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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.

Sperm capacitation is a crucial step towards the acquisition of fertilizing capacity. Despite the attempts to mimic the in vivo situation, there is still a lack of standardization in vitro techniques. Bicarbonate and serum albumin (BSA) are routinely used, although controversial results are reported regarding the optimal concentration of each compound. In addition, whether caffeine is needed on in vitro capacitation media in boar sperm remains to be elucidated. Here, 18 boar commercial artificial insemination doses were used to test different concentrations of bicarbonate (19, 37 or 56 mM) in experiment 1, BSA (1.5, 3, 4.5 mg/mL) in experiment 2 and the presence or absence of caffeine (5.15 mM) experiment 3. We analysed at 0, 30 and 120 min of incubation at 38.5°C, 5% CO2 : Total motility (TMOT), membrane integrity (VIAB), acrosomal exocytosis (rAcro; H33342/PI/PNA), capacitation status (chlortetracycline staining CTC) and mitochondrial membrane potential (JC-1). The higher concentrations of bicarbonate (37 and 56 mM) decreased TM and VIAB (p < .01) but increased rAcro (p < .01) after 120 min of incubation compared to the fresh control. In contrast, only the BSA concentration of 3 mg/mL reduced the VIAB at 120 min, but all the concentrations tested increased the average of JC-1 and decreased TM (p < .01) throughout incubation compared to the fresh control. Finally, in experiment 3, when boar sperm were incubated in the capacitating media with bicarbonate, BSA and with or without caffeine, the capacitated pattern measured by the CTC technique and rAcro increased after 120 min of incubation (p < .01) compared to fresh control, either in the presence or in the absence of caffeine. In summary, our results suggested that the combination of capacitating components, like bicarbonate and BSA, contributed to increasing the proportion of capacitated boar spermatozoa, mitochondrial membrane potential as well as acrosomal exocytosis. However, caffeine did not significantly infl

Rabbits constitute an interesting model to understand gamete interaction and test novel Artificial Reproductive Techniques, but in vitro fertilization (IVF) is particularly problematic in this species. We have conducted a series of experiments to develop a consistent IVF technique. Initially, we checked viability, acrosome integrity, capacitation and motility in ejaculated sperm purified by a density gradient and incubated at different times in three different media: Tyrode's Albumin Lactate Pyruvate (TALP), human tubal fluid (HTF), and Brackett and Oliphant (BO). Total and progressive motility at 10-24 h and linearity from 3 h onwards was significantly higher in BO medium compared to TALP and HTF. Subsequently, cumulus-oocyte complexes (COCs) collected 10 h after induction of ovulation were incubated with sperm in TALP, HTF or BO for 18 h with or without performing sperm pre-incubation for 6 h. Pronuclear formation rate at 18 h was significantly higher in BO compared to other media (∼84 % vs. 17-22 %) and was not improved by pre-incubation. As COCs recovery rate was low at 10 h after induction of ovulation, COCs were collected at 12 h and co-incubated with sperm in BO. Pronuclear formation rate was similar than those obtained in COCs collected at 10 h (∼85 %), and when embryos were allowed to develop in vitro, the protocol yielded high cleavage and blastocyst rates (91 and 59 %, respectively). In conclusion, ejaculated rabbit sperm purified in a density gradient fertilize efficiently COCs collected at 12 h in BO medium.

Thyroid hormones play a role in the control of seasonal reproduction in wild and domestic ruminants such as red deer and sheep. Horn growth shows an inverse relationship with reproductive activity, but the effect of thyroid function on the regulation of the seasonal pattern of horn growth is unknown. This work examines the seasonal pattern of plasma thyroxine concentration, and its effect on testosterone secretion and seasonal changes in horn growth, in male Iberian ibexes (Capra pyrenaica). Plasma testosterone, plasma thyroxine, and horn growth were measured over a year in four males kept under natural photoperiod conditions. In another four, thyroxine was administered coinciding with the period of physiological reduction in plasma testosterone (January–February). In the first four, seasonal changes in the plasma concentration of thyroxine were detected with the highest concentrations recorded in October, November, and December, coinciding with the greatest testosterone secretion. In the second four, thyroxine treatment affected neither plasma testosterone concentration nor horn growth. The plasma concentrations of testosterone and thyroxine appear to be closely related in the ibex, but the seasonal pattern of horn growth does not seem to be modulated by seasonal changes in thyroxine.

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.

The aims were to determine if the contact with estrous females stimulates testosterone and cortisol secretion in Iberian ibex bucks during or out of the breeding season and if their social rank affects this response. Nine Iberian ibex bucks (Capra pyrenaica), were challenged with estrous does during two periods: winter-spring and autumn (three tests at each season, separated 10 days). Individual success index (SI) was calculated in both season. Bucks were exposed to an estrous doe, their sexual and agonistic behavior was recorded, testosterone and cortisol concentrations were measured. There were no sexual interactions toward the estrous doe in any test. Testosterone concentration was unrelated to the SI in any season and was not modified with the tests. Cortisol concentration was greater in the first than in the second and third tests in both seasons (winter-spring: 99.7 ± 15.7 ng/mL, 65.9 ± 9.4 ng/mL and 62.3 ± 10.1 ng/mL P < 0.0001; autumn: 110.1 ± 15.8 ng/mL, 79.2 ± 13.4 ng/mL, and 87.6 ± 13.5 ng/mL; P < 0.01). Iberian ibex bucks allocated in captivity did not display sexual behaviors nor modify the testosterone secretion, which was not modified by social hierarchy, after estrous doe exposition, during or out of the pre-rutting period.

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.

The number of two-toed sloths (Choloepus hoffmanni) has significantly decreased in the last years. Deepening the knowledge of this tropical mammal's reproductive physiology is essential to improve captive breeding within conservation programs for this species. However, several aspects of its reproductive biology remain unexplored and have not been described sufficiently. The aim of this work was to describe the estrous cycle and reproductive physiology of two adult female C. hoffmanni by vaginal cytology, appearance of the external genitalia, and behavior. Vaginal cytology assay showed that the average duration of the estrous cycle was 15.1 ± 4.53 d. Positive correlations (P < 0.05) were found between the peak presence of superficial cells (estrous phase) and four parameters: aggressive behavior, pursuing behavior, vulvar swelling, and vaginal discharge. This pilot study, conducted on just two animals, forms a basis for a study design that may be employed for a more comprehensive assessment of the two-toed sloth reproductive physiology and behavior.

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

This study aimed to assess the suitability of egg yolk (EY) supplementation to a tris-citric acid-based extender on cryosurvival of guinea pig (Cavia porcellus) epididymal spermatozoa. Two synthetic-based extenders, tris-citric acid-glucose plus 20% EY (TCG-EY) and tris-citric acid-fructose (TCF) both with 5% glycerol, were compared. Thirty-two epididymides were recovered from 16 adult guinea pig males by gonadectomy, and then the sperm samples were retrieved by retrograde flushing using TCG-EY and TCF extenders for left or right epididymis, respectively. TCG-EY and TCF sperm samples were frozen in static liquid nitrogen vapors through a two-step cooling procedure. Before freezing, the percentage of progressive sperm motility and sperm with intact plasma and acrosome membranes from TCG-EY sperm samples were higher (p < 0.05) than those diluted with TCF. Post-thaw sperm kinematic variables and membrane integrity were drastically reduced (p < 0.001) compared with prefreezing samples, regardless of extender type. The post-thaw plasmatic and acrosome membrane integrity from TCG-EY sperm samples was higher (p < 0.05) than those from TCF samples. Except for the length, the morphometric head dimensions of sperm diluted with TCG-EY or TCF did not vary (p > 0.05) after the freezing-thawing process compared with the prefreezing samples. In conclusion, despite greater cell cryoinjury with both extenders, the EY supplementation exerted greater cell membrane protection before and after the freezing-thawing process. This research shows an in-depth analysis of guinea pig sperm cryopreservation; however, more studies are recommended.

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.

Previous studies of our group showed that subzero temperatures in supercooled boar semen are a serious detrimental factor during boar sperm cryopreservation. Like boar semen, rooster semen is quite sensitive to freezing and thawing, but the effects of supercooling on rooster sperm cells have not yet been investigated. The present work focused on studying the effect of subzero temperatures in supercooled rooster semen. Four pools (one pool per week) of 5 randomly selected roosters (different roosters per pool) were diluted four times with ASG extender. Half of the final volume of the diluted semen was added of ASG with 1.8 M DMA, giving a final concentration of 0.6 M DMA. After one hour of incubation, the semen was packed in 0.25-ml straws and introduced into a freezing chamber at -10 °C. At time points 2, 30 and 60 min, one straw was removed per time point for assessment of motility (CASA system), viability (DAPI staining) and DNA integrity (TUNEL). The results obtained were compared with the ones obtained from the fresh semen at time zero. The data were analyzed using the Wilcoxon¿s test. The differences between fresh semen versus ¿10°C were not significant, but there was a trend towards lower total motility and progressive motility in supercooled semen, as the P values were around 0.07. In conclusion, supercooling did not have a severe effect on viability, motility and DNA integrity of rooster sperm.