear velocity (P = 0.006), straight-line velocity (P = 0.003) and average path velocity (P = 0.004) were improved in the Quail Castellana breed. Valine addition increased the DNA integrity of cryopreserved semen (decreased post-thaw DNA fragmentation) in both breeds, with a significant effect (P = 0.002) in VF (36.3% VF-control vs 31%VF-valine). As expected, Quail Castellana cryopreserved sperm control showed higher fertility rate (34.4% 6 12.1) than VF cryopreserved sperm control (16.1% 1 6.2). Supplementing valine to the freezing extender doubled (P 5 0.026) the fertility rate of VF (32.6% 6 12.2) compared with the control (16.1% 1 6.2). In conclusion, supplementation of valine to chicken freezing extenders shows a positive effect on DNA fragmentation and fertilizing ability of frozen-thawed sperm, with a better response in a breed considered as the lowest freezer in our conservatory.

Simple Summary The Phoenicopteridae family is made up of six species of which 50% are in near-threatened status and 17% are in vulnerable status because their population is decreasing. The American flamingo is a useful model for development of successful semen cryopreservation procedures to be applied to threatened related species from the family Phoenicopteridae, and to permit genetic material banking. The current study sought to develop effective sperm cryopreservation protocols through examining the influences of two permeating cryoprotectants and the seminal plasma removal. In addition, morphometric and functional sperm characteristics were studied for the first time in this species. Head morphometric data provide relevant information in future studies about sperm cryobiology in these species because the head size is related with the ability of sperm to survive the freeze/thawed process. There was no apparent effect of cryoprotectants (DMA (dimethylacetamide) 6% and Me2SO (dimethylsulphoxide) 8%) on frozen–thawed flamingo sperm variables. The removal of the seminal plasma provided lower sperm quality after thawing than samples containing seminal plasma. This research demonstrated that there were no differences between Me2SO and DMA to successful freezing sperm of flamingos, but we recommend the use of Me2SO because the cell toxic effect of DMA. Abstract The American flamingo is a useful model for the development of successful semen cryopreservation procedures to be applied to threatened related species from the family Phoenicopteridae, and to permit genetic material banking. Current study sought to develop effective sperm cryopreservation protocols through examining the influences of two permeating cryoprotectants and the seminal plasma removal. During two consecutive years (April), semen samples were collected and frozen from American flamingos. In the first year, the effect of two permeating cryoprotectants, DMA (dimethylacetamide) (6%) or Me2SO (dimethylsulphoxide) (8%), on frozen–thawed sperm variables were compared in 21 males. No differences were seen between DMA and Me2SO for sperm motility, sperm viability, and DNA fragmentation after thawing. In the second year, the role of seminal plasma on sperm cryoresistance was investigated in 31 flamingos. Sperm samples were cryopreserved with and without seminal plasma, using Me2SO (8%) as a cryoprotectant. The results showed that samples with seminal plasma had higher values than samples without seminal plasma for the following sperm variables: Straight line velocity (22.40 µm/s vs. 16.64 µm/s), wobble (75.83% vs. 69.40%), (p < 0.05), linearity (62.73% vs. 52.01%) and straightness (82.38% vs. 73.79%) (p < 0.01); but acrosome integrity was lower (55.56% vs. 66.88%) (p < 0.05). The cryoresistance ratio (CR) was greater in samples frozen with seminal plasma than without seminal plasma for CR-progressive motility (138.72 vs. 54.59), CR-curvilinear velocity (105.98 vs. 89.32), CR-straight line velocity (152.77 vs. 112.58), CR-average path velocity (122.48 vs. 98.12), CR-wobble (111.75 vs. 102.04) (p < 0.05), CR-linearity (139.41 vs. 113.18), and CR-straightness (124.02 vs. 109.97) (p < 0.01). This research demonstrated that there were not differences between Me2SO and DMA to successful freezing sperm of flamingos; seminal plasma removal did not provide a benefit for sperm cryopreservation.

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.

9 Pág. Departamento de Reproducción animal. (INIA), Sperm vitrification is a simple, inexpensive method that allows the cryopreservation of sperm in the field and for endangered species is a useful alternative to conventional freezing. The study, therefore, is focused on the suitability of vitrification for cryopreserving Iberian wolf sperm and utilizing plasma testosterone concentration as a marker for procedure efficacy. Sperm and blood samples were collected from 17 wolves. There were 14 samples suitable for cryopreservation (12 ejaculated and two epididymal). Immediately after collection, these samples were proportioned into two aliquots for conventional freezing using a Tris-citric acid-glucose based extender (TCG) or vitrification utilizing an animal protein free extender (HTF®). Vitrification occurred by directly plunging a sperm suspension into liquid nitrogen. Sperm were assessed for motility, membrane integrity, acrosomal status and DNA integrity before and after cryopreservation. With both techniques, there were similar post-thaw/warming results (P > 0.05) with respect to progressive motility, kinetic variables VCL, VSL, VAP and BCF, DNA fragmentation, sperm membrane functionality and morphological abnormalities. Total motile sperm, progression ratios LIN, STR, and WOB, the ALH, sperm viability and sperm with intact membrane and acrosome were greater (P, This research was funded by MCINN/AEI/FEDER grant AGL2017-85753-R and by the Fundación Parques Reunidos INIA agreement CC19-096.

Sperm cryopreservation by ultra-rapid cooling based on dropping small volumes of sperm suspension directly into liquid nitrogen, has been successful in some wild ruminant species, including the Iberian ibex (Capra pyrenaica). In ultra-rapid cooling, the contents of these droplets are expected to enter a stable, glass-like state, but to the best of our knowledge no information exists regarding the presence or absence of ice formation in the extracellular milieu when using this technique. Different modifications to the extracellular milieu likely inflict different types of damage on the plasmalemma, the acrosome and mitochondrial membranes. The aims of the present work were: 1) to examine the physical state of the extracellular milieu after cryopreservation at slow and ultra-rapid cooling rates—and thus determine whether ultra-rapid cooling vitrifies the extracellular milieu; and 2) to compare, using conventional sperm analysis techniques and scanning and transmission electron microscopy, the damage to sperm caused by these two methods. Sperm samples were obtained by the transrectal ultrasound-guided massage method (TUMASG) from anesthetized Iberian ibexes, and cryopreserved using slow and ultra-rapid cooling techniques. Sperm motility (22.95 ± 3.22% vs 4.42 ± 0.86%), viability (25.64 ± 3.71% vs 12.8 ± 2.50%), acrosome integrity (41.45± 3.73% vs 27.00 ± 1.84%) and mitochondrial membrane integrity (16.52 ± 3.75% vs 4.00 ± 0.65%) were better after slow cooling (P

This article describes the urinogenital condition of three female Iberian ibexes (

10 Pág. Departamento de Reproducción animal, Seasonal endocrine changes may modify sperm cryoresistance in certain small ruminant species. The present work examines the effect of prolactin (PRL) on ram and buck sperm cryoresistance. A dopamine agonist (bromocriptine [BCR] 60 mg i.m. twice per week from May 15 to June 15, that is, approaching the summer solstice) or antagonist (sulpiride [SLP] 100 mg s.c. daily from December 15 to January 15, that is, around the winter solstice) was administered under solstice-appropriate photoperiod conditions to modify PRL secretion. Control animals received the vehicle only. Compared to the corresponding controls, BCR reduced PRL secretion to basal levels in both the rams and bucks. In rams, the cryoresistance ratios for sperm curvilinear velocity (P < 0.05) and lateral head displacement (P < 0.01) were higher for the BCR-treated animals. In bucks, neither the characteristics of fresh nor frozen-thawed sperm were affected by BCR treatment. After the administration of SLP, PRL levels increased and remained high for more than 5 h in the rams though they immediately began to fall in the bucks. By 24 h, PRL had returned to basal concentrations in both species. In rams treated with SLP, the cryoresistance ratios for sperm progressive motility, straight line velocity, sperm mean path velocity, cross beat frequency, and the progression ratios linearity, straightness and oscillation, were all lower compared to the controls (P < 0.05), while the amplitude of lateral head displacement was higher (P < 0.01). In bucks, sperm cryoresistance was not affected by SLP administration. Together, these results suggest that high levels of PRL negatively affect the cryoresistance of ram sperm, while buck sperm seems unaffected., This research was funded by MINECO/AEI/FEDER and EU grant AGL2017-85753-R. Flores-Gil VN was funded by FONDECYT-CONCYTEC (grant contract number 000245-2015-FONDECYT).

From birth to death, neurons are dynamically accompanied by neuroglial cells in a very close morphological and functional relationship. Three families have been classically considered within the CNS: astroglia, oligodendroglia and microglia. Many types/subtypes (including NGR2+ cells), with a wide variety of physiological and pathological effects on neurons, have been described using morphological and immunocytochemical criteria. Glio-glial, glio-neuronal and neuro-glial cell signaling and gliotransmission are phenomena that are essential to support brain functions. Morphofunctional changes resulting from the plasticity of all the glial cell types parallel the plastic neuronal changes that optimize the functionality of neuronal circuits. Moreover, neuroglia possesses the ability to adopt a reactive status (gliosis) in which, generally, new functions arise to improve and restore if needed the neural functionality. All these features make neuroglial cells elements of paramount importance when attempting to explain any physiological or pathological processes in the CNS, because they are involved in both, neuroprotection/neurorepair and neurodegeneration. There exist diverse and profound, regional and local, neuroglial changes in all involutive processes (physiological and pathological aging; neurodegenerative disorders, including Alzheimer¿s disease –AD-), but today, the exact meaning of such modifications (the modifications of the different neuroglial types, in time and place), is not well understood. In this review we consider the different neuroglial cells and their responses in order to understand the possible role they fulfill in pathogenesis, diagnosis and treatment (preventive or palliative) of AD. The existence of differentiated and/or concurrent pathogenic and neuro-protective/neuro-restorative astroglial and microglial responses is highlighted.

This study examines the influence of administering testosterone at the end of the mating season, on the responses (morphometric and functional) of ram and buck sperm to freezing-thawing. Five rams were administered 25 mg testosterone propionate (TP) subcutaneously in 2 mL of olive oil twice per week (Monday and Thursday) from October 1 to 31; 5 bucks received exactly the same treatment but from November 1 to 30. Control groups were administered 2 mL of olive oil without TP twice per week over the same period. In the rams, no significant differences were seen in plasma testosterone between the treated and control groups during treatment (0.8 ± 0.2 ng/mL vs 1.5 ± 0.5 ng/mL; P0.05). Significant differences were seen in this respect, however, in the bucks (4.3 ± 0.8 ng/mL and 6.9 ± 0.9 ng/mL; P0.05). In the rams, TP treatment increased (P0.05) the straight-line velocity (VSL), linearity (LIN), straightness (STR) and wobble (WOB) values in fresh sperm samples. Similarly, in the frozen-thawed samples, TP treatment increased the VSL, average path velocity (VAP), LIN and WOB values (P0.05) compared with controls. In the bucks, treatment with TP had no effect on any measured variable in fresh sperm; frozen-thawed sperm, however, returned greater VSL, LIN, STR, and WOB values (P0.05) than did controls. In the rams, treatment with TP led to a reduction in all fresh sperm head morphometric variables (P0.05). Freezing-thawing further reduced (P0.05) all morphometric variables in both the control and treated groups. In the bucks, treatment with TP increased (P0.05) the length, area, and perimeter of fresh sperm cells, unlike that seen in ram sperm. Compared with fresh sperm, freezing-thawing led to reduced (P0.05) morphometric variables in both the control and treated bucks, except for the sperm head width, which in the controls remained unchanged. In conclusion, TP treatment at the end of the mating season affected fresh sperm quality, in both Spanish Merino rams and Murciano-Granadina bucks, in a species-specific manner, but improved the sperm kinetic variables after freezing-thawing in both species, apparently improving sperm cryoresistance. Treatment with TP affects the dimensions of the sperm head in a species-specific manner.

In small ruminants, testosterone and prolactin plasma concentrations show circannual fluctuations as an adaptation mechanism to their seasonal breeding behavior. Sperm resistance to the freezing-thawing process shows seasonal fluctuation throughout the year, with lower sperm freezability at the beginning of the breeding season when prolactin and testosterone levels reach their maximum concentration. Nevertheless, whether these hormones directly affect post-thaw sperm quality parameters is still unclear. The objective was to study the effect of testosterone or prolactin added in vitro on sperm freezability in domestic ram (Ovis aries) and buck (Capra hircus). Sperm samples were incubated for 1 h with a range of testosterone (0, 2, 4, or 6 ng/mL; Exp. 1) or prolactin (0, 20, 100, 200, or 400 ng/mL; Exp. 2) concentrations. Samples were cryopreserved by slow freezing in straws at 0 h and after 1 h incubation. Sperm viability, acrosome integrity, motility, and kinetic parameters were assessed at 0 and 1 h in fresh and frozen-thawed samples. Results showed no hormone effect in fresh sperm, whereas these hormones affected post-thaw sperm parameters. In Exp. 1, in vitro incubation with testosterone decreased the post-thaw acrosome integrity of ram sperm (from 68.1 ± 6.3% to 49.6 ± 3.9%; P0.05). In Exp. 2, in vitro incubation with prolactin decreased the post-thaw acrosome integrity of ram (from 78.2 ± 3.4% to 66.3 ± 3.5%; P0.05) and buck sperm (from 81.7 ± 2.5% to 67.6 ± 3.5%; P0.05). Moreover, prolactin increased the post-thaw amplitude of lateral head displacement in ram sperm (from 3.3 ± 0.1 μm to 3.8 ± 0.2 μm; P0.05). In conclusion, either testosterone or prolactin added in vitro decreased the post-thaw acrosome integrity of ram and buck sperm. This suggests a destabilization process that could be decreasing sperm freezability when physiological levels of these hormones are high in vivo.

International audience; Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P

This article describes the urinogenital condition of three female Iberian ibexes (Capra pyrenaica-one infertile 3-yr-old adult and two prepubertal animals aged 1 (PP1) and 2 (PP2) yr, respectively, all raised in captivity. All showed constant urinal dribbling, leading to ulcerative dermatitis in the vulvar area. Housed in a stable with other females, the adult did not become pregnant after male contact in either of two consecutive mating seasons. Vaginoscopy and laparoscopic exploration performed on the prepubertal females revealed abnormalities of the vagina and urinary bladder. Ultrasound examination revealed atrophy of the left kidney in the adult female and PP1, and of the right kidney in PP2, with degeneration of the renal pelvis. A paraovarian cyst with hydrosalpinx was also detected in the left oviduct of the adult female. Postmortem analysis of the adult and PP2, which shared a mother, confirmed an extramural single ectopic ureter with vaginal insertion associated with atrophy of the ipsilateral kidney. Though PP1 was officially unrelated to the latter animals, all three might have had a common ancestor in their lineages.

In this study, there was an examination of the effect on the characteristics of cryopreserved black-footed (Spheniscus demersus) and gentoo (Pygoscelis papua) penguin semen, of thawing at 37 and 5 °C. For two consecutive years, semen was collected and frozen during the April-June period from six gentoo penguins, and during the October-November period from 13 black-footed penguins. After thawing, sperm motility variables were examined by computer-assisted sperm analysis. Propidium iodide and SYBR-14 were used as fluorochromes for the examination of membrane integrity. For the gentoo penguins, no differences were detected in the values of frozen-thawed semen characteristics after thawing at 37 or 5 °C. For the black-footed penguins, however, thawing at 5 °C resulted in greater values (P

The objective was to characterize the stress response and the seminal parameters obtained with electroejaculation (EE) or transrectal ultrasound-guided massage of the accessory sex glands (TUMASG) in two captive but nondomestic ruminants, the mouflons and the Iberian ibex under general anesthesia. In mouflons, the physiological responses (heart and respiratory rate, rectal temperature, cortisol, creatine kinase, potassium and glucose concentrations) changed similarly with both procedures. The TUMASG procedure was faster than EE in mouflons (21.7 ± 1.4 vs. 12.4 ± 1.2 minutes, P < 0.01). In ibexes, respiratory rate, cortisol and creatine kinase concentration changes were greater with EE than with TUMASG (final respiratory rate 62.7 ± 5.5 vs. 38.1 ± 5.6 breaths/min [P < 0.05]; final cortisol 51.4 ± 5.1 vs. 25.3 ± 5.6 ng/mL [P < 0.001]; and final creatine kinase 300.9 ± 99.9 vs. 87.1 ± 16.9 U/L [P < 0.001]). Electroejaculation provided better results in some sperm parameters (mouflons sperm score 3.4 ± 0.3 vs. 2.6 ± 0.2 [P < 0.01]; total number of sperm ejaculated 982.4 ± 299 vs. 710.0 ± 542.2 [P < 0.05]; ibexes sperm with progressive motility: 47.7 ± 6.2 vs. 20.5 ± 8.3 [P < 0.05]). The transrectal ultrasound-guided massage of the accessory sex glands appears to be an alternative technique to collect sperm from wild ruminants, reducing the need for electrical stimuli and thus decreasing the undesired responses of EE in the more sensitive species. On the other hand, better fresh sperm may be collected with EE. However, TUMASG provides practical advantages in animal welfare, firstly in these wild species more sensible to stress management and capture myopathy. © 2015 Elsevier Inc..

The rate at which lethal intracellular ice forms during sperm cryopreservation is highly dependent on the cooling protocol. The present work compares two cooling protocols for use with Iberian ibex (Capra pyrenaica) sperm by assessing the effects on the motility, viability, and size of frozen-thawed sperm cells. Ejaculates, obtained from six adult ibex males via transrectal, ultrasound-guided massage of the accessory sex glands plus electroejaculation if necessary, were cooled via either 1) Protocol 1 (decelerating cooling), involving cooling in liquid nitrogen vapor from 5 °C to -35 °C (40 °C/min), from -35 °C to -65 °C (17 °C/min), and then from -65 °C to -85 °C (3 °C/min); or 2) Protocol 2 (accelerating cooling) involving cooling in a biological freezer from 5 °C to -5 °C (4 °C/min), from -5 °C to -110 °C (25 °C/min), and then from -110 °C to -140 °C (35 °C/min). Compared to fresh ejaculates, sperm quality at thawing was found to be reduced by both protocols (p < .05), but especially by Protocol 1. Sperm head size was also significantly reduced by both protocols, although the Protocol 1 sperm heads were also significantly smaller than those of Protocol 2 sperms heads (p < .05). In fresh sperm samples, clustering analyses revealed two subpopulations of sperms with different morphometric characteristics, SP1 with larger cells, and SP2 with smaller cells. Both cooling protocols caused reduction in the proportion of SP1 cells, and an increase in the proportion of SP2 cells. In conclusion, the decelerating cooling protocol (Protocol 1) caused greater cryodamage to the sperm cells than the accelerating protocol (Protocol 2).

International audience; The aim of the present work was to examine the influence of access to pasture in an outdoor housing system on rooster sperm quality and response to cryopreservation and to examine the possible correlation between values for sperm quality variables and welfare indicators. Two groups of Black-barred Andaluza and Red-barred Vasca roosters were housed in an outdoor system, with one group given daily access to a grazing area containing plant species that typically grow on uncultivated Mediterranean land. Semen was collected once per week from each group, and the following sperm quality variables were assessed: sperm volume, appearance, concentration, motility, membrane integrity, acrosome integrity, and morphological abnormalities. In addition, two welfare indicators were examined: the heterophil/lymphocyte (H/L) ratio, and the duration of tonic immobility (TI). Ejaculates from the birds with access to pasture had higher percentages of sperm showing progressive motility (P = 0.019), and returned a higher motility index (P = 0.035). Unexpectedly, the H/L ratio was also higher in these birds. Virtually no differences were seen between the treatment groups with respect to sperm quality after freezing-thawing, although the semen of the Red-barred Vasca birds with access to pasture did show a higher percentage of progressive motility (P = 0.023) than the birds of the same breed with no such access. Significant correlations were detected between the H/L ratio and sperm motility (r = 0.420, P = 0.038), the sperm motility index (r = 0.526, P = 0.002), and progressive motility (r = 0.467, P = 0.003). No differences were seen between the treatment groups with respect to the duration of TI. In conclusion, access to pasture improved fresh sperm motility.

Background Recent evidence suggests that obesity, besides being a risk factor for cardiovascular events, also increases the risk of Alzheimer’s disease. Insulin resistance is common in all cases of obesity and appears to be the linkage between both diseases. Obesity, often associated with excessive fat and sugar intake, represents a preclinical stage toward insulin resistance during which nutrition intervention is likely to have maximum effect. Objective In this way, healthy lifestyles lifetime to prevent obesity-related modifiable risk factors such as inflammation, oxidative stress and metabolic disorders could be simultaneously beneficial for preserving cognition and controlling the Alzheimer’s disease. Method This review relates extensive research literature on facts linking nutrients and dietary patterns to obesity and Alzheimer’s disease. In addition briefly presents molecular mechanisms involved in obesity-induced insulin resistance and the contribution of peripheral inflammatory and defective insulin signalling pathways, as well as ectopic lipids accumulation to Alzheimer’s development through brain inflammation, neuronal insulin resistance, and cognitive dysfunction seen in Alzheimer’s disease. Results The work relates current and emerging pharmacological and non-pharmacological therapies for the management of obesity, insulin resistance and Alzheimer’s considering them as disorders with common molecular features. Conclusion The findings of this review validate the importance of some nutritional interventions as possible approach to prevent or delay simultaneously progression of Alzheimer’s disease and obesity. © 2017 Bentham Science Publishers.