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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.

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.

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

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.

Sperm cryopreservation is the most common procedure used to establish germplasm banks for endangered species - but sometimes sperm cells cannot be obtained. In such cases, freezing testicular tissue may be the only option. The testes contains germ cells at different stages of differentiation, including spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa, among which differences in cryoresistance might be expected. The present work compares the viability and DNA integrity of 'rounded' cells, and of elongated spermatids and spermatozoa, from the dog and wild boar, following the cryopreservation of testicular tissue by slow freezing or vitrification. Cell viability was analyzed by PI/SYBR14 staining, and DNA integrity via the TUNEL technique. For wild boar, no significant differences were seen between the two methods with respect to the percentage of viable cells, nor in the percentage of cells with DNA damage. In the dog, the percentage of viable rounded germ cells (65.0 ± 2.4%) was higher (P < 0.05) after vitrification than after slow freezing (45.1 ± 6.7%). No difference was found between the two methods in terms of the viability of elongated cells. For rounded cells, the percentage of intact DNA was greater (P < 0.05) after vitrification (90.5 ± 2.1%) than after slow freezing (42.6 ± 11.0%), while for elongated spermatids and spermatozoa it was higher (P < 0.05) after slow freezing (66.9 ± 6.1%) than after vitrification (50.7 ± 4.5%). Thus, the response to cryopreservation is cell type-, cryopreservation type-, and species-dependent. Vitrification would appear to be the most appropriate method for preserving dog testicular tissue given the associated high cell viability and low degree of DNA fragmentation, while in wild boar, either method might be used.

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.

Over the last century, several reproductive biotechnologies beyond the artificial incubation of eggs were developed to improve poultry breeding stocks and conserve their genetic diversity. These include artificial insemination (AI), semen storage, diploid primordial germ cell (PGC) methodologies, and gonad tissue storage and transplantation. Currently, AI is widely used for selection purposes in the poultry industry, in the breeding of turkeys and guinea fowl, and to solve fertility problems in duck interspecies crosses for the production of mule ducklings. The decline in some wild game species has also raised interest in reproductive technologies as a means of increasing the production of fertile eggs, and ultimately the number of birds that can be raised. AI requires viable sperm to be preserved in vitro for either short (fresh) or longer periods (chilling or freezing). Since spermatozoa are the most easily accessed sex cells, they are the cell type most commonly preserved by genetic resource banks. However, the cryopreservation of sperm only preserves half of the genome, and it cannot preserve the W chromosome. For avian species, the problem of preserving oocytes and zygotes may be solved via the cryopreservation and transplantation of PGCs and gonad tissue. The present review describes all these procedures and discusses how combining these different technologies allows poultry populations to be conserved and even rapidly reconstituted.

Black-footed penguins (Spheniscus demersus) are classified as endangered, and the populations of gentoo penguins (Pygoscelis papua) are rapidly decreasing. The optimization of semen cryopreservation in these species, for preserving their genetic diversity in genome resource banks, is essential for the success of captive breeding programs. This study compares the effectiveness of two permeating cryoprotectants, dimethylacetamide (DMA) and dimethylsulfoxide (DMSO), on frozen-thawed sperm characteristics. Semen samples were collected during each breeding season once a week during two consecutive years. Semen samples were packaged in 0.25 ml straws and frozen by placing them in nitrogen vapors. After thawing, sperm motility characteristics were examined by computer-assisted sperm analysis. Propidium iodide and SYBR-14 were used as fluorochromes for the examination of membrane integrity. DNA integrity was evaluated by TUNEL assay. Gentoo sperm characteristics after freeze-thawing did not show any differences when using DMSO or DMA. In black-footed samples, progressive motility, curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and straightness (STR) were greater using 8% DMSO (P < 0.05) than 6% DMA. The cryoresistance ratio (CR) using 8% DMSO was greater (P < 0.05) in gentoo than black-footed samples for CR-VCL and CR-VAP, and 6% DMA returned greater CR values (P < 0.05) than in black-footed samples for all characteristics evaluated. No differences were found in DNA fragmentation. In conclusion, the present results highlight the benefits of using 8% DMSO compared to 6% DMA in penguins. Sperm from black-footed showed a higher sensitivity to freezing-thawing process than gentoo sperm.

BoviPure® is a salt solution containing colloidal silica particles coated with silane used to select sperm (e.g., ruminants) by density-gradient centrifugation (DGC). This research assessed the suitability of the BoviPure-DGC and swim-up methods for selecting dog epididymal sperm in fresh, chilled and frozen-thawed samples on post-treatment sperm quality. Sperm samples (n = 60 epididymides) were recovered by retrograde flushing from thirty orchiectomized adult dogs. Thereafter, 20 sperm pools, containing sperm aliquots of three randomly selected animals, were used for chilling (at 5 ºC for 24 h) and freezing (in liquid nitrogen vapors). Sperm selection by BoviPure-DCG and swim-up was performed in both individual and pooled samples, including non-selected samples as controls. Overall, after BoviPure-DGC selection a higher sperm retrieval rate was obtained than the swim-up selection in both individual (P < 0.05) and pooled (P < 0.01) samples. BoviPure-DGC improved (P < 0.05) the total (TM) and progressive (PSM) sperm motilities, curvilinear (VCL) and straight-line (VSL) velocities, linearity (LIN), wobble (WOB), beat-cross frequency (BCF), and integrity of plasmatic (IPM) and acrosomal (IAM) membranes of individual samples in comparison with non-selected samples. In pooled samples, however, the BoviPure-DGC improved (P < 0.05) the PSM, VCL, WOB, and IPM of chilled and frozen-thawed samples. The swim-up method improved (P < 0.05) only some kinematic variables of the individual (VCL, WOB and BCF) and cryopreserved pooled samples (VCL and ALH) in comparison with non-selected samples. In conclusion, BoviPure-DGC was more effective for recovering and selecting both fresh and cryopreserved dog epididymal sperm than the swim-up procedure improving the kinematic variables, and membranes intactness.

During cryopreservation sperm encounter oxidative stress due to higher production of ROS molecules and insufficient natural antioxidant defence system. Therefore, present study was designed to identify the effects of various glutathione (GSH) concentrations on Indian red jungle fowl (Gallus gallus murghi) sperm quality and fertility pre-freezing and post-thaw incubation hours. Semen was collected from eight cocks and qualified semen ejaculates having motility >65% were pooled after initial evaluation. Semen was divided in four aliquots, diluted with red fowl extender (1:5) at 37 °C having GSH 0 mM (control), 0.1 mM, 0.5 mM and 1.0 mM, cryopreserved and stored at (-196 °C) in liquid nitrogen. Semen quality was assessed at post dilution, cooling, equilibration, and freeze-thawing at 0, 2 and 4 h of incubation at 37 °C. Sperm motility, plasma membrane integrity, viability, acrosome integrity and mitochondrial function were recorded highest (P < 0.05) with 0.5 mM GSH in extender at post-dilution, cooling, equilibration, freeze-thawing and 0, 2 and 4 h of incubation. Lipid peroxidation in sperm and seminal plasma were recorded lowest (P < 0.05) with 0.5 mM GSH during cryopreservation stages and post-thawing incubation. Moreover, antioxidant activities (total antioxidant potential and free radical scavenging capacity) were recorded highest (P < 0.05) in extender having 0.5 mM GSH. Fertility rates were recorded higher (P < 0.05) with 0.5 mM GSH compared to control. It is concluded that 0.5 mM GSH in extender improves sperm structural (sperm viability, plasma membrane integrity and acrosome integrity), functional integrity (motility, mitochondrial function) and fertility parameters of Indian red jungle fowl through enriching antioxidant potential and ameliorating the oxidative stress.

Epididymal sperm shows higher cryoresistance than ejaculated sperm. Although the sperm proteome seems to affect cell cryoresistance, studies aiming at identifying proteins involved in sperm freezing-tolerance are scarce. The aims of this study were to investigate differences of sperm freezability and proteome between epididymal and ejaculated sperm in three mountain ungulates: Iberian ibex, Mouflon and Chamois. Sperm samples were cryopreserved in straws by slow freezing. Tandem mass tag-labeled peptides from sperm samples were analyzed by high performance liquid chromatography coupled to a mass spectrometer in three technical replicates. The statistical analysis was done using the moderated t-test of the R package limma. Differences of freezability between both types of sperm were associated with differences of the proteome. Overall, epididymal sperm showed higher freezability than ejaculated sperm. Between 1490 and 1883 proteins were quantified in each species and type of sperm sample. Cross species comparisons revealed a total of 76 proteins that were more abundant in epididymal than in ejaculated sperm in the three species of study whereas 3 proteins were more abundant in ejaculated than epididymal sperm in the three species of study (adjusted P < 0.05; |log2| fold-change > 0.5). Many of the proteins that were associated with higher cryoresistance are involved in stress response and redox homeostasis. In conclusion, marked changes of sperm proteome were detected between epididymal and ejaculated sperm. This work contributes to update the sperm proteome of small ruminants and to identify candidate markers of sperm freezability.

The cryoresistance of bucks' semen collected by transrectal ultrasound-guided massage of the accessory sex glands (TUMASG) and electroejaculation (EE) was compared during the breeding season (Study 1) or the late non-breeding season (Study 2). Semen was collected from 10 Gabon bucks with both methods in each season and then frozen according to a standardized protocol. The time required for ejaculation, the number of electrical pulses applied, the sum of the electrical pulses applied∗voltage and the total number of vocalizations emitted during the collection by each animal were recorded. In Study 1, TUMASG required a longer time (P = 0.0006) but fewer electrical pulses and a lower sum of pulses∗voltage than EE (P < 0.0001 for both comparisons). Bucks vocalized fewer times during TUMASG than during EE (P < 0001). Semen collected with TUMASG had greater sperm concentration, sperm mass motility, total number of motile sperm and of sperm with progressive motility, and tended to have greater total number of sperm with functional membrane than semen collected with EE (P = 0.02; P = 0.003; P = 0.02; P = 0.02; P = 0.06, respectively). After the freezing-thawing process, sperm collected with TUMASG had a greater quality of the motility, a total number of motile sperm and sperm with progressive motility, a greater percentage of motile sperm and sperm with functional membrane, and tended to have a greater total number of sperm with normal morphology than semen collected with EE (P = 0.04; P = 0.04; P = 0.03; P = 0.02; P = 0.04; P = 0.06, respectively). In Study 1 sperm collected with TUMASG had greater cryoresistance for almost all the variables considered than when it was collected with EE. In Study 2, the number of electrical pulses and the sum of pulses∗voltage were greater with EE than with TUMASG (P < 0001; P = 0.0002, respectively). There were no differences in any sperm variable in fresh or thawed samples collected with TUMASG or EE. However, the other seminal characteri

Chicken semen cryopreservation is a tool for programs of genetic diversity management and endangered breeds conservation. Due to physiological features, the fertility rates of cryopreserved poultry sperm are lower than mammal species. Thus, improvement of the semen cryopreservation methods is required. A first study was performed by a 2 × 2 factorial design consisting of 2 methods of adding the cryoprotectant [Direct or Diluted (mixed with extender medium)] and 2 cryoprotectants (glycerol and dimethylacetamide). Then sperm quality indicators were evaluated after freezing. A second study with a 2 × 2 design was conducted to evaluate the effectiveness of bovine serum albumin (BSA) on the optimization of 2 different extenders (Lake and Animal Sciences Group [ASG]). Viability and motility variables were evaluated before and after freezing. There was no significant difference in sperm viability and motility variables between Direct or Diluted methods. Supplementation of extenders with BSA improved most of the sperm motility variables in both extenders before and after freezing. Progressive sperm, non-progressive sperm before freezing, and all post-thaw sperm motility parameters, except amplitude of lateral head displacement and beat-cross frequency, were increased in BSA-supplemented extenders (P < 0.05), and BSA improved sperm viability in ASG extender after thawing (P < 0.05). After thawing, the interaction between extender and BSA (P < 0.05), eliminated the differences between the 2 BSA-supplemented media in curvilinear velocity, straight-line velocity, average path velocity, and amplitude of lateral head displacement which were higher in non-supplemented ASG extender than nonsupplemented Lake medium. In conclusion, the direct or diluted methods of adding glycerol or dimethylacetamide, did not significantly affect the post-thaw sperm characteristics. BSA positively affected most of the post-thaw sperm motility indicators regardless of the type of extender and resulted

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.

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 < 0.05) in the conventional frozen-thawed sperm than vitrified-warmed sperm. Plasma testosterone concentrations varied from 0.0 ng/mL to 7.7 ng/mL. For epididymal sperm, sperm motility and viability following thawing were greater in vitrified-warmed samples than conventionally-frozen samples; however, small sample numbers precluded statistical analysis. When considered together, these results indicate vitrification may be a possible alternative for wolf sperm cryopreservation.

The present study investigates the efficacy of dimehtlyformamide (DMF) as a permeable cryoprotectant and its effect on quality and fertility of Indian red jungle fowl sperm. Semen was collected from eight mature roosters, pooled, divided into five aliquots and diluted with red fowl extender having DMF (0%, 4%, 6%, 8% and 10%). Diluted semen samples were cooled from 37 °C to 4 °C, 20% glycerol added to control (0% DMF), equilibrated for 10 min and filled in 0.5 mL French straws, kept over liquid nitrogen vapors for 10 min and plunged into liquid nitrogen. Sperm motility, plasma membrane functionality, viability and acrosome integrity were assessed at post dilution, cooling, equilibration and freeze-thawing stage of cryopreservation. Cryopreservation stages had negative effects (P < 0.05) on semen quality parameters. Percentages of sperm motility, plasma membrane functionality, viability and acrosome integrity were recorded highest in extender having 8% DMF at post-dilution, cooling, equilibration and freeze-thawing stage. Fertility results after artificial insemination were recorded higher (P < 0.05) with 8% DMF compared to 20% glycerol. Dimehtlyformamide (8%) in red fowl extender improves the post thaw semen quality and fertility in Indian red jungle fowl and can be used effectively to avoid the contraceptive effects of glycerol.

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; P > 0.05). Significant differences were seen in this respect, however, in the bucks (4.3 ± 0.8 ng/mL and 6.9 ± 0.9 ng/mL; P < 0.05). In the rams, TP treatment increased (P < 0.05) 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 (P < 0.05) 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 (P < 0.05) than did controls. In the rams, treatment with TP led to a reduction in all fresh sperm head morphometric variables (P < 0.05). Freezing-thawing further reduced (P < 0.05) all morphometric variables in both the control and treated groups. In the bucks, treatment with TP increased (P < 0.05) the length, area, and perimeter of fresh sperm cells, unlike that seen in ram sperm. Compared with fresh sperm, freezing-thawing led to reduced (P < 0.05) 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

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 = 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% ± 12.1) than VF cryopreserved sperm control (16.1% + 6.2). Supplementing valine to the freezing extender doubled (P = 0.026) the fertility rate of VF (32.6% ± 12.2) compared wit

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.

Density-gradients centrifugation (DGC) and filtration columns (FC) are used to separate deformed or dead sperm, debris, and other cells that may negatively affect the fertilizing capacity of sperm in fresh, chilled and frozen/thawed semen. The present study was conducted to evaluate the suitability of DGC (BoviPure®, Percoll® and Accudenz®) and FC (Sephadex G-15®) sperm selection procedures for fresh-extended and cold-stored ram semen by assessment of post-treatment sperm quality variables. Twenty normospermic ejaculates from ten adult Merino rams were used. Sperm concentration of recovered cells was greater (P < 0.001) after BoviPure treatment than other procedures in both fresh and cold semen. With the Sephadex method, there were more desirable values than with use of DGC procedures in several sperm motility variables measured by using the CASA system. In non-refrigerated semen samples, the percentage of progressive sperm motility (%PSM) after Sephadex filtration was greater (P < 0.05) than after BoviPure treatment; the straightline velocity (VSL) value after Sephadex filtration was greater (P < 0.01) than after Accudenz treatment; the amplitude of lateral head displacement (ALH) after Sephadex and Accudenz treatment was less than non-filtered semen (P < 0.001) and after Percoll (P < 0.01) and BoviPure (P < 0.05) treatments. In cold-stored semen samples, the %PSM after Sephadex filtration was greater than non-filtered (P < 0.05) semen and after BoviPure (P < 0.05), Percoll (P < 0.05) and Accudenz (P < 0.001) treatments. It is concluded that Sephadex column filtration can be used to select ram sperm in non-refrigerated and cooled semen, because percentage progressively motile sperm and some other sperm motility characteristics are greater with use of this techniques as compared with use of DGC methods.

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).

Seminal plasma removal is routine in goat sperm cryopreservation protocols. The classical washing procedure designed to accomplish this usually leaves the pellet resulting from use of this procedure contaminated with dead sperm, debris, and cells other than sperm. This contamination negatively affects viability of sperm after cryopreservation. The present research was conducted to compare the effect on chilled and frozen-thawed goat sperm of the classical washing method to that of a selective washing method involving density gradient centrifugation (DGC). In the first experiment, sperm variables were measured in freshly collected sperm, and again after its washing with both methods and chilling at 5 °C for 0, 3, 24, 48, 72 or 96 h. The DGC-washed sperm had greater (P < 0.01) straight line velocity (VSL), average path velocity (VAP) and progression ratio values at all chilling times. The amplitude of lateral head displacement (ALH) was, however, less (P < 0.001) in the DGC-washed sperm at all chilling times. There was a negative correlation (P < 0.05) between ALH and VSL. In the second experiment involving the freezing-thawing of sperm washed by using either method, aliquots were post-wash diluted with a Tris-citric acid/glucose/egg yolk/glycerol-based medium and frozen in liquid nitrogen for 5 days. After thawing, neither the VCL, VSL nor VAP of the DGC-washed samples were affected, whereas the traditionally washed samples had less motility. In conclusion, the use of DGC was associated with enhanced sperm motility variables after chilling and freezing-thawing. This procedure would, therefore, be a useful means of removing seminal plasma from goat semen and obtaining greater quality sperm for insemination purposes. © 2017

The fertilizing capacity of pure, fresh avian semen may disappear in just half an hour, hindering its successful use in artificial insemination (AI) projects. Longer storage requires the use of infra-physiological temperatures and of semen diluents that help preserve the spermatozoa but that do not interfere with their fertilizing capacity. This study examines the effect on sperm quality of storing red-legged partridge sperm for 3 h at 5°C with 2 different semen extenders: 1) a medium referred to as L&R-84, composed of sodium glutamate, glucose, magnesium acetate, potassium acetate, and polyvinylpyrrolidone, and 2) Lake 7.1 medium, composed of sodium glutamate, glucose, magnesium acetate, potassium citrate, and N,N-Bis(2-hydroxyethyl)taurine (BES). Extending with L&R-84 returned better curvilinear velocity (P < 0.01), straight-line velocity (P < 0.01), average path velocity (P < 0.01), linearity (P < 0.05), straightness (P < 0.05), and wobble (P < 0.05) values, while extending with the Lake 7.1 medium was associated with higher percentages (P < 0.001) of motile sperm. The fertility rate was higher (P < 0.05) when birds were inseminated with L&R-84-extended sperm than with Lake 7.1-extended sperm. The mean number of penetrations of perivitelline layer samples (taken from above the germinal disc) was also higher for the L&R-84-extended sperm (P < 0.05). These results show L&R-84 can be recommended as an extender for red-legged partridge semen to be stored for at least 3 h at 5°C.

A method for cryopreserving wild ibex sperm at high cooling rates was developed. To design a freezing solution based on Tris, citric acid, and glucose (TCG), two preliminary experiments were performed using glycerol (GLY) and dimethyl sulfoxide (DMSO) at different concentrations (5%, 10%, 20%). The 10% GLY + 10% DMSO combination reduced (P < 0.05) frozen-thawed sperm motility, which reached a minimum when 20% GLY + 20% DMSO was used. In the second experiment, sperm tolerance to three sucrose concentrations was evaluated (100-mM sucrose, 300-mM sucrose, 500-mM sucrose). Frozen-thawed sperm motility and sperm viability decreased (P < 0.05) at concentrations above 300 mM. The ultrarapid cooling procedure finally used involved a TCG egg yolk (ey)-based extender with 100-mM sucrose, either alone or with 5% GLY with or without BSA. Two warming procedures (37 °C vs. 60 °C) were also evaluated. The TCG ey with 100-mM sucrose but without GLY/BSA returned the best sperm quality variables. Slow warming at 37 °C strongly affected (P < 0.05) sperm motility and viability in all groups. Sperm selection by density gradient centrifugation produced no motile sperm when slow warming was performed. In contrast, when fast warming was used, sperm selection increased (P < 0.05) percentage of motility, viability, and the percentage of sperms with intact acrosomes. Heterologous in vivo fertilization involving domestic goats was performed to evaluate the in vivo fertilization capacity of the ultrarapidly cooled cryopreserved sperm (in TCG-ey + 100 mM sucrose), with warming undertaken at 60 °C. Inseminations of domestic goats resulted in three pregnancies (3 of 16, 18.7% fertility). In conclusion, ibex spermatozoa are strongly sensitive to high concentrations of permeable cryoprotectants and sucrose. However, the combination of ultrarapid cooling, using TCG-ey + 100-mM sucrose, and fast warming at 60 °C, followed by sperm selection by density gradient centrifugation to collect the motile sper

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..

Glycerol (GLY) and egg yolk (EY) are good cryoprotectants of avian and mammalian sperm, but in birds, they strongly inhibit the eventual fertilization of ova. Using the sperm penetration (SP-holes) assay and fertility trials, the present study investigates (1) thepossible mechanism by which this contraceptive effect occurs in chickens and (2) the maximum concentrations of GLY and EY tolerated by fresh rooster sperm. Seventy Black-Barred Andaluza hens (five per treatment) were inseminated four times (twice per week) with 0.1mL of fresh semen from roosters of the same breed diluted 11 (vv) with Lake and Ravie medium containing different concentrations of GLY or EY. No adverse effects on acrosome integrity, sperm motility, or viability were seen with any concentration of GLY or EY. The number of SP-holes on perivitelline layer samples taken from abovethe germinal disc became progressively lower at GLY concentrations of 1.5% or greater (P>0.05). No holes caused by sperms were seen in unfertilized eggs. The corresponding fertility results showed similar reductions when the GLY concentration was 1.5%or greater. No changes in the number of SP-holes were seen with increasing EY concentrations (0%-7.5%), nor were any differences in fertility observed, except for a reduction when 15% EY was used. The results therefore reveal that GLY affects the transit of sperms through the oviduct in their attempt to reach the infundibulum area, limiting their access to the ovum perivitelline layer. Egg yolk had no such effect, nor did it influence acrosome reaction capacity; its mechanism of contraceptive action therefore remains unknown. The maximum GLY and EY concentrations tolerated by the rooster sperm were 0.75% and 7.5%, respectively. © 2015 Elsevier Inc.

This study compares the effectiveness of two methods of sperm selection - Capripure® density-gradient centrifugation (DGC) and dextran swim-up (DSU) - in semen samples from Iberian ibex (Capra pyrenaica) and European mouflon (Ovis musimon). During the increasing photoperiod, Capripure® DGC improved the percentage of sperm with progressive motility (P<0.05) in ibexes, and selected 60.6% of the initial total number of spermatozoa contained in the ejaculate samples. In mouflon, Capripure® DGC selection was unaffected by photoperiod, had no influence on any sperm variable, and selected 47.8% of the initial total number of mouflon spermatozoa in ejaculate samples. Photoperiod had no influence on the effectiveness of DSU in either ibexes or mouflons. In the ibexes, DSU reduced (P<0.05) the percentage of sperm cells with morphological abnormalities, but only selected 11.3% of the initial total number of spermatozoa in ejaculate samples. In the mouflons, DSU had no significant influence on any sperm variable, and selected 27.8% of the initial total number. Capripure® DGC improved ibex and mouflon sperm motility (P<0.05) following 30min and 2h of post-centrifugation, stress-inducing incubation, respectively. In both ibexes and mouflon, sperm cells showing non-progressive motility were found after only 20h of post-centrifugation incubation following Capripure® DGC selection. In conclusion, Capripure® DGC would seem a useful method for selecting the best spermatozoa from both ibex and mouflon ejaculates. © 2014 Elsevier B.V.

Some breeds of goats originating from or adapted to subtropical latitudes display seasonal variations of their annual rhythm of reproduction. The reproductive seasonality causes a seasonal production of goat-derived products affecting producers, goats' meat and milk industry and consumers. A good knowledge of the annual rhythm of reproduction, as well as the identification of the major environmental cues controlling this rhythm in both males and females, is necessary to draw new and sustainable techniques to induce a synchronized reproductive activity outside the natural breeding season and therefore to increase the out-of-season availability of the goat-derived products. This knowledge allows to control caprine reproduction without the use of exogenous hormones, and to develop techniques adapted to the environmental, economic and social characteristics of the breeding systems. Thus, in this review we will describe how photoperiodic treatments can be used to induce the sexual activity of male goats during the period of sexual rest. Then, we will describe how these photo-stimulated males were used to induce and synchronize the sexual activity of anestrous goats maintained in intensive or extensive management conditions by means of the male effect. In subtropical latitudes, the use of the male effect performed by using photo-stimulated bucks constitutes an original and sustainable way to control goats' reproduction. © 2014 Elsevier B.V.

The aim of this work was to evaluate the protective effect of catalase (CAT) on frozen/thawed ibex epididymal sperm recovered post mortem, and to detect any harmful effect this might have on sperm fertilisation capacity. Epididymal spermatozoa were diluted using a Tris-citric acid-glucose medium (TCG) composed of 3.8% Tris (w/v), 2.2% citric acid (w/v), 0.6% glucose (w/v), 5% glycerol (v/v), and 6% egg yolk (v/v). Sperm masses from the right epididymis were diluted with TCG medium, while those from the left were diluted with TCG medium supplemented with 200. IU/mL CAT. Heterologous in vitro fertilisation (IVF) was used to assess the fertilisation capacity of this sperm. The addition of CAT to the extender did not improve frozen/thawed sperm variables. Moreover, a reduced fertilisation capacity was detected sperm diluted with TCG provided 25.5% 2PN zygotes, while just 13.2% was recorded for that diluted with TCG-CAT (P<. 0.01). The percentage of cleaved embryos at 48. hpi was higher (P<. 0.01) with the TCG sperm than with the TCG-CAT sperm (16.7% vs. 7.6%). The use of 200. IU/mL CAT as an additive cannot, therefore, be recommended for the preservation of ibex epididymal sperm. Other antioxidants should, however, be tested in both this and related wild mountain ungulates. © 2014 Elsevier Inc.

This study examines whether photoperiod and/or melatonin treatments can improve sperm variables outside the breeding season in the Iberian ibex-a model species for wild mountain ruminants-thus helping in the collection of high quality sperm beyond the normal breeding season for depositing in genetic resource banks. Adult Iberian ibex males (. n=. 17) were divided into four treatment groups (1) controls under the natural photoperiod (control group; n=. 4), (2) treatment with melatonin implants on December 22nd, February 22nd and April 22nd (group WS-M; n=. 5), (3) treatment with short photoperiod cycles, i.e.;2 months of long days followed by melatonin implants (to emulate 2 months of short days) throughout the year (group PHPld. +. M; n=. 4), and (4) treatment with melatonin implants on June 22nd and August 22nd (group SS-M; n=. 4). The interaction treatment x season had a strong influence on testis size (. P<. 0.05), the size of the seminal vesicles (. P<. 0.001), the percentage of abnormal sperms (. P<. 0.05), and percentage non-progressive (P. <. 0.05) and progressive (. P<. 0.001) sperm motility. In groups WS-M and PHPld. +. M, the normal springtime physiological reductions in testis size, non-progressive sperm motility and acrosome integrity were prevented. The values for the studied sperm variables were, however, reduced in the natural breeding season at the end of the experimental period in group PHPld. +. M, although not in group WS-M. The pattern of melatonin administration in group SS-M conferred no advantages on reproductive functionality. These results suggest that lengthening the short day period after the winter solstice (the WS-M treatment) extends reproductive activity in this species, allowing good quality sperm to be recovered for conservation purposes during the non-breeding season. © 2013 Elsevier B.V.

Mouflon and domestic Manchega sheep differ in the timing of their reproductive season under natural photoperiod (NP) conditions. This study examines whether they also differ in their reproductive responses to artificial photoperiod cues. For this, mouflons (n= 24) and ewes (n= 24) were exposed between the winter and summer solstices to artificial long days (LD 16. h light/day), to short days (SD) simulated via the use of melatonin implants, or to NP conditions (controls), and their ovulatory activity monitored. The effects of these treatments on annual changes in prolactin concentration were also recorded. In the LD mouflon ewes, the offset and onset (7 March ± 5 and 2 October ± 4, respectively) of cyclic ovulatory activity occurred earlier (P< 0.001) than in the NP animals (26 April ± 6 and 20 October ± 2, respectively), but no differences were seen (P> 0.05) between the SD and NP mouflon ewes in either the onset of anoestrus (12 May ± 6 and 26 April ± 6, respectively) or the onset of subsequent ovulatory activity (13 October ± 8 and 20 October ± 2, respectively); however the duration of the anoestrus period was significantly reduced in the SD. In LD Manchega ewes, the onset of anoestrus was advanced (2 February ± 5 vs 15 March ± 11), but ovulatory activity started at the same time as in NP Manchega ewes (16 July ± 4 vs 5 July ± 8). In the SD Manchega ewes, two animals showed continuous cyclic ovulatory activity over the course of the experiment while the remainder entered anoestrus two months later (16 May ± 6, P< 0.001) than their NP counterparts. In these SD ewes, the onset of cyclic ovarian activity was very variable. An annual rhythm of plasma prolactin concentration was seen in both the mouflon and Manchega ewes under all three photoperiod conditions. However, the amplitudes of the changes seen in prolactin concentration were smaller in both the LD and SD animals than in the corresponding NP animals (P< 0.001). In conclusion, the results show that these two t

The present work examines the relationship between seasonal changes in testicular function, accessory gland size, and horn growth in Iberian ibexes, as well as the relationship between these changes and the resistance of ibex spermatozoa to freezing-thawing. The size of the bulbourethral glands and seminal vesicles showed pronounced monthly variation (P < 0.001), which was correlated positively with the plasma testosterone concentration (P < 0.001) and scrotal circumference (P < 0.001). The size of the accessory sex glands peaked during the autumn. Overall, semen quality was markedly improved during autumn and winter. When horn growth was at a minimum during autumn and winter, semen quality and accessory gland size were all increased compared to in spring and summer. However, increased plasma testosterone levels in the autumn were strongly associated with reduced sperm freezability; thus, the cryosurvival of spermatozoa collected during the autumn was poorer than at other times of the year. In winter, however, when the plasma testosterone concentration fell to baseline, the negative effects of cryopreservation on the percentage of motile spermatozoa and on the integrity of the plasma membrane of frozen-thawed sperm cells were significantly less intense (P < 0.05). These findings show a clear relationship between the functional and morphological status of the different parts of the reproductive tract that optimises reproductive function during the breeding season in the ibex male. They also show that winter is the most suitable season for the collection and cryopreservation of ibex spermatozoa. © 2011 Elsevier Inc.

A sperm cryopreservation protocol requiring dimethylacetamide (DMA, 6%) as a cryoprotectant was optimized via assays involving different prefreezing equilibration times (1, 10, 30, 60, and 120 min at 5°C) and different freezing rates achieved by the following 1) using nitrogen vapor to reduce the temperature from 5°C to -85°C at 10°C/min (slow freezing rate); 2) using a biological freezer unit in a 2-step method to reduce the temperature from 5°C to -35°C at 7°C/min and then from -35°C to -140°C at 60°C/min (medium freezing rate); or 3) using a biological freezer unit in a 1-step freezing method to reduce the temperature from 5°C to -180°C at 60°C/min (rapid freezing rate). Heterospermic semen samples from chicken breeds raised as part of a Spanish genetic resource conservation program were used in all assays. The 1-min equilibration treatment was associated with a lower percentage of viable thawed spermatozoa than the 30-min treatment (P < 0.05). The remaining sperm variables studied were not affected by equilibration time. The medium-rate 2-step freezing method was associated with a higher percentage of motile spermatozoa after thawing and with greater acrosome integrity (P < 0.05) than the slow nitrogen vapor or rapid 1-step methods. Thawed sperm movement quality and plasma membrane integrity (as assessed by the hypoosmotic swelling test) were better (P < 0.05) in samples frozen by the mediumrate 2-step freezing method than in those subjected to the slow nitrogen vapor method. Fertility was not influenced by freezing method, although that achieved with the medium rate 2-step freezing method showed a trend toward being greater than that achieved with the rapid 1-step method (P = 0.07). Together, the present results suggest that slow cooling rates are not recommendable when using dimethylacetamide. The 2-step freezing method may be useful in the establishment of a germplasm bank for Spanish chicken breeds. © 2011 Poultry Science Association Inc.

This study examines the patterns of melatonin secretion during postnatal development in wild (mouflon; n = 7) and domestic (Spanish Merino; n = 6) female lambs under their natural photoperiod conditions. The aim was to determine whether these types of sheep which differ in the timing of puberty, also differ in the establishment of daily melatonin secretory rhythms and/or in the nocturnal plasma melatonin secretion. In addition, the time when the lambs first reached nocturnal levels of melatonin similar to adults was also evaluated. For this purpose, the melatonin secretion in lambs was compared with those of their mothers. A day/night (D/N) difference in plasma melatonin concentration was noticed as early as 1 day after birth in the Merino female lambs (D 5.9 ± 1.0 pg/ml compared to N 22.0 ± 3.3 pg/ml; p < 0.05), and by 1 week after birth in the mouflon female lambs (D 4.9 ± 0.3 pg/ml compared to N 56.9 ± 15.3 pg/ml; p < 0.05). An effect of the genotype (p < 0.05) and age (p < 0.01) was detected on mean nocturnal plasma melatonin concentrations, which were lower in the Merino than in the mouflon lambs. Night-time plasma concentration of melatonin was also high in mouflon than in Merino mothers (p < 0.05). No differences were detected between the wild genotype and the domestic one in the time when the lambs first reached nocturnal levels of melatonin similar to those of their mothers. © 2009 Elsevier B.V. All rights reserved.

Extenders containing low concentrations of egg yolk are recommended for cryopreserving ibex spermatozoa. However, the phylogenetic relationship of the Spanish ibex (Capra pyrenaica) with domestic goats suggests that phospholipases in the seminal plasma may have a negative effect on the response to freezing-thawing when egg yolk-based diluents are employed. The aim of the current work was to determine how seminal plasma removal from Spanish ibex semen, collected by electroejaculation over a period of 1 yr, affects its response to freezing-thawing. Semen was collected from six adult ibexes maintained in captivity. The negative effects of freezing-thawing on the quality of sperm motility and on the integrity of the acrosome and plasma membrane were more serious in the nonwashed semen samples than in those from which the seminal plasma had been removed (P < 0.01, P < 0.05, and P < 0.05 respectively). The beneficial effect of removing the seminal plasma was particularly noticeable during the time of decreasing photoperiod. This suggests that ibex semen shows increased phospholipase activity during the rutting season. © 2010 Elsevier Inc. All rights reserved.

Ibex spermatozoa can be successfully frozen using glycerolated media. However, no information is available regarding the most effective method of glycerol addition in this species. The aim of the present work was to evaluate the effect of the glycerolization temperature on the response to freezing-thawing of ibex spermatozoa collected by electroejaculation. The effect of the interaction glycerolization temperature x plasma testosterone concentration was also evaluated. The spermatozoa used in this work came from six adult ibexes maintained in captivity. Each ejaculate was divided into two aliquots in a Tris-egg yolk-based medium. One fraction was subjected to single step dilution with 5% glycerol at room temperature (23°C). The other fraction was diluted in two steps, first by dilution at room temperature with an extender identical to that described above but without glycerol, followed by the addition of glycerol after cooling to 5°C. The glycerolization temperature did not affect any sperm variable after thawing. Heterospecific artificial insemination involving domestic goats, revealed no differences in the fertilization rate for frozen-thawed spermatozoa diluted by the one or two step procedures (18.2% vs. 20.0%). The interaction glycerolization temperature x plasma testosterone concentration had no affect on the freezing-thawing of the sperm cells. The results revealed, however, that high plasma testosterone levels during the pre-rutting season may interfere with the freezing-thawing process, having a negative influence on sperm cryosurvival. © 2010 Elsevier Inc.

The season may affect the values of fresh semen variables and therefore influence the success of cryopreservation. The aim of this study was to improve the evaluation of seasonal changes in semen quality in Spanish Black Castellana roosters maintained under natural environmental conditions. Semen was collected from 11 Black Castellana roosters (housed under natural photoperiod and temperature conditions) by massage twice every month for 12 mo. In addition to determining ejaculate volume, sperm concentration, and sperm motility (the classic sperm variables), we used the hypo-osmotic swelling test to examine the membrane integrity of the spermatozoa. Further, morphological abnormalities and acrosome integrity were assessed via aniline blue staining. Semen volume (P < 0.05), sperm concentration (P < 0.01), and the percentage of spermatozoa with an intact acrosome (P < 0.01) were significantly affected by the season of the year. The annual profile of the percentage of spermatozoa showing acrosome integrity followed a trend roughly parallel to annual variations in temperature (Spearman rank correlation = 0.77, P < 0.01). According to the hypo-osmotic swelling test, membrane integrity fell in July (P < 0.05 compared with all other months), the month of highest temperatures. Aniline blue staining and the hypo-osmotic swelling test provide an easy and useful means of evaluating sperm abnormalities, including acrosome morphology and membrane integrity, and could be easily introduced into routine avian semen quality assessments. The results show that high semen quality is associated with long day photoperiods. Extreme heat or cold appear to exert a negative influence on sperm quality. © 2009 Poultry Science Association Inc.

Two experiments were conducted to determine the effects of supplementing hCG at insemination on the luteal function and reproductive performance in estrus-induced mature Manchega ewes. The first experiment was carried out under field conditions with 1560 ewes on 27 farms. After estrous synchronisation with progestagen sponges and eCG, cervical inseminations were performed between October and February (breeding season) on 665 ewes on 12 farms and between March and June (non-breeding season) on 895 ewes on 15 farms. In each period and within each farm, approximately half of the ewes received an i.m. injection of 500 IU hCG at the time of insemination. The other half remained untreated and served as controls. Neither fertility (hCG 44.2%; control 42.0%) nor prolificacy (hCG 1.57; control 1.54) were affected by hCG treatment. However, on farms where fertility in the control group was persistently low, fertility of the hCG treated ewes was increased by 7.6% relative to those not treated (P < 0.1). The second experiment was carried out on 64 ewes from an experimental flock. Estrus and ovulation were induced in all ewes as in experiment 1. Thirty-two ewes received a single i.m. injection of 500 IU hCG at the time of intrauterine insemination, while the other half were not treated. Plasma progesterone (P 4) concentrations was measured every 2 days over a 28 day period following insemination. Progesterone secretion in the inseminated pregnant ewes was not modified by the hCG administration. However, a tendency (P < 0.1) for higher P 4 concentrations from days 8 to 14 was observed in those ewes that had been treated with hCG, but that did not conceive at AI. Pregnancy (62.5% and 59.4%), fertility or number of ewes lambing/ewes inseminated (56.3% and 50.0%) and prolificacy (1.56 and 1.50) of the inseminated pregnant ewes did not differ between the hCG-treated and control ewes. In ewes that failed to conceive at AI and were mated at the next oestrus (return cycle), fertil

Seasonal variations in the horn development and testicular activity of the Spanish ibex (Capra pyrenaica hispanica) (n = 6) and European mouflon (Ovis orientalis musimon) (n = 5) were monitored to determine the role of increasing testosterone concentration on the arrest of horn growth during the rutting season. Marked seasonal variations in the rate of horn growth (P < 0.01) and testicular activity (P < 0.001) were seen in both species, although the magnitude and timing of these changes were different (P < 0.01). Horn growth rate was inversely correlated to seasonal levels in testosterone plasma concentration in both species (ibex R = -0.45, P < 0.01; mouflon R = -0.51, P < 0.01). In the mouflon, the increase in plasma testosterone concentration recorded in September (P < 0.05 compared with the lowest concentration) coincided with a significant reduction in horn growth (P < 0.05). In the ibex, the increase in plasma testosterone concentration in October (P < 0.05 compared with the lowest concentration) was associated with a significant arrest of horn growth in November (P < 0.05). These results appear to support the hypothesis that high peripheral plasma levels of testosterone are linked with the seasonal arrest of horn growth during the rutting period. © 2006 Elsevier B.V. All rights reserved.

The horns are secondary sexual characteristics used by males of many ungulate species for intra-sexual fights during the rut. Thus, the dominant males with most developed horns are naturally selected for reproduction. Several studies have suggested that the quality of the horn, in many wild ruminants, may be correlated with semen quality. The aim of the present study was to determine whether inter-individual differences in levels of horn asymmetry and horn size are related to differences in sperm quality in a wild population of Spanish ibex by the assay of epididymal spermatozoa collected postmortem. In order to test this hypothesis we collected morphometric horns data from a total of 59 mature males (9-15 years of age) that were legally hunted during rutting season. The testicles were recovered, and the collection of epididymal spermatozoa was done at different times after death (2-60 h). The percentage of motile spermatozoa, motility rate, plasma membrane integrity, sperm viability, sperm morphology, and acrosome integrity were evaluated. Our findings showed that viable epididymal spermatozoa may be retrieved from dead animals many hours after death. However, sperm parameters were affected by the elapsed time between the death of the animal and spermatozoa collection. The study revealed that the horn quality was firstly associated with sperm motility. © 2006 Elsevier B.V. All rights reserved.

Tris-egg yolk based diluents provide adequate cryoprotection for the sperm of most wild species in which they have been tested. The objective of the current study was to evaluate various Tris-based diluents containing different concentrations of egg yolk, for the fertilizing ability of epididymal spermatozoa of the Spanish ibex (Capra pyrenaica) after freezing and thawing. For this purpose, we used heterologous in vivo fertilization by intrauterine insemination of domestic goats (Capra hircus). In Experiment 1, a Tris-citric acid-glucose (TCG) diluent containing 6% (v/v) egg yolk and a TCG extender containing 20% egg yolk were compared. In Experiment 2, a TCG-6% egg yolk extender was compared with Triladyl-20% egg yolk. Diluted samples were cooled slowly to 5 °C over 1 h and equilibrated at that temperature for 2 h. At that point, aliquots of samples were loaded into 0.25 ml straws, and frozen in nitrogen vapor for 10 min. The fertility of spermatozoa frozen in TCG-6% egg yolk was higher (P < 0.05) than for those extended with TCG-20% egg yolk, and tended to be higher than for those frozen with Triladyl-20% egg yolk. >From the results of this study, the use of Tris-based extenders containing low concentrations of egg yolk (6%) is recommended for cryopreserving Spanish ibex epididymal spermatozoa. © 2006 Elsevier Inc. All rights reserved.

As a consequence of increasing limitations to maintaining genetic variability in endangered wildlife species, methods of assisted reproduction widely used in domestic animals are being applied to nondomestic species. However, practical efforts have met limited success to date. The Spanish ibex (Capra pyrenaica hispanica) is a wild caprine originating exclusively in the mountains of Spain. This study was designed to evaluate the fertilizing capability of cryopreserved Spanish ibex epididymal spermatozoa recovered postmortem. For this purpose, we have previously evaluated the effect of time elapsed between death and sperm recovery on spermatic parameters, and the fertilization ability of frozen-thawed spermatozoa using heterologous in vivo fertilization by intrauterine insemination in domestic goat (Capra hircus). The time of death significantly affected most sperm quality parameters (motility, viability and intact acrosomes). The fertility obtained by heterologous artificial insemination was 18.7%, and only goats inseminated with spermatozoa recovered within 8 h after death became pregnant. Our findings showed that heterologous in vivo fertilization is a useful method to evaluate the fertilizing capacity of sperm samples in rare or wild species. Sperm samples, with verified fertilization ability in the previous trial, were used to inseminate a total of six ibex females. Inseminations resulted in one pregnancy. The study demonstrated for the first time the feasibility of applying artificial insemination in Spanish ibex. © 2005 Elsevier Inc. All rights reserved.

This study compares morphological changes, performed with real-time transrectal ultrasonography, of testis and accessory glands and seasonal endocrine changes in a wild (Mouflon) and a domesticated (Spanish Merino) breed of sheep. In Mouflons, the maximum plasma testosterone concentrations, testicular diameter and vesicular gland size occurred synchronously during autumn (P < 0.001). In Merino rams the highest circulating levels of testosterone (P < 0.05) and maximum testicular diameter (P < 0.001) occurred during summer, with no seasonal variations in vesicular and bulbourethral glands. The seasonal changes in plasma concentrations of prolactin were not correlated with annual variations in testicular and glandular size in neither wild nor domestic species. No differences were observed between both species for the seasonal pattern of prolactin secretion and mean amplitude of melatonin. Wide differences in reproductive patterns between wild and domestic types of rams do not appear to be attributed to seasonal changes in prolactin and melatonin secretion. © 2005 Elsevier Inc. All rights reserved.

Annual variations in the growth of horns, and their correlation with seasonal changes of testicular size, and prolactin (PRL) and melatonin secretion were monitored in six pubertal mouflon rams living in their original latitude (40°N). Mouflons born and maintained under captive conditions were classified in two age classes sub-adult (2 years; n = 3) and adult (≥3 years; n = 3). The rate of horn growth was greater (P < 0.001) in sub-adult than in adult mouflon rams. Horn growth was influenced by season in both adult and sub-adult mouflons (P < 0.05) with largest monthly growth occurring in spring and summer. Seasonal variations of plasma PRL concentrations were correlated with horn growth in adult, but not in sub-adult mouflon rams. The rate of horn growth was inversely correlated with testicular size (r = -0.5, P = 0.07). Seasonal changes in the amplitude of the daily melatonin rhythm in solstices and equinoxes were observed, which were not correlated with variations in the rate of horn growth. These results provide support for a possible role of PRL in the control of growth of horns in the adult mouflon. © 2004 Elsevier B.V. All rights reserved.

The aim of this study was to determine, for goats, the effects of daily doses of GnRH antagonist on ovarian endocrine and follicular function. Ten does were given 45mg FGA intravaginal sponges and then five were treated with daily injections of 0.5mg of the GnRH antagonist Teverelix for 11 days from 2 days after the day of sponge insertion, while five does acted as controls. Pituitary activity was monitored by measuring plasma FSH and LH daily from 2 days before the first GnRH injection to Day 12. Follicular activity was determined by ultrasonographic monitoring and by assessing plasma inhibin A levels during the same period. In treated does, the FSH levels decreased linearly (0.8±0.1ng/ml to 0.5±0.1ng/ml, P<0.01) and remained lower than the mean concentration in control goats (0.8±0.1ng/ml, P<0.005). LH levels were also lower during the period of antagonist treatment (0.6±0.2ng/ml versus 0.4±0.1ng/ml, P<0.0005). During GnRH antagonist treatment, there was a significant decrease in the number of large follicles (≥6mm) from Day 3 of treatment (1.2±0.6, P<0.0001), with no large follicles from Day 9. The number of medium follicles (4-5mm in size) also decrease during the period of treatment (4.2±0.7 to 1.0±0.6, P<0.0001), leading to a significant decrease in inhibin A levels when compared to the control (143.7±31.3pg/ml versus 65.2±19.1pg/ml, P<0.00005). In contrast, the number of small follicles (2-3mm) increased in treated goats from Day 4 of treatment (9.6±2.9 to 20.2±6.3, P<0.005). Such data indicate that GnRH antagonist reduced plasma levels of FSH and LH with suppression of the growth of large dominant ovarian follicles and a two-fold increase in number of smaller follicles. The results confirm that GnRH antagonist treatment can be used in goats to control gonadotrophin secretion and ovarian follicle growth in superovulatory regimes. © 2003 Elsevier Inc. All rights reserved.

The aims of this study were to examine whether mouflons exposed to constant long and short day photoperiods are able to exhibit an annual cycle of hair growth and moult, and prolactin (PRL) secretion. Mouflon ewes were assigned to three groups of treatment. Ewes were maintained, either under natural photoperiod (control, n=9), or received a series of subcutaneous melatonin implants from December to April (n=8), or were exposed to a constant long day photoperiod (16-hlight8-hdark; 16L8D) during 18 months (n=7). Blood was collected weekly to determine PRL concentrations, and hair samples were clipped weekly from the base of the neck to measure the length of predominant hair. Under constant long days and with melatonin implants, mouflons expressed an annual rhythm of PRL secretion, even though these treatments modified the times of rise or falling of PRL concentrations throughout the year. Hair growth initiation was almost coincident with the summer solstice in both control and melatonin-implanted mouflons but occurred two months earlier in long day hold mouflons (P<0.001). Long day hold mouflons had a lower hair growth rate than control and melatonin-implanted mouflons (P<0.001), and at the end of the experiment, a shorter hair length (3.4±0.24cm; P<0.01) than control (4.3±0.17cm), and melatonin-implanted mouflons (4.2±0.12cm). Our data support the conclusion that in mouflon, an endogenous circannual rhythm of PRL secretion exists, and that the seasonal cycle of hair growth and moult appears to depend, at least in part, on circulating levels of PRL. © 2004 Elsevier Inc. All rights reserved.

Daily transrectal ultrasonographies were conducted to study development of all follicles with antral diameters ≥2 mm during the follicular phase of oestrous cycle in Mouflon, a strictly monovular wild-sheep. A total of 14 follicular phases was studied after oestrus synchronization with two cloprostenol doses, 9 days apart, in five cyclic Mouflon ewes. In 13 cycles (92.8%), the ovulatory follicle arose from those antral follicles present in both ovaries when luteolysis was induced, being the largest one with a mean size of 4.4±0.3 mm at that moment in 10 cycles (76.9%). The remaining cycles had a larger follicle, but it was decreasing in size. Appearance of new follicles ≥2 mm in size remained unaffected during the follicular phase (3.7±0.2), but there was found a linear decrease in the number of those growing to ≥3 mm (2.5±0.4 to 1.1±0.2, P < 0.05) and ≥4 mm (0.6 ±0.2 to 0.1±0.1, P < 0.005), detection of new follicles growing to ≥5 mm was negligible. Then, number of medium (4-5 mm) growing follicles present in both ovaries decreased from 1.5 ± 0.3 at 0 h to 0.3 ± 0.1 at 72 h (P < 0.005). In conclusion, the single ovulatory follicle is the largest growing follicle present in both ovaries at the moment of luteolysis. This follicle is selected to grow and ovulate while development of other follicles is inhibited. © 2001 Elsevier Science B.V.

Ovarian follicular development was characterized in 24 Spanish Merino ewes to study effects of the follicular status and the FSH commercial product used on follicular growth and subsequent superovulatory response. Estrus was synchronized using 40 mg fluorogestone acetate sponges. The superovulatory treatment consisted in 2 daily im injections of FSH from 48 h before to 12 h after sponge removal. Sheep were assigned randomly to 2 groups treated with 6 decreasing doses (4, 4, 3, 3, 2, 2 mg) of FSH-P(TM) or with 6 doses of 1.25 mL of OVAGE(TM) Growth and regression of all follicles ≥2 mm were observed by transrectal ultrasonography, and recorded daily from Day 6 before sponge insertion to the first FSH injection, and then twice daily until estrus was detected with vasectomized rams. Differences were detected in follicular development from the first FSH injection to detection of estrus (-48 to 36 h from sponge removal) between groups. Administration of FSH-P increased the appearance of new follicles with respect to OVAGEN (6.3 ± 0.7 vs 4.8 ± 0.4; P < 0.05), and the mean number of medium (4 to 5 mm) follicles (8.9 ± 1.2 vs 6.6 ± 0.9; P < 0.05). However, the mean number of follicles that regressed in size after sponge removal (5.9 ± 0.4 vs 3.3 ± 0.4) and the number of preovulatory sized follicles that did not ovulate (60 vs 42.4%) were also higher in FSH-P treated ewes (P < 0.05). So, finally, there were no differences in ovulation rate, as determined by laparoscopy on Day 7 after sponge removal, between ewes treated with FSH-P or OVAGEN (6.3 ± 1.9 vs 7.0 ± 1.7 CL). In all the ewes, the ovulatory response was related (P < 0.05) both to the number of small follicles (2 to 3 mm in diameter) present in the ovaries at the start of treatment with exogenous FSH and to the number of follicles that reached ≥4 mm in size at estrus, despite differences in the pattern of follicular development when using different commercial products. (C) 2000 by Elsevier Science Inc.