Flavone and 3-hydroxyflavone supplementation in cryopreservation medium protects canine sperm against apoptosis and lipid peroxidation.

Cryopreservation is a pivotal technique in safeguarding genetic material across diverse species, despite its inherent challenges linked to induced spermatozoa damage, notably apoptosis and lipid peroxidation (LPO). Given the insufficient antioxidant defense of spermatozoa against LPO, there is a rising interest in integrating additional additives into extenders to ameliorate mammalian semen quality. Among these additives, flavonoids have garnered considerable attention due to their potent antioxidative properties. Hence, our study aimed to assess the efficacy of flavone (FL) and 3-hydroxyflavone (3-OH = ) supplementation in the cryopreservation medium to protect canine sperm against the damaging impacts of freezing and ensure the preservation of their reproductive potential. Semen was collected from five Beagle stud dogs and then pooled. Then, the sample was divided into 7 groups, each treated with 1) 0 mM, 2) 0.1 mM FL, 3) 0.2 mM FL, 4) 0.4 mM FL, 5) 0.1 mM 3-OH = , 6) 0.2 mM 3-OH = , 7) 0.4 mM 3-OH = . Semen samples were subjected to cryopreservation in French straws and glycerol as a cryoprotectant. In the frozen thawed semen, sperm motility parameters by CASA system and sperm membrane integrity, acrosome status, mitochondrial activity, DNA fragmentation, early apoptosis with capacitation, and LPO were assessed using flow cytometry just after thawing (0 h) and 4 h post thaw. Results reveal significant increase in the proportion of live spermatozoa with undamaged acrosomes in the FL 0.1 and 3-OH = 0.2 groups at 0 h post thaw. At this time point, 3-OH = 0.1 significantly reduced the DNA fragmentation index (DFI) compared to the FL 0.1 and 0.2 groups. However, after the next 4 h, 3-OH = 0.4 exhibited the lowest (P < 0.05) DFI compared to FL 0.2 and 3-OH = 0.1. Additionally, 3-OH = 0.4 showed the highest (P < 0.05) proportion of non apoptotic and non capacitated spermatozoa compared to FL 0.1 0 h post-thaw. Simultaneously, the same group demonstrated significant reduction in apoptotic and capacitated sperm cells, at 0 h and 4 h post-thaw. Moreover, 3-OH = at 0.1 (0 h and 4 h) and 0.2 mM (4 h) significantly enhances the proportion of live sperm without LPO post thaw. Whitin the FL groups, only 0.4 FL significantly increased the percentage of live sperm without LPO. No significant effect of the tested substances was observed on sperm motility, cell membrane integrity, or mitochondrial activity. These findings highlight the promising role of flavone and 3-hydroxyflavone in enhancing sperm resilience during cryopreservation, suggesting their protective function against acrosome damages, capacitation, apoptosis and lipid peroxidation.

The neonicotinoid, imidacloprid, disrupt the chicken sperm quality through calcium efflux.

Imidacloprid (IMI), an insecticide from the neonicotinoid group widely used in agriculture, has drawn attention due to its potential harmful effects on non-target species, including bird populations. In the present work, we investigated the effect of IMI on avian semen by in vitro exposure of rooster spermatozoa to this pesticide. The semen was collected twice a week. Samples collected on one day were pooled and incubated with the following IMI concentrations: 0 mM, 0.5 mM, 5 mM, 10 mM, and 50 mM at 36°C for 3 h. Comprehensive semen analysis was carried out after 1 h and 3 h of incubation, evaluating sperm motility parameters with the CASA system and using flow cytometry to assess membrane integrity, mitochondrial activity, acrosome integrity, chromatin structure, intracellular calcium level and apoptosis markers such as: early apoptosis and caspase activation and lipid peroxidation. The results of the first experiment suggest that low concentrations of IMI have a different effect on sperm motility compared to higher concentrations. In IMI samples, we also observed a lower percentage of cells with a high level of calcium ions compared to the control, and a lower level of lipid peroxidation. We concluded that IMI may act as a blocker of calcium channels, preventing the influx of these ions into the cell. To confirm this mechanism, we conducted a second experiment with calcium channel blockers: SNX 325, MRS-1845, and Nifedipine. The results of this experiment confirmed that the mechanism of action of IMI largely relies on the blockade of calcium channels in rooster sperm. Blocking the influx of calcium ions into the cell prevents the formation of Ca²âº-dependent pores, thereby preventing an increase in cell membrane permeability, ultimately blocking early apoptosis and lipid peroxidation in chicken spermatozoa.

Breeding behavior analysis in a large captive colony of African penguins (Spheniscus demersus) and its implications for population management and conservation.

The African penguin Spheniscus demersus, frequently housed in zoos, holds potential for future reintroduction efforts due to its declining wild population. This paper aims to explore various aspects of reproductive performance in African penguins within a large ex situ colony at Zoo Wroclaw in Poland, covering 9 years of breeding behaviors. The analysis reveals parallels in colony growth and partner change patterns with those observed in the wild. Positive correlations were found between breeding success and pair-bond duration, with the increasing colony size influencing reproductive performance. Contrary to their wild counterparts, captive African penguins initiate breeding attempt and produce a fertilized egg at a younger age. However, successful breeding still requires gaining experience or forming pairs with more experienced partners. Our research indicates that providing captive African penguins with unlimited food resources and sufficient nesting space results in rapid colony growth. The increased colony size facilitates breeding behaviors that positively influence population dynamics, particularly through the maintenance of long-term pair bond relationships and the potential for partner changes when necessary or desirable to enhance breeding success. We present compelling case studies in pair fidelity, offering valuable insights and implications for the management of captive populations and conservation efforts.

Prolonged cold-preservation of domestic cat ovarian tissue is improved by extracellular solution but impaired by the fragmentation of ovary.

For domestic cats ovaries, recommended cold-storage limit is 24 h in Phosphate Buffered Saline (PBS) or Dulbecco`s PBS (DPBS). Here, we attempted to verify wheatear cat ovaries may benefit from more complex solutions during prolonged cold-storage (>24 h). First, the preservation capabilities of extracellular (SP+), intracellular (UW) solutions and DPBS supplemented with glutathione (DPBS+GSH) were compared using ovary fragments from the same ovary (n=10). Intact ovary stored in DPBS served as a control. Ovaries were kept at 4 °C for 48 h, and 72 h. In the second experiment, first ovary was stored in DPBS, second in SP+ or UW solution for 48 h (n = 12). Ovaries pairs stored in DPBS for 24 h served as a control (n=8). Tissue samples were evaluated directly after cold-storage and after following 24 h in vitro culture. Ovarian follicle morphology, apoptosis rates (cleaved caspase-3, TUNEL), and follicular growth activation (Ki-67) were assessed. Ovary fragmentation impaired follicular morphology preservation upon cold-storage comparing to intact ovary. However, ovarian fragments stored in UW for 48 h and in SP+ for 72 h presented better morphology than DPBS+GSH group. Comparison of intact ovaries cold-storage for 48 h showed that SP+ provided superior follicular morphology over DPBS, and it was comparable to the outcome of 24-hour storage. No follicular activation after in vitro culture was observed. Nevertheless, tissue culture increased considerably caspase-3 cleavage and TUNEL detection. The ovary fragmentation prior to cold-storage is not recommended in domestic cats. Replacement of DPBS with SP+ solution for whole ovary and UW solution for ovarian tissue fragments improves follicular structure preservation during 48-hour cold-storage.

Addition of low concentration of cholesterol-loaded cyclodextrin (CLC) has a positive effect on cryopreserved canine spermatozoa evaluated by andrological and biophysical methods.

BACKGROUND: This study was conducted to find the best concentration of cholesterol-loaded cyclodextrin (CLC) which has a positive impact on canine post thaw semen quality. Three different concentrations of CLC (0.83 mg/ml; 1.66 mg/ml; 3.32 mg/ml) and 2-hydroxylpropyl-beta-cyclodextrin (HBCD) (1.66 mg/ml) were used in addition to cryopreservation extender and compared with the control after thawing. Samples were assessed using computer-assisted semen analyzer (CASA), flow cytometry, fluorimeter by measuring the fluorescence anisotropy (ANISO) and determining the generalized membrane polarization (GP). RESULTS: An addition of 0.83 mg/ml CLC significantly increased the percentage of progressive motile (PROG) and rapid spermatozoa (RAP) (P < 0.05). 1.66 mg/ml HBCD decreased progressive motility of spermatozoa and population with rapid movement relative to the control (P < 0.05). Furthermore, the groups with an addition of 1.66 mg/ml and 3.32 mg/ml of CLC, as well as the group with only cyclodextrin, increased percentage of dead spermatozoa without lipid peroxidation and decreased percentage of viable spermatozoa without LPO which was lower in these groups than in the control (P < 0.05). Other sperm parameters assessed on flow cytometer were not significantly different. The addition of CLC at 0.83 mg/ml and 3.32 mg/ml concentrations and 1.66 mg/ml of HBCD caused an increase in ANISO measured at 23 ºC (P < 0.05). CONCLUSIONS: In conclusion, the results suggest that increasing cholesterol in the plasma membrane of canine spermatozoa can improve their freezability. However, only low concentrations of CLC may improve semen quality after thawing without adversely affecting other parameters.

The morphology, morphometry and functionality of fresh and cryopreserved wisent (Bison bonasus) epididymal spermatozoa.

Epididymal spermatozoa obtained post mortem are considered a valuable source of genetic material which is often irrevocably lost. This makes these gametes constitute a key element in protection and restitution programs. The wisent (Bison bonasus, Linnaeus 1758) is a species that survived in zoos after extinction from its natural habitat. This resulted in a narrowing of the genetic pool of the whole population, which is at present derived from only 12 ancestors. Currently, wisent protection programs are aimed at preserving the genetic diversity by establishing a germplasm bank. The objective of this study was to comprehensively characterize the morphology, morphometry and functionality of wisent epididymal spermatozoa and evaluate the effectiveness of their cryopreservation in extender based on Tris buffer and chicken egg yolk. The median total number of spermatozoa obtained from one individual was 1985.0 × 106 (62.5 × 106-7452.0 × 106). These gametes were characterized by median: 40.0% (0.5-70.0%) subjective motility, 69.8% (32.5-90.0%) viability and 54.3% (10.5-83.3%) normal morphology. The sperm head had a median size of 5.0 µm (3.5-6.7 µm) width, 8.5 µm (6.4-11.3 µm) length and 36.9 µm2 (23.7-48.6 µm2) surface area. The viable population of the obtained gametes was characterized by median values 53.2% (4.5-80.3%) of intact sperm membrane, 50.8 (26.0-76.6%) of intact acrosome, 0.4% (0-98.7%) of fragmented chromatin, 5.9% (0.0-88.8%) of cells with high mitochondrial potential and 42.1% (8.3-63.7%) without lipid peroxidation. The viable population of the frozen/thawed gametes was characterized by median values: 18.4% (2.4-57.9%) of intact sperm membrane, 35.1 (11.9-56.7%) of intact acrosome, 0.07% (0-89.2%) of fragmented chromatin, 12.8% (0.0-49.7%) of cells with high mitochondrial potential and 16.3% (2.2-53.6%) without lipid peroxidation. Due to the material originating from a relatively large number of wild individuals, the research presented here contributed to the description of certain species standards for the assessment of wisent epididymal spermatozoa. The presented effect of cryopreservation on these gametes justifies the use of an extender based on Tris buffer with the addition of chicken egg yolk. The obtained effects are satisfactory from the point of view of preserving valuable genetic material and their use in ART.

Effect of male age on semen quality in domestic animals: potential for advanced functional and translational research?

Age and other factors like season and breed are often associated with sperm quality and fertility in domestic animals. Even though many studies assessed the relationship between the age of the male and sperm parameters, the effects have not been comprehensively evaluated. Changes in semen quality from pubertal (young) to adult and old age were identified in the bull, ram, buck, boar, dog, and stallion, respectively. The review discusses the association between male age and semen volume, the total number of spermatozoa per ejaculate, sperm concentration, motility, morphology, sperm cell function, sperm DNA integrity, oxidative stress, and antioxidant activity in these species of animals. Generally, semen characteristics improve to a certain age, which declines as the animal ages. Only a few studies evaluated the impact of advanced age or employed advanced functional sperm assessment methods to assess age-related changes in sperm quality and male fertility. Such studies in the dog or stallion, for instance, may contribute to advancing knowledge in human-assisted reproductive techniques used in patients of advanced paternal and maternal age.

Does Better Post-Thaw Motility of Dog Sperm Frozen with CLC Mean Better Zona Pellucida Binding Ability?

Even though the search for methods improving cryopreservation of canine spermatozoa led to an improvement of post-thaw quality, fertilizing results after insemination with frozen-thawed semen are still not satisfying. In this study, we focused on modification of spermatozoa membrane fluidity and investigated whether kinematic parameters as assessed by computer-assisted semen analyzer (CASA) can be improved. The primary aim of our study was to investigate whether the use of cholesterol-loaded cyclodextrins (CLC; 0.5 mg, 1 mg, 2 mg) and 2-Hydroxypropyl-ß-cyclodextrin (HBCD; 1 mg) positively influence capacitation status as examined by tyrosinphosphorylation, cholesterol efflux and zona binding assay (ZBA) of spermatozoa. The use of 0.5 mg of CLC increased the percentage of motile, progressive and rapid spermatozoa compared to the control. Addition of HBCD decreased motility and progressive motility of spermatozoa and the population with rapid movement in comparison to the control. The percentage of live spermatozoa without efflux of cholesterol compared to the control was increased when extender with 0.5 mg of CLC was used. There was no change in capacitation status. The zona binding ability of spermatozoa was significantly lower in the group with 0.5 mg of CLC than in the control. In conclusion, these results suggest that improvement of kinematic parameters does not necessarily coincide with better zona pellucida binding ability of spermatozoa.

Fluorescence Microscopy and Flow-Cytometry Assessment of Substructures in European Red Deer Epididymal Spermatozoa after Cryopreservation.

Thawed spermatozoa, sampled post mortem from the fresh epididymides of European red deer and epididymides stored for up to 12 h at 2-4 °C, were evaluated by fluorescence microscopy (FM) and flow cytometry (FC). The sperm samples were extended and cryopreserved. The sperm motility (CASA), sperm viability (SYBR+/PI-), acrosome integrity, mitochondrial activity, apoptotic changes, and chromatin stability were assessed. Sperm were analyzed by FM before cryopreservation, and by FM and FC after thawing. Epididymal storage time (for 12 h) had no significant effect (p > 0.05) on the examined variables before cryopreservation. After thawing, the storage variants differed (p ˂ 0.05) in the percentage of apoptotic sperm (FM and FC) and DNA integrity (FC). The results of FM and FC differed (p ˂ 0.05) in all the analyzed parameters, excluding SYBR+/PI. Significant correlations (p ˂ 0.01) were observed between the sperm viability, acrosome integrity, and the percentage of non-apoptotic spermatozoa, regardless of the applied technique. In FM, the above parameters were also significantly correlated with mitochondrial activity. The study demonstrated that European red deer spermatozoa stored in the epididymides at 2-4 °C for 12 h can be used for cryopreservation. Both techniques were equally reliable, but FM was better suited for evaluating mitochondrial activity whereas FC was more useful in the evaluation of DNA fragmentation.

Effect of Dimethylacetamide Concentration on Motility, Quality, Antioxidant Biomarkers, Anti-Freeze Gene Expression, and Fertilizing Ability of Frozen/Thawed Rooster Sperm.

Sperm cryopreservation is of great importance for the poultry industry but still needs to be optimized. The high susceptibility of poultry sperm to cryodamage leads to low fertility rates after cryopreservation. Therefore, the present study aimed at evaluating the effect of including a cryoprotectant, dimethylacetamide (DMA), in the chicken semen freezing extenders at a final concentration of 3%, 6%, or 9% on the post-thawed sperm motility, quality, antioxidant biomarkers, anti-freeze gene expression, and fertilizing ability. Results showed that the total motile sperm, progressivity, and viability were quadratically increased (p < 0.05) in the 6% DMA group. The antioxidant enzyme activity and lipid peroxidation were negatively (p < 0.05) affected by the increase in DMA concentration. Furthermore, some anti-freeze-associated genes such as heat shock protein 70 (HSP70) and ras homolog family member A (RHOA) were linearly and quadratically down-regulated (p < 0.05) with the high concentration of DMA. Finally, the fertility and hatchability rates did not indicate statistical differences between DMA groups. It can be concluded that using the low concentration of 3−6% DMA in the freezing semen extender is preferable to obtain acceptable results in the post-thawed sperm quality and fertility.

Comparison of Commercial Poultry Semen Extenders Modified for Cryopreservation Procedure in the Genetic Resource Program of Czech Golden Spotted Hen.

Spermatozoa cryoconservation represents an important strategy for partial in vitro or rescue programs designed for threatened livestock populations. The procedure for the semen cryopreservation of the Czech Golden Spotted Hen was proposed due to the lower fertilization rate of poultry semen compared to mammalian species. The aim of this study was to compare commercial extenders designed for liquid storage preservation with the use of a predefined cryoprotectant, and, thus, to propose an important tool for the procedure of the semen cryopreservation of the Czech Golden Spotted Hen. Ejaculates were sampled from four roosters during five semen collection days. The samples were frozen in Poultry media®, Raptac® and NeXcell® extenders supplemented with a 9% N-methylacetamide (NMA) cryoprotectant. Sperm parameters of the total motility (MOT; %), plasma membrane and acrosome intactness (PAI; %), plasma membrane damage (%), acrosome damage (%) and cells with plasma membrane and acrosome damage (%) were assessed using a mobile mCASA analyzer and flow cytometer after the cryopreservation of the insemination doses (IDs). For Poultry media® (PAI = 51.11%; MOT = 23.58%) and Raptac® (PAI = 52.04%; MOT = 23.13%) extenders with the addition of an NMA cryoprotectant, the comparable results were detected after thawing. For NexCell® media, the results were poor (PAI = 7.07%; MOT = 3.83%). Our results indicated two extenders suitable for the cryopreservation procedure, with the applied modification.

Advances in storage of poultry semen.

Semen cryopreservation is a key biotechnological strategy used to preserve and protect genetic resources, which are subject to increasingly serious reductions in some species, and to protect animal biodiversity. Assisted reproductive techniques, however, are still not utilized to the same extent in avian species to the extent that occurs in mammals. The reasons for this situation are described in this review. The content of this paper is focused on current poultry preservation systems, published since 2010, and new strategies that are very promising for preserving avian genetic resources. Two major types of storage technologies which are utilized for avian sperm preservation, liquid storage and cryopreservation, are emphasized. The issues on which there is a focus includes supplementation of avian extenders with various compounds prior to the preservation period, use of cryoprotectants and fertility results when there were in vitro sperm evaluations. Results from recent studies indicate there are opportunities to improve the quality of bird semen after preservation. It is obvious that cryo-diluent composition may be the most important factor for development of efficacious cryopreservation methods for avian semen.

Current Status and Advances in Semen Preservation.

Recent advances in assisted reproductive technology (ART) have increased the effectiveness of fertility treatments [...].

Supplementation of Avian Semen Extenders with Antioxidants to Improve Semen Quality-Is It an Effective Strategy?

Oxidative stress in sperm is a phenomenon related to the increasing rate of oxidation of cellular components and the excessive production of reactive oxygen species (ROS). The high content of polyunsaturated fatty acids in bird sperm cell membranes renders these cells particularly susceptible to lipid peroxidation (LPO). Therefore, to ensure the proper functioning of cells, it is necessary to have a balance between the formation of ROS and the protective action of the antioxidant system. This review aims firstly to briefly introduce the antioxidant system characteristics of avian semen. Secondly, we summarize the recent knowledge regarding progress in extender supplementation using antioxidants and other compounds to improve avian semen quality parameters and fertility rates. The review focuses on enzymes, vitamins, amino acids, proteins, some plant extracts, and other compounds that can be used to supplement the extenders to reduce the formation of oxidants in poultry semen and maintain its quality and enhance its fertility.

Long-term stiripentol administration, an anticonvulsant drug, does not impair sperm parameters in rats.

In this study, we investigated the influence of long-term administration of stiripentol on sex hormones and semen quality in young Wistar rats. Investigated animals received for 6 months either stiripentol or saline solution. After one month, stiripentol increased temporarily serum level of testosterone (p < 0.05) and FSH (p < 0.01). However, after 6 months levels of testosterone, FSH, LH, prolactin and SHBG were comparable in both groups. After 6 months, semen analysis did not reveal differences in sperm concentration, total sperm count and sperm motility between groups. However, stiripentol increased the rate of head defect (p < 0.001) and midpiece abnormalities (p < 0.05). Flow cytometry revealed higher percentage of live cells without lipid peroxidation (p < 0.00001) and higher percentage of live spermatozoa with intact acrosomes (p < 0.000001) in rats receiving stiripentol. There was no significant difference between groups in sperm mitochondrial activity and DNA fragmentation index. However, percentage of high DNA stainability cells was increased in stiripentol group (p < 0.001). The data showed that stiripentol does not cause obvious disturbances in young rat's semen. Detected changes in semen morphology and chromatin structure need further explanation, and their influence on rat's fertility should be evaluated.

Expression of Apoptosis-Related Genes in Cat Testicular Tissue in Relation to Sperm Morphology and Seasonality-A Preliminary Study.

Apoptosis is a crucial process in spermatogenesis, responsible for the elimination of abnormal sperm cells and testicular regression out of breeding season. The aim of this study was to assess if the expression of apoptosis-related genes in testicular tissue of domestic cats differed: (1) between normozoospermic and teratozoospermic donors, and (2) between reproductive and non-reproductive season. The expression of genes: BCL2L1, BCL2, BAX, BAD, FAS, FASLG, and caspases (CASP3, CASP8, CASP9, and CASP10) was analyzed by qRT-PCR in testicular tissue samples. During non-reproductive season significantly higher expression of two anti-apoptotic genes (BCL2L1 and BCL2) was observed. Additionally, there was a significant higher expression of CASP10 in teratozoospermic cats during non-reproductive than during reproductive season. No differences were noted between normozoospermic and teratozoospermic groups. Upregulation of some genes during the non-reproductive season indicates engagement of apoptotic mechanisms in the seasonal changes of semen quality in cats, however further studies on protein levels and analysis of changes on distinct testicular germinal layers are required. At the same time, teratozoospermia in the general population of cats seems to be not connected with dysregulation of apoptosis in the testes.

Comparison of the Morphology and Developmental Potential of Oocytes Obtained from Prepubertal and Adult Domestic and Wild Cats.

The aim of the study was to compare the morphology and developmental potential of oocytes obtained from adult and prepubertal domestic cats (Felis catus) and wild cats (Lynx lynx, Leptailurus serval, Felis manul, Panthera tigris altaica). The average number of oocytes obtained from an adult domestic cat was 23 ± 11, which was significantly lower than from kittens (43 ± 29). A similar number of oocytes was derived from adult Pallas's cats (28 ± 8), and serval (30). The lowest number of oocytes was collected from the lynx (5 ± 3). No oocytes were obtained from newborn Amur tiger while in the case of older domestic and Pallas's cat and lynx kittens (1-3 months) 43, 48 and 41 oocytes were collected, respectively. Significant differences (p < 0.001) were observed between the number of oocytes with dark cytoplasm from adult and prepubertal animals of all analyzed species. The diameter of oocytes from adult and prepubertal animals was similar in all species, and was on average 161 ± 4 µm for oocytes with dark cytoplasm and 150 ± 18 µm for oocytes with light cytoplasm. In all species, oocytes with light cytoplasm were significantly smaller (p < 0.05) than dark ones, and their population was more diverse. Results of in vitro maturation of the domestic and wild cat's oocytes obtained from adult and prepubertal females were similar (47-52%). The cleavage rate after in vitro fertilization (IVF) was lower for prepubertal than adult domestic cats (42 vs. 51%; p < 0.05%). Moreover, we observed differences in the quantity (28 vs. 39%; p < 0.05) and quality of blastocysts and even greater problems with hatching blastocysts from prepubertal kittens (8 vs. 19%; p < 0.001). More blastomeres were detected in blastocysts of adult cats. They also demonstrated significantly higher number of inner cell mass (ICM) (p < 0.001) and higher number of trophoblast cells (TE) (p < 0.05).

Chemerin Impairs In Vitro Testosterone Production, Sperm Motility, and Fertility in Chicken: Possible Involvement of Its Receptor CMKLR1.

The chemokine chemerin is a novel adipokine involved in the regulation of energy metabolism but also female reproductive functions in mammals. Its effects on male fertility are less studied. Here, we investigated the involvement of chemerin in chicken male reproduction. Indeed, the improvement of the sperm of roosters is a challenge for the breeders since the sperm quantity and quality have largely decreased for several years. By using specific chicken antibodies, here we show that chemerin and its main receptor CMKLR1 (chemokine-like receptor 1) are expressed within the chicken testis with the lowest expression in adults as compared to the embryo or postnatal stages. Chemerin and CMKLR1 are present in all testicular cells, including Leydig, Sertoli, and germinal cells. Using in vitro testis explants, we observed that recombinant chicken chemerin through CMKLR1 inhibits hCG (human chorionic gonadotropin) stimulated testosterone production and this was associated to lower 3ßHSD (3beta-hydroxysteroid dehydrogenase) and StAR (steroidogenic acute regulatory protein) expression and MAPK ERK2 (Mitogen-Activated Protein Kinase Extracellular signal-regulated kinase 2) phosphorylation. Furthermore, we demonstrate that chemerin in seminal plasma is lower than in blood plasma, but it is negatively correlated with the percentage of motility and the spermatozoa concentration in vivo in roosters. In vitro, we show that recombinant chicken chemerin reduces sperm mass and individual motility in roosters, and this effect is abolished when sperm is pre-incubated with an anti-CMKLR1 antibody. Moreover, we demonstrate that fresh chicken sperm treated with chemerin and used for artificial insemination (AI) in hen presented a lower efficiency in terms of eggs fertility for the four first days after AI. Taken together, seminal chemerin levels are negatively associated with the rooster fertility, and chemerin produced locally by the testis or male tract could negatively affect in vivo sperm quality and testosterone production through CMKLR1.

Characteristics of capercaillie (Tetrao urogallus) semen analysed with flow cytometry combined with fertility results.

In order to increase the reproductive indices of capercaillie kept in closed breeding facilities, it is necessary to constantly expand the methods of better understanding the characteristics of sperm and their fertilizing potency. The aim of the study was to analyse selected features of capercaillie sperm using flow cytometry and their connection with fertility results. The study included five males, three of which were kept in a family group with eight females and two were kept alone. For sperm viability, acrosome integrity, mitochondrial potential and DNA defragmentation were assessed. Paternity analyses were performed in order to confirm the paternity of the individual and to link the evaluated semen traits with reproductive success. Analyses carried out in the flow cytometer showed any significant differences between males in sperm characteristics. In the semen of male No. 101, the father of all chicks from the analysed family group, 91.3% of live sperm, 91.5% with intact acrosome, 83.6% with active mitochondria and 2.0% with DNA defragmentation were observed. The average fertility rate was 71.0%, and chick hatchability was 100%. Using flow cytometry in the analysis of capercaillie semen and its connection with the results of natural mating, we were able to obtain deeper knowledge about new sperm characteristics that were not examined before and which in the future may be helpful in selecting males for the reproductive flocks and developing assisted reproduction techniques.

Metformin Improves Quality of Post-Thaw Canine Semen.

Sperm cryopreservation is an assisted reproductive technique routinely used in canine species for genetic conservation. However, during cryopreservation, the DNA damages are still elevated, limiting the fertilization rate. The present study was conducted to evaluate whether supplementation of canine semen extender with a molecule limiting the metabolic activities can improve the quality of frozen-thawed canine spermatozoa. We used metformin, known to limit the mitochondrial respiratory and limit the oxidative stress. Before and during the freezing procedure, metformin (50µM and 500µM) has been added to the extender. After thawing, sperm exposed to metformin conserved the same viability without alteration in the membrane integrity or acrosome reaction. Interestingly, 50µM metformin improved the sperm motility in comparison to the control, subsequently increasing mitochondrial activity and NAD+ content. In addition, the oxidative stress level was reduced in sperm treated with metformin improving the sperm quality as measured by a different molecular marker. In conclusion, we have shown that metformin is able to improve the quality of frozen-thawed dog semen when it is used during the cryopreservative procedure.