Sperm chromatin condensation as an in vivo fertility biomarker in bulls:a flow cytometry approach

Background:Genetic selection in cattle has been directed to increase milk production.This,coupled to the fact that the vast majority of bovine artificial inseminations(AI)are performed using cryopreserved sperm,have led to a reduction of fertility rates over the years.Thus,seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive efficiency.In humans,sperm chromatin condensation evaluated through chromomycin A3(CMA3)has recently been purported to be a powerful biomarker for sperm functional status and male infertility.The objectives of the present study were:a)to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability,and b)to test whether this parameter could be used as a predictor of in vivo fertility in bulls.The study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein males.Reproductive outcomes of each sire were determined by non-return rates,which were used to classify bulls into two groups(highly fertile and subfertile).Results:Chromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow cytometry.Sperm quality parameters(morphology,viability,total and progressive motility)were also assessed.Pearson correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo fertility.Sperm morphology,viability and total motility presented an area under the ROC curve(AUC)of 0.54,0.64 and 0.68,respectively(P>0.05),and thus were not able to discriminate between fertile and subfertile individuals.Alternatively,while the percentage of progressively motile sperm showed a significant predictive value,with an AUC of 0.73(P=0.05),CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in bulls.Specifically,the percentage of viable sperm with poor chromatin condensation showed better accuracy and precision to predict in vivo fertility,with an AUC of 0.78(P=0.02).Conclusions:Chromatin condensation evaluated through CMA3/Yo-Pro-1 and flow cytometry is defined here as a more powerful tool than conventional sperm parameters to predict bull in vivo fertility,with a potential ability to maximising the efficiency of dairy breeding industry.     

Effect of low oxygen tension atmosphere and maturation media supplementation on nuclear maturation, cortical granules migration and sperm penetration in swine in vitro fertilization

The aim of this study was to evaluate the efficiency of low oxygen tension (5% CO(2) , 5% O(2) and 90% N(2) ) on in vitro oocyte maturation using defined media (0.1% polyvinyl alcohol - PVA) or 10% porcine follicular fluid (PFF)-supplemented media. To achieve this goal, oocytes were evaluated regarding cortical granules (GCs) migration, nuclear maturation and sperm penetration. Oocytes were in vitro matured under different conditions: 5% or 20% O(2) atmosphere and 0.1% PVA- or 10% PFF-supplemented media and evaluated at 0 and 44 h of maturation. To evaluate the migration of CGs and nuclear maturation, by confocal microscopy, oocytes were incubated with 100 μg of FITC-PNA/ml and 10 μg/ml of propidium iodide. To address sperm penetration, after maturation, in vitro fertilization for 6 h and in vitro culture for 18 h, zygotes were incubated with 10 mg/ml Hoechst 33342. Pronuclei and polar bodies were quantified using an epifluorescence microscope. Atmosphere conditions did not affect the CGs migration, but media supplementation did. Oocytes matured in 10% PFF media had a higher percentage of CGs in the oocyte periphery than oocytes matured in PVA-supplemented media. However, this fact did not have effect on in vitro sperm penetration levels. No effect of atmosphere conditions and media supplementation was observed on the rates of metaphase II oocytes. Therefore, the use of low oxygen tension in association with PVA maturation media does not improve the in vitro maturation system of porcine oocytes, because its use did not improve nuclear maturation, CGs migration and zygotes monospermic rates.     

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.     

Acid-base and biochemical stabilization and quality of recovery in male cats with urethral obstruction and anesthetized with propofol or a combination of ketamine and diazepam

This study compared acid-base and biochemical changes and quality of recovery in male cats with experimentally induced urethral obstruction and anesthetized with either propofol or a combination of ketamine and diazepam for urethral catheterization. Ten male cats with urethral obstruction were enrolled for urethral catheterization and anesthetized with either ketamine-diazepam (KD) or propofol (P). Lactated Ringer’s solution was administered by intravenous (IV) beginning 15 min before and continuing for 48 h after relief of urethral obstruction. Quality of recovery and time to standing were evaluated. The urethral catheter was maintained to measure urinary output. Hematocrit (Hct), total plasma protein (TPP), albumin, total protein (TP), blood urea nitrogen (BUN), creatinine, pH, bicarbonate (HCO₃⁻), chloride, base excess, anion gap, sodium, potassium, and partial pressure of carbon dioxide in mixed venous blood (pvCO₂) were measured before urethral obstruction, at start of fluid therapy (0 h), and at subsequent intervals. The quality of recovery and time to standing were respectively 4 and 75 min in the KD group and 5 and 16 min in the P group. The blood urea nitrogen values were increased at 0, 2, and 8 h in both groups. Serum creatinine increased at 0 and 2 h in cats administered KD and at 0, 2, and 8 h in cats receiving P, although the values were above the reference range in both groups until 8 h. Acidosis occurred for up to 2 h in both groups. Acid-base and biochemical stabilization were similar in cats anesthetized with propofol or with ketamine-diazepam. Cats that received propofol recovered much faster, but the ketamine-diazepam combination was shown to be more advantageous when treating uncooperative cats as it can be administered by intramuscular (IM) injection.     

Differences in helminth infections between captive and wild spur-thighed tortoises Testudo graeca in southern Spain: a potential risk of reintroductions of this species

Although the spur-thighed tortoise, Testudo graeca, is one of the most widely distributed species of tortoises, its natural populations are threatened through its whole range. Particularly at south-eastern Spain, the species is mainly threatened by habitat destruction and over-collection, given that this chelonian has been traditionally considered an appreciate pet. As south-eastern Spanish wildlife recovery centers shelter hundreds of captive animals mainly coming from illegal trade or captive-bred, there is a strong debate about what to do with these animals: maintaining them in captivity all along their lives or reintroducing them to wildlife. It is well known that the reintroduction of captive animals supposes a risk for the wild population due to the uncertainty of their genetic origin and to the possible spread of infectious diseases. However, despite the increasing evidence that infectious agents are a potential health hazard for wildlife, little is known about the risk that introduced parasites could suppose for the wild populations of spur-thighed tortoise. The present study investigates for the first time the presence of helminth eggs and worms in faeces from 107 wild and captive individuals collected from mid-March to mid-June 2010, and relates the findings to different environmental and host variables. Sixteen oxyurid species and the ascarid Angusticaecum holopterum were identified. This last nematode and the oxyurid species Tachygonetria palearticus and T. seurati had not been reported in Spanish wild T. graeca previously. The prevalence of oxyurid eggs and worms were 94% and 70%, respectively; while, ascarid eggs and worms were found in 26% and 5% of tortoises, respectively. Ascarid infections affected mostly captive animals and were associated to caparace deformities and symptoms of upper respiratory tract disease (p<0.05). Oxyurid infections were not associated to negative health traits and prevalence increased with age. In free-living tortoises, the distribution of pharingodonid genera also varied according to habitat; moreover, T. longicollis, T. pusilla, T. conica, T. robusta and Mehdiella stylosa where significantly more frequent in wild compared to captive tortoises (p<0.05). Study results highlight important differences in the nematode fauna of captive and free-living tortoises and questions one more time if the reintroductions of captive animals suppose a risk for the wild population since the former ones can harbor and distribute among free populations pathogens like ascarid nematodes.     

Humoral and cellular immune responses in pigs immunized intranasally with crude rhoptry proteins of Toxoplasma gondii plus Quil-A

We evaluated the humoral and cellular immune responses in pigs immunized intranasally with crude rhoptry proteins of Toxoplasma gondii plus Quil-A. The experiment used 13 mixed-breed pigs divided into the following three groups: G1 (vaccinated-challenged, n=6), which received the rhoptry vaccine (200(g/dose); G2 (adjuvant-challenged, n=4), which received PBS plus Quil-A; and G3 (unvaccinated-challenged, n=3), which was the control group. The treatments were performed intranasally at days 0, 21, and 42. Three pigs from G1 produced IgG and IgM antibody levels above the cut-off in the ELISA on the challenge day. Partial protection was observed in G1 at the chronic phase of infection when compared with G3. The preventable fractions were 41.6% and 6.5%, in G1 and G2, respectively. The results of this study suggest that rhoptry proteins plus Quil-A stimulated humoral, local, and systemic immune responses, which were able to partially protect the brain from cyst formation.     

Is it possible to store spotted wolffish (Anarhichas minor) sperm by refrigeration?

Fish Physiology and Biochemistry - Spotted wolffish Anarhichas minor reproduction in captivity is dependent on in vitro fertilization. However, it is often challenging to acquire sufficient fresh...     

Anatomicohistological Characteristics of the Female Genital Organs of the White‐lipped Peccary (Tayassu pecari) in the Peruvian Amazon

This study examined the anatomical and histological characteristics of the genital organs of the female white‐lipped peccary in the wild in different reproductive stages, collected by rural hunters in the North‐eastern Peruvian Amazon. Mean ovulation rate was 2.12 ± 0.83 follicles and litter size was 1.78 ± 0.41 embryos or fetuses per pregnant female, resulting in a low rate of reproductive wastage, averaging 0.33 ± 0.66 (16.04%) oocytes or embryos per pregnancy. The ovulation rate and the anatomical performance of the uterus could limit the prolificacy of this species. Females in follicular phase showed follicular waves suggesting the synchronous growth of a cohort of follicles. Different uterine and vaginal epithelium features changed in accordance with the reproductive state of the female. Pregnant females and females in the luteal phase presented a significant proliferation of endometrial uterine glands, characterized by hyperplasia and branching of endometrial glands, and increase in the proportion of cervical epithelial cells with periodic acid‐schiff (PAS)‐positive granules compared with that in females in the follicular phase. Females in the follicular phase showed a more developed vaginal epithelium (in thickness and in layer composition) than females in the luteal phase and pregnant females.     

Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection

Neospora caninum is an intracellular parasite that causes major economic impact on cattle raising farms, and infects a wide range of warm-blooded hosts worldwide. Innate immune mechanisms that lead to protection against this parasite are still unknown. In order to investigate whether myeloid differentiation factor 88 (MyD88) is required for resistance against N. caninum, genetically deficient mice (MyD88^−/−) and wild type littermates were infected with live tachyzoites and the resistance to infection was evaluated. We found that sub-lethal tachyzoite doses induced acute mortality of MyD88^−/− mice, which succumbed to infection due to uncontrolled parasite replication. Higher parasitism in MyD88^−/− mice was associated with the lack of IL-12 production by dendritic cells, delayed IFN-γ responses by NKT, CD4⁺ and CD8⁺ T lymphocytes, and production of high levels of IL-10. MyD88^−/− mice replenished with IL-12 and IFN-γ abolished susceptibility as the animals survived throughout the experimental period. We conclude that protective IFN-γ-mediated immunity to N. caninum is dependent on initial MyD88 signaling, in a mechanism triggered by production of IL-12 by dendritic cells. Further knowledge on Toll-like receptor recognition of N. caninum antigens is encouraged, since it could generate new prophylactic and therapeutic tools to control parasite burden.