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The freeze-thawing process induces osmotic changes that may affect the membrane domain location of aquaporins’ (AQP) in spermatozoa. Recent studies suggest that changes in AQP3 localization allows better sperm osmo-adaptation, improving the cryoresistance. Ultra-rapid freezing is an alternative cryopreservation technique that requires less equipment than conventional freezing, and it is faster, simpler and can be used in the field. This study aimed to determine the influence of freezing-thawing rates (slow (control) vs. ultra-rapid) on AQP3 expression and location in the spermatozoa from small ruminants (sheep and goats) and its relationship with sperm cryo-damage. Spermatozoa were collected from 10 Merino rams and 10 Murciano-Granadina bucks. The presence and distribution of AQP3 were assessed by Western blotting and immunocytochemistry (ICC), employing a commercial rabbit polyclonal antibody. Sperm motility was CASA system-analyzed, and membrane and acrosome integrity assessed by fluorescence (PI/PNA-FITC). Western blotting did not detect a significant effect of freezing-thawing rate on the amount of AQP3 while ICC found freezing-thawing rate affecting AQP3 location (P<0.05). In both species, the percentages of spermatozoa showing AQP3 in the post-acrosome region, mid-piece, and principal piece of the tail were greater in samples cryopreserved by slow freezing-thawing (control) than ultra-rapid freezing-thawing rates (P<0.05). Spermatozoa cryopreserved using ultra-rapid freezing-thawing showed decrease motility, plasma membrane, and acrosome integrity (P<0.05), which might be related, at least in part, to a lower expression of AQP3. In conclusion, the cooling rate modifies the location of AQP3 in spermatozoa of sheep and goat, which might be associated with sperm cryosurvival.

Beca predoctoral de LP financiada por fondos FEDER (PP.TRA.2016.0010 FEDER funds). Beca de colaboración de estudiantes de pregrado de E Flix financiada por el Ministerio de Ciencia e Innovación., This study examines the relationship between seasonal changes in plasma testosterone and thyroxine concentrations and the relationship between these changes and the sperm resistance to the freezing-thawing process in Merino x Merino Precoz crossbreed rams and Florida breed bucks. Plasma testosterone and thyroxine concentration, sperm head area, sperm variables and their cryoresistance ratios were analyzed throughout a year. Both species showed a rise in thyroxine preceding testosterone fall. Plasma thyroxine concentrations in rams were higher during autumn than in spring and summer (P<0.05), whereas testosterone concentrations were not influenced by season. Conversely, bucks showed seasonal differences in plasma testosterone levels, with higher concentrations during summer and autumn than winter and spring (P<0.05), whereas thyroxine concentrations were not influenced by season. In bucks, a negative correlation was detected between plasma testosterone concentrations and the cryoresistance ratio for membrane integrity (R=-0.88; P<0.05) from April to September and between plasma thyroxine concentrations and the cryoresistance ratios for total motility (R=-0.66; P<0.05), curvilinear velocity (R=-0.61; P<0.05), the amplitude of lateral head displacement (R=-0.77; P<0.05) and the beat-cross frequency (R=-0.74; P<0.05), throughout the year. The results suggest that the annual variation in sperm cryoresistance may be related to seasonal endocrine changes, at least in goat. Winter and spring are the most appropriate seasons to collect and freeze sperm from Florida goat, Agencia Estatal de Investigación (AEI), MCINN (AGL2017-85753-R. ), FEDER/UE (PP.TRA.2016.0010 fondos FEDER), Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

J. Santiago-Moreno recibió una beca del Ministerio de Ciencia, Innovación y Universidades para visita de investigadores seniors a centros extranjeros (PRX19/00149). El Consejo Sueco de Investigación FORMAS (2017-00946 y 2019-00288), Estocolmo, Suecia, proporcionó apoyo analítico., 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., Agencia Estatal de Investigación (AEI) (AGL2017-85753-R y PID2020-113288RB-I00/AEI/10.13039/501100011033), MCINN (AGL2017-85753-R y PID2020-113288RB-I00/AEI/10.13039/501100011033), FEDER/EU, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Author contributions JSM: Conceptualisation, Writing – review and editing. ATD, CC, RV, PB, PPF, BP, EO, BMM, and MCE: Writing – original draft., Reproductive technologies can help to protect wild ruminant species from becoming extinct. In addition, the decline in some wild game species has also raised interest in reproductive technologies to increase the number of animals that can be produced. Most biobanking efforts have focused on developing effective protocols for preserving sperm, oocytes, and embryos. Cryopreservation of sperm remains the least invasive method and the cheapest procedure for germplasm storage. Over the last few years, several reproductive biotechnologies have been developed beyond the conventional freezing of spermatozoa. These include ultra-rapid freezing techniques. Nevertheless, fertility results after artificial insemination using frozen-thawed spermatozoa are not always acceptable in wild small ruminants. Moreover, these technological efforts have met variable success related to the sample’s origin (epididymal retrieved postmortem or ejaculated) and the season of sperm sample collection and storage. Epididymal sperm shows higher cryoresistance than ejaculated sperm. Changes in sperm proteome between epididymal and ejaculated sperm seem to contribute to this different cryotolerance. The role of endocrine status has been studied in some wild species to better understand the underlying mechanism of the annual variation in ruminant sperm cryoresistance. Seasonal changes in testosterone and prolactin are involved in sperm cryoresistance; sperm recovery and cryopreservation are recommended around the end of the rutting season, when good quality sperm samples can still be obtained, testosterone levels have already decreased, and prolactin concentrations remain low. The mechanisms of hormone action on sperm freezability are not well known. Still, it has been suggested that testosterone affects cell proliferation in the testis, during spermatogenesis, and membrane properties of sperm cells during their transit through the reproductive tract, which might influence their cryotolerance. Recent, Agencia Estatal de Investigación (AEI), Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Pequeño B was the recipient of a grant for pre-doctoral researchers from AEI (PRE2018–085637)., Spermatozoa collected from the cauda epididymis of wild ruminants are more cryoresistant than ejaculated spermatozoa. Changes in the membrane location of aquaporins (AQPs) follow the osmotic changes that occur during freeze-thawing, and might influence the cryosurvival of spermatozoa depending on their source. This work reports the location of AQP3, AQP7 and AQP10 in the cauda epididymal and post-ejaculation spermatozoa of three wild mountain ungulate species (Iberian ibex, mouflon, and chamois), as determined by Western blotting (WB) and immunocytochemistry (ICC) using commercial rabbit polyclonal primary antibodies. WB confirmed the presence of all three AQPs in the spermatozoa of all the studied species, while ICC showed AQP3 to be mainly located in the sperm acrosome, mid-piece, principal piece, and end piece, both in cauda epididymal and ejaculated cells. The percentage of ejaculated spermatozoa showing AQP3 in the principal piece was higher in the ibex than in the chamois (P < 0.05), and higher in epididymal spermatozoa in the mouflon than in the chamois (P < 0.05). AQP7 was located in the acrosome of both epididymal and ejaculated spermatozoa, as well as in the cytoplasmic droplet of the epididymal spermatozoa of all three species. No differences were seen between the species with respect to the percentage of spermatozoa showing AQP7. AQP10 was located mainly in the mid-piece, principal piece and end piece of the sperm tail in both epididymal and ejaculated spermatozoa. The percentage of mouflon spermatozoa with AQP10 in the end piece was higher in the cauda epididymal than in the ejaculated spermatozoa (P < 0.05). In conclusion, except for AQP10 in the mouflon, the locations of the studied AQPs are similar in epididymal and ejaculated spermatozoa, with inter-species differences seen only for AQP3. Further studies are needed to determine what this might mean with respect to sperm cryopreservation., Agencia Estatal de Investigación (AEI), Ministerio de Ciencia e Innovación, FEDER, Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Beca predoctoral de Pequeño financiada por la AEI (PRE2018-085637), Context: In the epididymis, epithelial cells manage changes in the luminal environment for proper sperm maturation. Moreover, aquaglyceroporins, a subgroup of aquaporins (AQP), modulate the transport of water, glycerol and other small molecules in epithelial cells. Aims: We aim to characterise the lining epithelium, quantify its cell composition and immunolocalise the aquaglyceroporins AQP3, AQP7, AQP9 and AQP10 alongside the epididymal ductus of three wild ruminant species, and to determine if species-specific differences could be associated with cauda sperm cryoresistance variations. Methods: Epididymides from Iberian ibex (n =5), mouflon (n =5) and chamois (n =6) were obtained. Cauda spermatozoa were collected and sperm parameters were analysed before and after freezing. Histology and immunohistochemistry of AQP3, 7, 9, 10 and T-CD3 were performed in the caput, corpus and cauda epididymal regions. Key results: This work first describes the lining epithelium in Iberian ibex, mouflon and chamois epididymis along the three anatomical regions, consisting of principal, basal, apical, clear and halo cells. However, the percentage of each cell type differed in ibex compared to mouflon and chamois. The positive T-CD3 immunolabeling of all the halo cells confirmed their T-lymphocyte nature. Aquaglyceroporin expression patterns were similar among species, except for differences in AQP7 and AQP10 immunolocalisation in ibex. Species-specific differences in epididymal sperm cryoresistance were confirmed. Conclusions: The epididymal epithelium of the three wild ruminants differ in their relative number of cell types and AQP immunolocalisation, which ultimately appears to affect cauda epidydimal spermatozoa cryoresistance. Implications: Our study provides information on the relevance of the quantitative composition and AQP pattern expression in epididymal lining epithelium on sperm cryoresistance., Agencia Estatal de Investigación (AEI), Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Este trabajo también fue financiado por un contrato entre Zoitechlab S.L. (Arquimea Group) y el INIA (CON18-141), 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., Agencia Estatal de Investigación (AEI) (AGL2017-85753-R y PID2020-113288RB-100/AEI/10.13039/501100011033), MCINN/AEI/FEDER (AGL2017-85753-R y PID2020-113288RB-100/AEI/10.13039/501100011033), EU (AGL2017-85753-R y PID2020-113288RB-100/AEI/10.13039/501100011033), Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Osmotic changes during the process of freeze-thawing involve changes in the location of aquaporins (AQPs) in membrane domains of spermatozoa. Some AQPs, like aquaporin 3 (AQP3), are linked to sperm cryotolerance in the porcine species. Conspicuous individual variability exists between rams and their ejaculates, which may be classified as displaying good freezability (GFE) or poor freezability (PFE), depending on several endogenous and environmental factors. The present work aimed to examine whether differences in freezability could even involve changes in location and expression of AQP3 in ram spermatozoa.

This work examines the effect of equilibration time with extender on ultra-rapidly frozen-thawed wild ruminant epididymal (origin: Iberian ibex) and ejaculated (origin: mouflon) sperm variables. Sperm samples were prepared either without prior equilibration, or equilibrated for 30 min before freezing. Higher quality (p < 0.05) frozen-thawed spermatozoa were obtained when equilibration was allowed, for ejaculated sperm in terms of sperm motility, acrosome apical ridge integrity, sperm viability, and percentage of normal cells, and for epididymal sperm in terms of linearity and straightness of sperm movement. The sperm head area, head perimeter, head length and head width were smaller (p < 0.01) in the equilibrated than non-equilibrated frozen-thawed epididymal sperm; no such dimensional changes were recorded for ejaculated sperm. In conclusion, equilibration prior to ultra-rapid freezing improves the cryoresistance of sperm cells, although viable sperm cells can be obtained without equilibration. The epididymal sperm showed greater cryoresistance, supporting the idea that it is more resistant to freeze-thawing than ejaculated sperm.

Spermatozoa collected from the cauda epididymis of wild ruminants are more cryoresistant than ejaculated spermatozoa. Changes in the membrane location of aquaporins (AQPs) follow the osmotic changes that occur during freeze-thawing, and might influence the cryosurvival of spermatozoa depending on their source. This work reports the location of AQP3, AQP7 and AQP10 in the cauda epididymal and post-ejaculation spermatozoa of three wild mountain ungulate species (Iberian ibex, mouflon, and chamois), as determined by Western blotting (WB) and immunocytochemistry (ICC) using commercial rabbit polyclonal primary antibodies. WB confirmed the presence of all three AQPs in the spermatozoa of all the studied species, while ICC showed AQP3 to be mainly located in the sperm acrosome, mid-piece, principal piece, and end piece, both in cauda epididymal and ejaculated cells. The percentage of ejaculated spermatozoa showing AQP3 in the principal piece was higher in the ibex than in the chamois (P < 0.05), and higher in epididymal spermatozoa in the mouflon than in the chamois (P < 0.05). AQP7 was located in the acrosome of both epididymal and ejaculated spermatozoa, as well as in the cytoplasmic droplet of the epididymal spermatozoa of all three species. No differences were seen between the species with respect to the percentage of spermatozoa showing AQP7. AQP10 was located mainly in the mid-piece, principal piece and end piece of the sperm tail in both epididymal and ejaculated spermatozoa. The percentage of mouflon spermatozoa with AQP10 in the end piece was higher in the cauda epididymal than in the ejaculated spermatozoa (P < 0.05). In conclusion, except for AQP10 in the mouflon, the locations of the studied AQPs are similar in epididymal and ejaculated spermatozoa, with inter-species differences seen only for AQP3. Further studies are needed to determine what this might mean with respect to sperm cryopreservation.

The western capercaillie (Tetrao urogallus) is endangered in many Western and Central European countries, where its numbers have fallen sharply over the last 100 years. Climate change, habitat fragmentation and loss of connectivity pose continuing challenges for most of its populations. In Spain, the Cantabrian population is at great risk of extinction. Estimates suggest only 191 individuals remain, inhabiting some 350 km2 of the Cantabrian Range [1]. The Pyrenean population, which is disconnected from the Cantabrian population, has decreased in size by some 50 % in the last 20 years; the latest population estimates stand at just 404 birds [2].

Técnicas reproductivas en las especies animales aborda todas las actuaciones que se llevan a cabo en el campo de la biotecnología de la reproducción. Sus contenidos se estructuran en secciones temáticas bien diferenciadas que incluyen el control del ciclo estral, la obtención de semen, la conservación del esperma, la inseminación artificial, la transferencia y criopreservación de embriones, y las técnicas de producción in vitro. Dentro de cada sección, y con objeto de facilitar la consulta, cada capítulo se centra en una sola especie animal: equinos, bovinos, pequeños rumiantes, cerdos, perros y gatos. Adicionalmente incluye una sección dedicada a las técnicas reproductivas de mayor interés en conejos, camélidos y gallinas, especies de gran importancia pero a menudo pasadas por alto en otros manuales de la especialidad

Reproductive technologies can help to protect wild ruminant species from becoming extinct. In addition, the decline in some wild game species has also raised interest in reproductive technologies to increase the number of animals that can be produced. Most biobanking efforts have focused on developing effective protocols for preserving sperm, oocytes, and embryos. Cryopreservation of sperm remains the least invasive method and the cheapest procedure for germplasm storage. Over the last few years, several reproductive biotechnologies have been developed beyond the conventional freezing of spermatozoa. These include ultra-rapid freezing techniques. Nevertheless, fertility results after artificial insemination using frozen-thawed spermatozoa are not always acceptable in wild small ruminants. Moreover, these technological efforts have met variable success related to the sample's origin (epididymal retrieved postmortem or ejaculated) and the season of sperm sample collection and storage. Epididymal sperm shows higher cryoresistance than ejaculated sperm. Changes in sperm proteome between epididymal and ejaculated sperm seem to contribute to this different cryotolerance. The role of endocrine status has been studied in some wild species to better understand the underlying mechanism of the annual variation in ruminant sperm cryoresistance. Seasonal changes in testosterone and prolactin are involved in sperm cryoresistance; sperm recovery and cryopreservation are recommended around the end of the rutting season, when good quality sperm samples can still be obtained, testosterone levels have already decreased, and prolactin concentrations remain low. The mechanisms of hormone action on sperm freezability are not well known. Still, it has been suggested that testosterone affects cell proliferation in the testis, during spermatogenesis, and membrane properties of sperm cells during their transit through the reproductive tract, which might influence their cryotolerance. Recent

In the epididymis, epithelial cells manage changes in the luminal environment for proper sperm maturation. Moreover, aquaglyceroporins, a subgroup of aquaporins (AQP), modulate the transport of water, glycerol and other small molecules in epithelial cells.

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.

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 < 0.05) were found between breeders in sperm viability (58.0 ± 1.90 breeder D vs. 35.2 ± 7.41 breeder A, 37.2 ± 4.09 breeder B and 22.3 ± 5.92 breeder C) and DNA fragmentation (62.4 ± 9.91 breeder C vs. 31.8 ± 7.08 breeder B and 24.5 ± 5.49 breeder D). The lowest DNA fragmentation values for semen from breeder D birds were coincident with higher integrity of the mitochondrial membrane.4. The results revealed higher sperm cryoresistance in the cockerels from one of the breeders, possibly due to differences in management system (e.g. diet, housing, control of stress elements and pathogens, reproduction practices or maintenance of genetic diversity). These differences may determine the sperm freezability, and thus the effectiveness of developing a germplasm bank., r

The Cantabrian capercaillie (Tetrao urogallus cantabricus) is critically endangered. This subspecies has the lowest genetic variability and it is in regression. It belongs to Phasianidae family; therefore, the domestic chicken (Gallus gallus domesticus) could be a good model for developing reproductive technologies for use in capercaillie populations with low availability of animals.

Introduction and objective: Osmotic changes during the process of freeze-thawing involve changes in the location of aquaporins (AQPs) in membrane domains of spermatozoa. Some AQPs, like aquaporin 3 (AQP3), are linked to sperm cryotolerance in the porcine species. Conspicuous individual variability exists between rams and their ejaculates, which may be classified as displaying good freezability (GFE) or poor freezability (PFE), depending on several endogenous and environmental factors. The present work aimed to examine whether differences in freezability could even involve changes in location and expression of AQP3 in ram spermatozoa. Methods: Thirty ejaculates from 10 rams (three of each) were evaluated and subsequently classified as GFE (n = 13) or PFE (n = 17) through a principal component analysis (PCA) and k-means cluster analysis. Spermatozoa were examined for the presence, abundance and distribution of AQP3 by western blot and immunocytochemistry, employing a commercial rabbit polyclonal antibody (AQP3 - ab125219). Results and discussion: Although AQP3 was found in the sperm acrosome, midpiece, principal and end piece of the tail in both fresh and after frozen-thawedsamples, its highest immunolabeling was found in the mid- and principal piece. In the GFE group, the expression of AQP3 in the mid- and principal piece was greater (P < 0.05) in frozen-thawed samples than in fresh specimens while such differences were not detected in the PFE group. Sperm cryotolerance relates to changes in AQP3 expression and thus AQP3 could be used as a biomarker for cryotolerance. Conclusion: A greater capacity of AQP3 localization in mid- and principal piece of the spermatozoa could be linked to an increase the osmo-adaptative capacity of ejaculates with better capacity to withstand freeze-thawing processes.

Cryopreservation of testicular tissues offers new possibilities to protect endangered species, genetically valuable individuals or even the fertility potential of prepubertal individuals who have died unexpectedly. However, the use of this technique still remains a challenge. In this study, slow freezing and vitrification of testicular tissue was investigated to find out which cryopreservation method could better preserve the viability and DNA integrity of testicular germ cells in diverse wild species.

This study examines the relationship between seasonal changes in plasma testosterone and thyroxine concentrations and the relationship between these changes and the sperm resistance to the freezing-thawing process in Merino x Merino Precoz crossbreed rams and Florida breed bucks. Plasma testosterone and thyroxine concentration, sperm head area, sperm variables and their cryoresistance ratios were analyzed throughout a year. Both species showed a rise in thyroxine preceding testosterone fall. Plasma thyroxine concentrations in rams were higher during autumn than in spring and summer (P<0.05), whereas testosterone concentrations were not influenced by season. Conversely, bucks showed seasonal differences in plasma testosterone levels, with higher concentrations during summer and autumn than winter and spring (P<0.05), whereas thyroxine concentrations were not influenced by season. In bucks, a negative correlation was detected between plasma testosterone concentrations and the cryoresistance ratio for membrane integrity (R=-0.88; P<0.05) from April to September and between plasma thyroxine concentrations and the cryoresistance ratios for total motility (R=-0.66; P<0.05), curvilinear velocity (R=-0.61; P<0.05), the amplitude of lateral head displacement (R=-0.77; P<0.05) and the beat-cross frequency (R=-0.74; P<0.05), throughout the year. The results suggest that the annual variation in sperm cryoresistance may be related to seasonal endocrine changes, at least in goat. Winter and spring are the most appropriate seasons to collect and freeze sperm from Florida goat.

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.

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 < .05). No differences were seen between the methods with respect to sperm motility variables; most sperm (>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.

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 spe

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.

Semen cryopreservation is an increasingly demanded technique in canids, particularly in order to preserve and spread high genetic value material. Sperm vitrification may represent an interesting alternative to costly and time consuming conventional freezing. The objective of this study was to evaluate the effect of sperm vitrification on sperm morphometry and ultrastructure compared to conventional freezing. Pools of nine beagle dogs were both frozen and vitrified. Computerized morphological parameters (length, wide, area and perimeter) and sperm ultrastructure, using scanning and transmission microscopy, were analysed in both fresh and in thawed/warmed samples. There were no differences (p > 0.05) between post-thaw and fresh morphometric variables of the sperm heads. However, cluster analysis revealed that sperm-heads turned out to be smaller after thawing (p < 0.05) in two of the four subpopulations. Vitrification-warming process led to an overall increase in sperm-head size. Furthermore, the sperm head size increased after warming in two subpopulations (p < 0.05). In conclusion, the variations in the sperm head area depended on the cryopreservation procedure (conventional freezing or vitrification). Conventional freezing tended to decrease the head dimensions, at least in some subpopulations, and vitrification led to an overall increase in the sperm head size. Decondensation of chromatin and plasma membrane blebbing in the head region was observed by transmission electron microscopy in several vitrified sperm, which might explain the increase of head dimensions detected by CASA-Morph system.