PPARG is dispensable for bovine embryo development up to tubular stages.

Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors (PPARs) mediate the signaling actions of prostaglandins and other lipids and, between them, PPARG has been pointed out to play a relevant role on conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG KO embryos in vitro by two independent gene ablation strategies and assess their developmental ability. In vitro development to Day (D) 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass and trophectoderm cell numbers were similar between WT and KO D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, and embryonic disc formation and hypoblast migration rates were unaffected by the ablation. The development to tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disc was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages.

Birchen and Blue Leonesa sperm cryopreservation: a new technique for evaluating the integrity of cockerel sperm membranes.

1. Birchen and Blue Leonesa are two endangered chicken breeds mainly raised in Curueño Valley in North Spain. The establishment of a germplasm bank to guarantee the preservation of these breeds is needed. However, cockerels from different breeder flocks can show variance in semen cryoresistance.2. The following work focused on the sperm characterisation and cryopreservation of Birchen and Blue Leonesa cockerels from four different breeders. A total of 30 semen pools were analysed. Besides conventional sperm analysis, including motility by computer-aided sperm analysis (CASA) and DNA fragmentation by TUNEL, the present study tested a double staining method (MitoTrackerTM Green FM/propidium iodide). This gave simultaneous assessment of plasma and acrosomal and mitochondrial membranes, which were previously validated by SYBR-14/PI, CASA, aniline blue and TUNEL.3. No significant differences were found among fresh semen variables between breeds and breeders. For post-thawed variables, significant differences (P < 0.05) were found between breeders in sperm viability (58.0 ± 1.90 breeder D vs. 35.2 ± 7.41 breeder A, 37.2 ± 4.09 breeder B and 22.3 ± 5.92 breeder C) and DNA fragmentation (62.4 ± 9.91 breeder C vs. 31.8 ± 7.08 breeder B and 24.5 ± 5.49 breeder D). The lowest DNA fragmentation values for semen from breeder D birds were coincident with higher integrity of the mitochondrial membrane.4. The results revealed higher sperm cryoresistance in the cockerels from one of the breeders, possibly due to differences in management system (e.g. diet, housing, control of stress elements and pathogens, reproduction practices or maintenance of genetic diversity). These differences may determine the sperm freezability, and thus the effectiveness of developing a germplasm bank.

Conventional slow freezing cryopreserves mouflon spermatozoa better than vitrification.

This work examines the effectiveness of a TCG (Tris, citric acid, glucose, 6% egg yolk and 5% glycerol) and a TEST (TES, Tris, glucose, 6% egg yolk and 5% glycerol) sperm extender in the freezing of mouflon spermatozoa at slow cooling rates, using different pre-freezing equilibration times (2-3 hr). It also examines the tolerance of mouflon spermatozoa to different concentrations of cryoprotectants (5, 10, 20% glycerol; 5%, 10%, 20% dimethyl sulfoxide; 6% polyvinylpyrrolidone) and/or sucrose (100, 300, 500 mm). The highest quality (p < .01) thawed spermatozoa were obtained when using the TEST extender and an equilibration time of 3 hr. Sperm motility and membrane integrity were strongly reduced when using rapid freezing rates (60-85°C min-1 ), independent of the concentration of cryoprotectants. The lowest sucrose concentration (100 mm) provided the highest (p < .05) percentage of motile spermatozoa and live spermatozoa with an intact acrosome. Vitrified-warmed sperm variables were at their best when the spermatozoa was diluted in TCG-6% egg yolk + 100 mm sucrose and warmed at 60°C. Slow warming at 37°C strongly reduced (p < .05) sperm motility and viability. However, sperm vitrification returned lower fertility, sperm motility and sperm viability values than conventional sperm freezing.

Spermiotoxicity of commercial condoms made from polyurethane, polyisoprene and latex, using domestic ruminants as an experimental animal model.

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.

Giant panda (Ailuropoda melanoleuca) sperm morphometry and function after repeated freezing and thawing.

This work examines the effects of subsequent cycles of freezing-thawing on giant panda (Ailuropoda melanoleuca) sperm morphometry and function, and assesses whether density-gradient centrifugation (DGC) can increase the number of freezing-thawing cycles this sperm can withstand. A sperm sample was collected by electroejaculation from a mature giant panda and subjected to five freezing-thawing cycles. Although repeated freezing-thawing negatively affected (P < 0.05) sperm motility and membrane integrity, in both nonselected and DCG-selected sperm samples, >60% of the sperm cells in both treatments showed acrosome integrity even after the fifth freezing cycle. In fresh semen, the sperm head length was 4.7 µm, the head width 3.6 µm, area 14.3 µm(2) and perimeter length 14.1 µm. The present results suggest that giant panda sperm trends to be resistant to repeated freezing-thawing, even without DGC selection.

Influence of Staining Method on the Values of Avian Sperm Head Morphometric Variables.

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.

Effect of the interaction between cryoprotectant concentration and cryopreservation method on frozen/thawed chicken sperm variables.

This work examines the effect of the interaction between different concentrations of two cryoprotectants - glycerol (GLY) and dimethylacetamide (DMA) - and two methods of cryopreservation - pellets produced by plunging into liquid nitrogen and gradual in-straw freezing - on frozen/thawed chicken sperm variables. Sperm was cryopreserved using: (i) 6% DMA, following the in-straw and the pellet methods (ii) 11% GLY, following the in-straw and the pellet methods; and (iii) 8% GLY in the in-straw method and 3% DMA in the pellet method (i.e. reduced cryoprotectant concentrations). When 6% DMA was used as the cryoprotectant, no differences were seen between the in-straw and pellet methods in terms of frozen/thawed sperm variables or fertility (10.8% and 12.8%, respectively). The viability and motility variables of the frozen/thawed sperm produced using the in-straw method with 11% GLY were higher (p < 0.05) than those recorded for the sperm preserved using the same cryoprotectant and concentration in the pellet method. However, fertility was extremely low in both groups (2.1% and 4.2% for the in-straw and pellet methods, respectively). Finally, the use of 8% GLY in the in-straw method returned higher sperm viability, intact acrosome and motility values than the use of 3% DMA in the pellet method (p < 0.01). No differences were seen, however, in the fertility results obtained (28.8% and 25.0%, respectively). These results suggest that cryoprotectant concentrations can be reduced and still provide acceptable fertility rates.

Characterization of red-legged partridge (Alectoris rufa) sperm: Seasonal changes and influence of genetic purity.

The general decline in wild Iberian populations of the red-legged partridge (Alectoris rufa) has been accompanied by an increase in game-farm facilities producing hybrids with chukar partridges (Alectoris chukar). Genetic introgression from chukar partridges is thought to modify male red-legged partridge reproductive indicators. The aim of the present study was to determine the effects of such genetic introgression on seasonal reproductive patterns by comparing the sperm and plasma testosterone concentrations of males from pure red-legged and hybrid red-legged/chukar populations. Semen was collected twice monthly over a 12-mo period using a massage technique. Both types of bird showed a clear seasonal pattern of spermatogenic activity. The proportion of males ejaculating sperm was higher (P<0.05) among the pure red-legged birds. The greatest sperm production was recorded in March to May among the pure birds and April to May among the hybrids. Reproductive activity in both groups decreased in June, to reach a minimum in August to December among the hybrids and in September to December among the pure birds. Spermatogenic activity resumed in January in both groups. The sperm concentration produced by the pure birds was smaller than that of the hybrids (P<0.001), but the percentage of motile sperm was higher in the pure birds (P<0.001). The sperm of the hybrids showed greater straight-line velocity (P<0.05), linearity (P<0.001), straightness (P<0.001), sperm wobble (P<0.05), and beat-cross frequency values (P<0.001). The length and area of the sperm head were smaller in the pure birds (P<0.05). The seasonal plasma testosterone concentration pattern followed a trend roughly parallel to the ejaculatory response. The present results suggest that genetic introgression influences the reproductive variables of the red-legged partridge.

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.

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.

Role of sperm velocity variables associated with poultry breed in 'last male precedence'.

It is well known that when a hen mates with multiple roosters, it is the sperm of the last male that usually fertilizes most of the eggs ('last male precedence'). Sperm quality varies between males within a breed, but also between breeds, and thus, sperm competitiveness after mating may depend on the breeds of the roosters involved. The aim of the present work was to identify differences in sperm competitiveness between breeds, especially with respect to motility. A multibreed mating model was used. Blue Andaluza (BA) and Black Castellana (BC) hens left for 21 days with BA and BC roosters, respectively, were then left with Black-barred Andaluza (Bb) roosters for another 21 days (experimental groups hBA-rBC-rBb and hBC-rBA-rBb). Bb roosters (as the second breed replacing the first) fertilized the majority of eggs in both the hBC-rBA-rBb and hBA-rBC-rBb groups. The percentage of offspring sired by BA roosters (8.0%) was higher (p < 0.05) than the percentage of chicks sired by BC roosters (2.1%). The fertility of the BC hens in the hBC-rBA-rBb group was higher (p < 0.01) than that of the BA hens in the hBA-rBC-rBb group. No difference in sperm concentration was seen between the breeds. Within the rapid sperm subpopulation (sperm velocity, >50 µm/s), Bb sperm showed a higher straight-line velocity (VSL) and average path velocity (VAP) (p < 0.05) than BC sperm. The VSL and VAP values for Bb and BA sperm were similar. In conclusion, the present results show that the sperm of the BA breed, traditionally regarded as of moderate fertility, compensates for this drawback via sperm movement characteristics that afford it an advantage in competition scenarios involving males of other breeds. The VSL and VAP of the rapid sperm subpopulation may play the most important role in securing last male precedence.

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.

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.

Role of changes in plasma prolactin concentrations on ram and buck sperm cryoresistance.

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.

Evidence that refractoriness to long and short daylengths regulates seasonal reproductive transitions in Mediterranean goats.

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.

Influence of testosterone administration at the end of the breeding season on sperm cryoresistance in rams (Ovis aries) and bucks (Capra hircus).

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 Merino rams and Murciano-Granadina bucks, in a species-specific manner, but improved the sperm kinetic variables after freezing-thawing in both species, apparently improving sperm cryoresistance. Treatment with TP affects the dimensions of the sperm head in a species-specific manner.

Effect of supplementation of valine to chicken extender on sperm cryoresistance and post-thaw fertilization capacity.

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 with the control (16.1% + 6.2). In conclusion, supplementation of valine to chicken freezing extenders shows a positive effect on DNA fragmentation and fertilizing ability of frozen-thawed sperm, with a better response in a breed considered as the lowest freezer in our conservatory.

In vitro supplementation of testosterone or prolactin affects spermatozoa freezability in small ruminants.

In small ruminants, testosterone and prolactin plasma concentrations show circannual fluctuations as an adaptation mechanism to their seasonal breeding behavior. Sperm resistance to the freezing-thawing process shows seasonal fluctuation throughout the year, with lower sperm freezability at the beginning of the breeding season when prolactin and testosterone levels reach their maximum concentration. Nevertheless, whether these hormones directly affect post-thaw sperm quality parameters is still unclear. The objective was to study the effect of testosterone or prolactin added in vitro on sperm freezability in domestic ram (Ovis aries) and buck (Capra hircus). Sperm samples were incubated for 1 h with a range of testosterone (0, 2, 4, or 6 ng/mL; Exp. 1) or prolactin (0, 20, 100, 200, or 400 ng/mL; Exp. 2) concentrations. Samples were cryopreserved by slow freezing in straws at 0 h and after 1 h incubation. Sperm viability, acrosome integrity, motility, and kinetic parameters were assessed at 0 and 1 h in fresh and frozen-thawed samples. Results showed no hormone effect in fresh sperm, whereas these hormones affected post-thaw sperm parameters. In Exp. 1, in vitro incubation with testosterone decreased the post-thaw acrosome integrity of ram sperm (from 68.1 ± 6.3% to 49.6 ± 3.9%; P < 0.05). In Exp. 2, in vitro incubation with prolactin decreased the post-thaw acrosome integrity of ram (from 78.2 ± 3.4% to 66.3 ± 3.5%; P < 0.05) and buck sperm (from 81.7 ± 2.5% to 67.6 ± 3.5%; P < 0.05). Moreover, prolactin increased the post-thaw amplitude of lateral head displacement in ram sperm (from 3.3 ± 0.1 µm to 3.8 ± 0.2 µm; P < 0.05). In conclusion, either testosterone or prolactin added in vitro decreased the post-thaw acrosome integrity of ram and buck sperm. This suggests a destabilization process that could be decreasing sperm freezability when physiological levels of these hormones are high in vivo.

Crystal structure of 1-ferrocenyl-2-(4-nitro-phen-yl)ethyne.

The title ferrocene derivative, [Fe(C5H5)2(C8NO2)], including an alkyne bonded to a para-nitro-phenyl substituent, which was synthesized from a copper-free Sonogashira cross-coupling reaction between ethynylferrocene and 4-bromo-1-nitro-benzene, crystallizes in the P21/n space group. In the ferrocene unit, the penta-dienyl (Cps) rings are in an eclipsed conformation. The angle of rotation between the substituted cyclo-penta-dienyl ring and the p-nitro-phenyl group is 6.19 (10)°, yielding a quasi-linear extension of the ferrocenyl substitution. Important inter-molecular inter-actions arise from π-π stacking between the Cp rings and the p-nitro-phenyl, from corners of the Cp rings that are perpendicularly aligned, and between the O atoms from the nitro substituent and carbons at the corners of the Cp rings, propagating along all three crystallographic axes.

Efficiency of estrous synchronization in tropical sheep by combining short-interval cloprostenol-based protocols and "male effect".

This study assessed the efficacy of a protocol combining short-interval cloprostenol-based protocols and "male effect" for estrous synchronization in hair sheep. In Experiment 1, 24 ewes were randomly assigned to three groups (n=8) and treated with cloprostenol on Days 3, 5 and 7 after ovulation, respectively. Estradiol secretion during the follicular phase was similar among groups. Onset of estrus (P<0.001) and the timing of maximum LH concentration (P<0.01) were earlier in group D3 than in D5 and D7 groups. During the subsequent cycle, the number and size of corpora lutea were higher (P<0.05) in ewes of the groups D3 (1.9+/-0.3 and 115.1+/-14.3mm(2)) and D5 (1.8+/-0.2 and 100.2+/-11.2mm(2)) than in group D7 (1.3+/-0.2 and 75.6+/-6.4mm(2)) group. In Experiment 2, 24 ewes were treated with two cloprostenol injections (7 days apart). Twelve ewes were exposed to "male effect" previous to an isolation period (ME group), whereas the remaining ewes were controls without male exposure (CTR group). Male effect induced earlier preovulatory LH surge (P<0.05) and ovulation (P<0.001) than CTR group. In Experiment 3, the estrus was synchronized in 68 ewes. Nineteen of them (group FGA) were treated using intravaginal sponges impregnated with fluorogestone acetate for 12 days and inseminated at 55h. Forty-nine females (group ME) were treated like ME group. Twenty-four (ME48 group) and 25 ewes (ME55 group) were inseminated at 48 and 55h after treatment, respectively. The fertility rate was numerically higher in ME48 than ME55 and FGA groups (62.5, 44.0 and 47.4%, respectively). In conclusions, the combined use of short-interval cloprostenol treatment and "male effect" may be an adequate alternative for synchronizing estrus and applying artificial insemination in hair sheep throughout the entire year.