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

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

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

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

Recent studies have noted that the circulating testosterone concentration may affect the ability of spermatozoa to survive cryopreservation. However, few attempts to confirm such a relationship have been made. Wild ruminant species have very marked seasonal changes in their reproductive function and strong annual changes in their plasma testosterone concentration.

The domestic ferret (Mustela putorius furo) provides a good model for developing new reproductive technologies for use with threatened related species. Such technologies could also be used in the reproductive management of this pet species. The present work reports an improved freezing protocol for ferret sperm. Semen was collected by electroejaculation plus rectal massage (in an attempt to reduce the electrical stimulation necessary) from five adult male ferrets, and then subjected to one of two freezing protocols: (a) from 5 to -35°C at 40°C/min, then from -35 to -65°C at 17°C/min, and finally from -65 to -85°C at 3°C/min-a decelerating freezing rate; and (b) from 5 to - 10°C at 5°C/min, and then from -10 to -130°C at 60°C/min-an accelerating freezing rate. After thawing, the viability and acrosomal integrity of the sperm frozen via the two-step accelerating method were better than those frozen via the three-step decelerating method (43.3 ± 3.5% and 71.2 ± 3.4% compared with 29.7 ± 3.7% and 58.8 ± 3.4% respectively; p < .05). No differences were seen between the methods with respect to sperm motility variables; most sperm (>90%) remained static with both freezing methods. In conclusion, although the method with accelerating freezing rate was associated with better post-thaw sperm viability and acrosome integrity values, neither of the two freezing methods tested provided adequate motility results after thawing. Combining rectal massage with electrical stimuli seemed to reduce the number of the latter required for successful sperm collection.

This study examines the effect of L-carnitine (LC) on chilled ram semen stored for up to 96 hr. Semen samples were collected, placed in a skimmed milk + 6% egg yolk extender, pooled, aliquoted and diluted with the same extender supplemented with different LC concentration: 0 (control), 1 mM (LC1), 2.5 mM (LC2.5), 5 mM (LC5), 7.5 mM (LC7.5) or10 mM (LC10). Sperm kinetics and membranes (plasma, acrosome and mitochondrial) were examined using the CASA system and triple fluorescence staining (PI/ PNA-FITC/Mitotracker). The progressive motility was greater (p < .05) with LC7.5 treatment than the control sperm at 96 hr. The curvilinear velocity (p < .01) and the percentage of sperm with intact membranes (plasma/acrosome/mitochondria) (p < .01) were greater with all LC treatments than the control group at all times. Straight line velocity was greater (p < .01) with LC5 and LC7.5 treatments than the control group after 48 hr. The LC5 group also returned lower ALH values (p < .05) than these seen for the control groups after 48 hr. The fertilizing capacity of LC5 samples stored at 15°C for 2 hr (LC5-15°C-2h) and at 5°C for 24 hr (LC5-5°C-24h) was tested in three ewe groups via cervical fixed-time artificial insemination. In two groups, the fertilizing capacity of the LC5-5°C-24h was reduced (p < .001). In the remaining group, however, no significant difference was seen between the LC5-15°C-2h and LC5-5°C-24h sperm in this respect (pregnancy rates 52.4% versus 42.8%; p > .05). Overall, the present results suggest that supplementing skimmed milk-egg yolk-based extenders with LC has a positive effect on chilled sperm variables and fertilizing capacity.

The use of condoms could provide a means of collecting high-quality spermatozoa from different species under physiological ejaculation conditions. However, certain condom materials may affect sperm functionality. This study examined the spermiotoxicity of different commercial condom materials towards ram and goat spermatozoa. Sperm samples were diluted in Tyrode's medium and placed in contact with a piece of condom material (polyurethane, polyisoprene or latex) and incubated for 30 or 90 min. Contact time in the polyisoprene and latex treatments affected some sperm variables; no such effects were seen, however, in the polyurethane treatments. For ram spermatozoa in contact with polyisoprene, the percentage of dead spermatozoa with a damaged acrosome increased at 90 min, while for spermatozoa in contact with latex, the percentage of live spermatozoa with an intact acrosome decreased. For goat spermatozoa in contact with both polyisoprene and latex, the percentage of dead spermatozoa with a damaged acrosome increased at 90 min, while for spermatozoa in contact with polyisoprene, the percentage of live spermatozoa with an intact acrosome decreased. In conclusion, latex and polyisoprene contain components that affect sperm motility, plasma membrane integrity and acrosome function. Polyurethane does not seem to reduce the quality of semen. © 2016 Blackwell Verlag GmbH.

Contents Computer-assisted systems for the assessment of sperm morphometry (ASMA systems) have been used successfully with several mammalian species. Unfortunately, they have so far been of little use for assessing bird semen, a consequence of the filiform shape of avian spermatozoa. This study compares two staining techniques (Hemacolor® and aniline blue staining) for the morphometric analysis of rooster and red-legged partridge spermatozoa as part of a computer-assisted light microscopy method. For both species, Hemacolor® staining provided a significantly higher percentage of measurable cells (93.7 ± 11.7% in roosters and 71.9 ± 15.3% in red-legged partridges). Hemacolor® also showed greater repeatability (lower coefficients of variation) for length and area in roosters' sperm and for width in the case of red-legged partridge's sperm. In the roosters, the Hemacolor® technique returned significantly (p < 0.05) larger sperm head width and area values than did the aniline blue technique, while the latter resulted in greater sperm head length values (p < 0.05). In the red-legged partridge, no differences were seen in the results for sperm head width and area provided by the two techniques, but aniline blue staining was associated with longer length measurements. In conclusion, the morphometric values recorded differed depending on the staining method and species. However, the Hemacolor® technique might be deemed the more appropriate for computerized sperm assessment systems as it provides larger percentages of measureable cells and shows greater repeatability. © 2015 Blackwell Verlag GmbH.

Contents The purpose of the present study was to examine the seasonal variation in freezing damage in free-range rooster sperm. Over a period of 1year, 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. © 2011 Blackwell Verlag GmbH.

Contents Two experiments were performed to assess the role of refractoriness to long and short daylengths in the timing of seasonal reproductive transitions in Mediterranean Malagueña goats. In the first experiment, intact adult does were either maintained under natural photoperiod conditions (NP; n = 5) or exposed to a constant long day photoperiod (LD - 16 h light/day; n = 5) from June to December 2005. In the second experiment, does were maintained either under NP conditions (n = 5) or received a series of subcutaneous melatonin implants (MI) from December 2005 to June 2006 to mimic a short day signal (n = 5). In both experiments, ovulatory activity was assayed by determining progesterone concentrations in blood samples collected twice per week. The onset of seasonal ovulatory activity did not differ (p > 0.05) between the LD and NP does (3 October ±5 days and 23 September ±3 days respectively). The simulated short days did not modify (p > 0.05) the timing of the end of ovulatory activity. The MI does become anoestrus at the same time (1 March ±9 days) as their NP counterparts (23 February ±8 days). These results indicate that, in Malagueña does, the onset and offset of anoestrus are not driven by photoperiod changes but appear to be the result of long and short day refractoriness respectively. An endogenous circannual rhythm may therefore be involved in regulating seasonal reproductive changes in these animals. © 2010 Blackwell Verlag GmbH.

The methods of assisted reproduction commonly used in domestic animals are currently being applied to non-domestic species. This is because of the limitation of maintaining the genetic variability in the wild, as it occurs in wild ruminants of the Mediterranean Basin. Despite the apparent progress of the technology, success of the offspring to grow and become healthy adult individuals has remained low in wild species. Difficulties usually arise from insufficient information about basic reproductive biology, such as the seasonal changes in ovarian and testicular activity. Directional adaptive evolution involves development of specie-specific physiological reproductive patterns to cope with various environmental factors. Thus, species originating and living at the same latitude display different reproductive strategies to entrain the breeding activity at an optimal time of the year. The aim of this paper was to present current knowledge on reproductive physiology of Mediterranean wild ruminants as a basic prerequisite for the successful use of assisted reproduction methods. Special emphasis is given to seasonal endocrine changes, ovarian cycles and testicular activity of Iberian wild ruminants, together with the role of social interactions on the regulation of these events. © 2006 The Authors.

Objectives of this study were to characterize patterns of follicular development in sheep superovulated with purified follicle stimulating hormone (FSH) (OVAGEN™, ICP, Auckland, New Zealand) and to determine its influence on preovulatory events (onset of the oestrus behaviour and timing of the preovulatory luteinizing hormone surge) and ovarian response (ovulation rate and embryo yield). Number and size of all ≥ 23 mm follicles from the first FSH injection to withdrawal of progestagen sponges was determined by transrectal ultrasonography just prior to every FSH injection in nine Manchega ewes superovulated with eight decreasing doses (ml) (1.5 × 3, 1.25 × 2 and 1 × 3) of OVAGEN injected twice daily from 60 h before to 24 h after the withdrawal of 40 mg fluorogestone acetate sponges. Oestrous detection and jugular blood sampling for LH radioimmunoassay were performed every 3 h from 14 to 53 h after sponge removal and ovulation rate and number of embryos were determined 4 days after progestagen withdrawal. Administration of OVAGEN induced a significant rise (p < 0.0005) in the number of follicles ≥ 4 mm in size because of an increased growth in size of follicles from the first FSH injection to sponge removal, an increase in the number of newly detected follicles from 12 to 36 h of the first FSH dose (p < 0.005) and a decrease in regression rate from 24 h (p < 0.001). The number of follicles 2-3 mm in size at first FSH dose (10.4 ± 1.5) was positively correlated with the number of ≥ 4 mm follicles at 0 h (19.0 ± 2.7, p < 0.01). A higher number of ≥ 4 mm follicles at 0 h was related with an earlier appearance of oestrus (31.5 ± 1.5 h, p = 0.08) and LH surge (45.0 ± 2.3 h, p < 0.005), and a higher ovulation rate (18.2 ± 3.8, p < 0.005). On the other hand, the rate of embryo recovery was decreased in ewes with earlier preovulatory LH peaks (p < 0.005), with a shorter interval between oestrus and LH peak (p < 0.05).

The onset of puberty was assessed from plasma progesterone profiles for Mouflon (n = 8) and Manchega (n = 7) female lambs born in April. Four Mouflons exhibited their first ovulation at a mean date of 27 November ± 1.4 days, the mean age being 248.5 ± 3.9 days and the mean body weight 23.8 ± 0.6 kg. The remaining four Mouflons, which had a slower growth rate, attained puberty during the autumn of the following year. Five of seven Manchega lambs attained puberty in their first year at a mean date of 5 Oct ± 3.3, the mean age being 185.6 ± 2.6 days and the mean body weight 41.8 ± 2.0 kg. The mean live-weight at the onset of puberty was 65.1 and 82% (p < 0.05) of adult body weights for Manchega and Mouflons, respectively. Overall, the results indicate that there is a threshold of body-weight necessary for the attainment of puberty in the first breeding season for spring-born lambs, which is very different between both Mouflon and Manchega. When body weight was below 23.8 ± 0.6 and 41.8 ± 2.0 kg for Mouflon and Manchega lambs, respectively, first ovulation did not occur until the beginning of the next breeding season. The onset of puberty during the second year of life occurred within the specific reproductive seasons for Mouflon (October-April) and Manchega (July-March) ewes, despite minimal further growth.

Ultrasonographic observations of the corpus luteum (CL) and collection of blood samples for progesterone radioimmunoassay were performed daily during 15 oestrous cycles in Spanish Merino ewes, a consistently monovular breed. Ultrasonographic image of the CL changed during the oestrous cycle, increasing its echogenic pattern from ovulation to luteolysis. The size of the CL and mean progesterone levels were significantly affected by day of cycle (p < 0.05 and p < 0.001, respectively). Both increased their values from day 1 to day 12 (from 49.6 ± 7.4 to 154.6 ± 11.8 mm2 and from 0.2 ± 0.0 to 2.8 ± 0.5 ng/ml, respectively) and then declined sharply until day 0 (28.2 ± 5.3 mm2 and 0.1 ± 0.0 ng/ml, respectively). There was a significant correlation between CL area and plasma progesterone concentrations during the entire oestrous cycle, taking the developing and regressing phases of the CL separately (p < 0.05). A central cavity was observed in 33.3% of the CL studied. The presence of this cavity had no effect in total luteal-tissue area of the CL nor on oestrous cycle length or on progesterone concentrations. Likewise, the cavity did not affect the correlations observed between CL size and progesterone levels, CL size and day of cycle and progesterone levels and day of cycle. It is concluded that ultrasonographic assessment of CL area is a reliable method for estimating peripheral plasma progesterone levels, regardless to the presence or absence of a cavity in the CL.