Fertilization capacity of cryopreserved Iberian ibex epididymal sperm in a heterologous in vitro fertilization assay.

In vitro fertilization (IVF) can be used to assess the fertilization capacity of sperm. Heterologous IVF may be useful when assessing that of wild animals as it is often difficult to obtain adequate numbers of naturally corresponding oocytes. The aim of the present study was to assess the fertilization capacity of frozen-thawed ibex epididymal spermatozoa via heterologous IVF involving the oocytes of prepubertal domestic goats. The effect on fertilization and embryo development of adding oestrous sheep serum (ESS) to the fertilization medium was also examined. Cumulus-oocyte complexes (COCs) were matured in TCM-199 for 24-27 h at 38.5°C in a 5% CO2 in air atmosphere. Frozen-thawed epididymal spermatozoa were selected by density gradient centrifugation. After maturation, the oocytes were co-incubated with spermatozoa in synthetic oviductal fluid (SOF) with different concentrations of ESS: SOF-C (0%), SOF-2 (2%) and SOF-20 (20%). At 17 h post-insemination (hpi), zygotes with one female and one male pronucleus (2PN) were categorised as normal; zygotes with 3PN were recorded as polyspermic, and oocytes with 1PN as asynchronous. Cleavage and blastocyst development were assessed at 48 and 168 hpi respectively. The percentage of zygotes with 2PN was higher in the SOF-2 than in the SOF-20 treatment group (27.7% versus 2.9% P < 0.05). The percentage of blastocysts formed with the SOF-C, SOF-2 and SOF-20 treatments were 1.1%, 7.5% and 0% respectively. These results show that the presence of 2% ESS achieves better results than the use of no serum or the standard 20% concentration. Heterologous IVF may be an effective method for predicting the fertilization capacity of ibex spermatozoa, and therefore perhaps that of other wild mountain ungulates.

Hormone-free protocols for the control of reproduction and artificial insemination in goats.

The dairy goat industry is of great economic importance to certain rural areas of the European Union (EU), especially the Mediterranean region. Its sustainability, however, is severely affected by the seasonality of goat reproduction, which leads to fluctuations in the availability of final products. Classical hormone treatments based on progestagens and eCG are the main tools employed in the effort to achieve fertility outside of the normal breeding season. They are also used to induce and synchronize oestrus and ovulation in artificial insemination programs. The food safety policy of the EU is becoming ever stricter with regard to the use of hormonal treatments for reproductive purposes, pushing livestock-raising towards ever cleaner and greener production systems. Recent advances in the use of natural methods able to generate endocrine signals that induce the ovulatory process have improved our capacity to foster reproduction in the non-breeding season. When used in a fashion appropriate for the latitude at which animals live, their breed, and the management system under which they are raised, environmental (photoperiod), nutritional and sociosexual (the male effect) signals offer alternatives to classic hormonal techniques. This affords the fragile and heterogeneous goat production sector with new opportunities. This article describes the most representative advances made in the use of the male effect as a natural method of inducing ovulation during seasonal anoestrus. Its association with other methods for optimizing responses and synchronizing induced ovulation is also discussed; such associations allow it to be used as an alternative to hormonal treatment in artificial insemination programs.

Sperm characteristics and heterologous in vitro fertilisation capacity of Iberian ibex (Capra pyrenaica) epididymal sperm, frozen in the presence of the enzymatic antioxidant catalase.

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 200IU/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 48hpi was higher (P<0.01) with the TCG sperm than with the TCG-CAT sperm (16.7% vs. 7.6%). The use of 200IU/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.

Photoperiod and melatonin treatments for controlling sperm parameters, testicular and accessory sex glands size in male Iberian ibex: A model for captive mountain ruminants.

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.

Influence of season on the freezability of free-range poultry semen.

The purpose of the present study was to examine the seasonal variation in freezing damage in free-range rooster sperm. Over a period of 1 year, heterospermic semen samples were collected weekly by massage from the roosters of 14 Spanish chicken breeds, all housed under natural photoperiod and climatic conditions. All samples were frozen in straws using DMA as a cryoprotectant, placing them first in nitrogen vapour and then plunging them into liquid nitrogen. No seasonal effects on fresh sperm quality were found. Neither did season affect the percentage of viable frozen-thawed spermatozoa nor the percentage with an intact acrosome. However, the collection season influenced (p < 0.05) most frozen-thawed sperm motility values. The percentage of immotile frozen-thawed spermatozoa was lower (p < 0.05) in spring-collected sperm than in summer- or autumn-collected samples. The percentage of spermatozoa showing progressive motility was higher in spring-collected sperm compared with winter-, summer- or autumn-collected samples (p < 0.05). The curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP) values of spring-collected sperm were also higher (p < 0.05). In conclusion, spring would appear to be the best season for collecting and freezing the semen of free-range Mediterranean chicken breeds.

Effect of post-mortem time on post-thaw characteristics of Spanish ibex (Capra pyrenaica) spermatozoa.

Viable epididymal sperm can be obtained in the Spanish ibex during 24h after death, but it has been observed a significant effect of the post-mortem time on fertility success, so only goats inseminated with semen recovery during the first 8h became pregnant. The aim of this study was to determine the effect of post-mortem time on epididymal semen samples from of Spanish ibex. For this purpose, sperm samples from 36 males were collected at different post-mortem times, from 2 to 24h, and cryopreserved. Thawed samples were incubated for 2h at 37°C without dilution or after dilution in a modified Tyrode medium, in order to study the sperm resistance to dilution. Moreover, flow cytometry was used to assess the sperm viability (PI), phospolipid disorder of the plasma membrane (M540), mitochondrial membrane potential (Mitotracker Deep Red), indirect apoptosis markers (YOPRO-1) and sperm chromatin stability (SCSA(®)). Sperm motility was evaluated by computer-assisted sperm analysis (CASA). Our results have shown that post-mortem time caused a reduction in mitochondrial membrane potential. In this regard, the loss of energy could be responsible for the loss of maintenance of the membrane with a consequent increase in permeability leading to a decrease in sperm viability and motility, losing linearity and speed. Moreover, the loss of maintenance of the membrane influence the extent to which sperm will survive the cryopreservation process, as it shows the results obtained from the dilution-incubation resistance test. Finally, one important finding of this study is the demonstration of no effect of post-mortem time on post-thaw DNA integrity, giving us the possibility of using sperm samples from valuable males, even if it was not possible to process during the first 8h.

Semen cryopreservation for the creation of a Spanish poultry breeds cryobank: optimization of freezing rate and equilibration time.

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 medium-rate 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.

Seasonal variation in reproductive physiological status in the Iberian ibex (Capra pyrenaica) and its relationship with sperm freezability.

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.

The influence of washing Spanish ibex (Capra pyrenaica) sperm on the effects of cryopreservation in dependency of the photoperiod.

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.

Assisted reproduction in Mediterranean wild ruminants: lessons from the Spanish ibex (Capra pyrenaica).

Despite apparent progress in reproductive technology as applied to wild ruminants, the success achieved in terms of the number of offspring that become healthy adults has remained low. Difficulties often arise through a lack of knowledge regarding appropriate cryopreservation techniques, and indeed through a lack of detailed information on the reproductive physiology of the species in question. The Spanish ibex (Capra pyrenaica) is a wild caprid found exclusively in the mountains of Iberia; only two of the original four subspecies still exist. Great efforts need to be made to preserve this species. The endocrine and environmental mechanisms that control its seasonal reproduction need to be properly understood, reproductive technologies (particularly the cryopreservation of gametes) optimised, and genetic resource banks developed. The experience obtained with the Spanish ibex may be useful in ex situ conservation strategies designed to preserve other threatened Mediterranean wild ruminants.

Use of the hypo-osmotic swelling test and aniline blue staining to improve the evaluation of seasonal sperm variation in native Spanish free-range poultry.

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.

Cryopreservation of Spanish ibex (Capra pyrenaica) sperm obtained by electroejaculation outside the rutting season.

The effectiveness of electroejaculation for obtaining Spanish ibex sperm samples for freeze preserving outside the rutting season was evaluated-the aim being to optimise biological resources for the establishment of germplasm banks. The effect of different egg yolk concentrations (6% or 12%, v/v) in diluents of different buffer composition (Tris-citric acid buffer or Tes-Tris buffer) on frozen-thawed samples of the above also investigated. Experiments were undertaken with six ibex males in February-May, and involved four different semen samples from each animal with four combination of extender, respectively: Tes-Tris-glucose (TTG)-6% egg yolk, TTG-12% egg yolk, Tris-citric acid-glucose (TCG)-12% egg yolk, TCG-6% egg yolk. The results show that electroejaculation is a useful way of obtaining sperm samples from Spanish ibex outside the rutting season (i.e., at a time coinciding with plasma testosterone levels of <0.4ng/ml). According to the results of the eosin-nigrosin staining and the hypo-osmotic swelling test, the freezing-thawing process significantly reduced the viability and membrane integrity of the spermatozoa extended with TTG-6% egg yolk, TTG-12% egg yolk, and TCG-12% egg yolk, but did not affect these variables in spermatozoa extended with TCG-6% egg yolk. Therefore, the use of Tris-citric acid-based extenders containing low concentrations of egg yolk is recommended for cryopreserving Spanish ibex spermatozoa obtained by electroejaculation outside the rutting season.