Letrozole administration effects on the P450aromatase expression and the reproductive parameters in male sheep (Ovis aries).

Letrozole comprises a non-steroid aromatase inhibitor that has been applied as a preventive for many uses, such as breast cancer prevention, treatment of hormonal dysfunction, and male infertility. Precisely in Northeast Brazil, ovine consist of the leading livestock produced, and their reproduction in captivity has been demonstrated difficult. Thus, we hypothesized whether the application of letrozole will improve male sheep reproduction. One group of 6 animals received a daily dosage of 0.5mg/Kg of letrozole for 60 days, while the other six animals were used as control. Samples were collected from control and treated animals after 30 and 60 days of the experiment. Blood samples were collected to measure the steroid hormone levels. Semen was collected from control and treated groups using an artificial vagina and cryopreserved for spermatozoa morphology and CASA analysis. The testicles were collected for histological analysis, gene expression, and immunohistochemistry of P450aromatase protein. Hormone levels demonstrate no differences in the estradiol/testosterone levels among the control and both treated groups. Immunohistochemistry analysis revealed the presence of P450aromatase protein in spermatogonia cells and Leydig cells in the control and treated groups in both periods analyzed. Moreover, there were no differences in the P450aromase gene expression in the control and treated group. Morphological analysis of the spermatozoa revealed that letrozole treatment did not affect mitochondrial activity or cause any deformities. In addition, motility parameters in the sperm from the treated group were not affected by letrozole treatment compared to the control group. Morphological analysis of the testis demonstrated that letrozole treatment increase the seminiferous tubule area but no signs of germ cell damage. Our results show that letrozole has a morphological effect on the testicles in the ovine model but no pathological or severe effect caused by hormone level imbalance. Overall, letrozole comprises a non-steroid aromatase inhibitor, which can be applied to ovine reproduction.

Lipid peroxidation in bull semen influences sperm traits and oxidative potential of Percoll®-selected sperm.

Although bovine embryo in vitro production (IVP) is a common assisted reproductive technology, critical points warrant further study, including sperm traits and oxidative status of sperm for in vitro fertilization (IVF). Our aim was to evaluate whether the lipid peroxidation index of commercial bull semen is influenced by sperm traits and oxidative status of sperm populations selected using Percoll® gradient. Semen straws from 48 batches from 14 Nelore bulls were thawed individually, analyzed for motility and subjected to Percoll selection. After Percoll, the lipid peroxidation index of the extender was evaluated, whereas selected sperm were analyzed for motility, acrosome and membrane integrity, mitochondrial membrane potential, chromatin resistance and oxidative potential under IVF conditions. Batches were divided retrospectively in four groups according to lipid peroxidation index. Sperm from Group 4 with the lowest index of lipid peroxidation had, after Percoll selection, greater plasma membrane integrity (81.3%; P = 0.004), higher mitochondrial potential (81.1%; P = 0.009) and lower oxidative potential (135.3 ng thiobarbituric acid reactive substances (TBARS)/ml; P = 0.026) compared with Group 1 with highest lipid peroxidation index (74.3%, 73% and 213.1 ng TBARS/ml, respectively). Furthermore, we observed negative correlations for the lipid peroxidation index with motility, membrane integrity and mitochondrial potential, and positive correlations with oxidative potential. In conclusion, oxidative stress in semen straws, as determined using lipid peroxidation in the extender, is associated with sperm traits and their oxidative potential under IVF conditions. These results provided further insights regarding the importance of preventing oxidative stress during semen handling and cryopreservation, as this could affect sperm selected for IVF. Finally, Percoll selection did not completely remove sperm with oxidative markers.

Reproductive and endocrinological effects of Benign Prostatic Hyperplasia and finasteride therapy in dogs.

Benign prostatic hyperplasia (BPH) is one of the most important reproductive disorders in aging dogs. Therapeutic measures include orchiectomy and pharmacological treatment, leading to reduction of prostate volume and clinical signs. One of the most common drugs used in BPH treatment is finasteride, but data regarding its possible side effects are scarce. Thus, the aim of this study was to evaluate the effects of BPH and short-term (2 months) finasteride therapy on clinical, endocrinological, and reproductive parameters in dogs. Dogs were allocated into four experimental groups: Non-affected (n = 5), BPH (n = 5), Non-Affected-Finasteride (n = 5) and BPH-Finasteride (n = 5) groups. Dogs were evaluated monthly during 2 months by a complete breeding soundness examination, B-mode ultrasound and Doppler ultrasonography of the testicular artery, hormonal profile (testosterone, estrogen and dihydrotestosterone) and oxidative profile of the prostatic fluid. After 2 months, dogs were gonadectomized and testicles were subjected to histologic analysis. Finasteride treatment reduced dihydrotestosterone concentrations, without negative influence on semen quality and also reverted testicular hemodynamics changes of BPH. On the other hand, BPH was accompanied by significant changes in testosterone and estrogen concentrations and semen quality, mainly related to sperm kinetics alterations. In conclusion, BPH dogs have important hormonal and sperm alterations, however, short-term finasteride treatment (2 months) was able to reduce overall effects of BPH, thus representing a method of therapy for BPH treatment.

Serological response against bovine herpesvirus and bovine viral diarrhea virus induced by commercial vaccines in Holstein heifers / Resposta sorológica contra herpesvírus bovino e vírus da diarreia viral bovina induzida por vacinas comerciais em novilhas Holandesas

Vaccination is a strategy to the prevention and control of reproductive diseases caused by bovine viral diarrhea virus (BVDV) and bovine herpesvirus type 1 (BoHV-1), however the various compositions of commercial vaccines should be evaluated for their ability to induce protection mediated by antibodies. The objective of this research was to evaluate the production of specific neutralizing Abs against BVDV-1 and 2, and BoHV-1 induced by commercial vaccines composed by different adjuvants. Holstein heifers were vaccinated and distributed in three experimental groups: Group I (G1) was vaccinated with a commercial vaccine containing inactivated BVDV-1, BVDV-2 and BoHV-1 diluted in alum hydroxide as adjuvant (n=9); Group II (G2) was vaccinated with an product containing inactivated strains of BVDV-1, BVDV-2, BoHV-1 and BoHV-5 diluted in oil emulsion as adjuvant (n=10); Group III (G3) was vaccinated with a commercial vaccine containing inactivated BVDV-1 and BVDV-2, besides live modified thermosensitive BoHV-1, diluted in Quil A, amphigen and cholesterol (n=10); A control, non-vaccinated group (n=6) was mock vaccinated with saline. Heifers received two subcutaneous doses of 5mL of each commercial vaccine on the right side of the neck, with 21 days interval. Humoral immune response was assessed by the virus neutralization test (VN) against BVDV-1 (NADL and Singer strains), BVDV-2 (SV253 strain) and BoHV-1 (Los Angeles strain) in serum samples collected on vaccination days zero (D0), 21 (D21) and 42 (D42; 21 days after boosting). Neutralizing Abs against BVDV-1 NADL was detected only in D42, regardless of the vaccine used. Similar geometric mean titers (GMT) for BVDV-1 NADL were observed between G1 (log2=5.1) and G3 (log2=5.1). The seroconversion rate (%) was higher in G1 (78%) when compared to G2 (10%) and G3 (40%). For BVDV-1 Singer, it was also possible to detect Abs production in G1 (log2=5.8, 100% seroconversion rate) and G3 (log2=3.5, seroconversion rate = 60%), only after the booster dose (D42). Neutralizing Abs to BVDV-2 (SV253) were detected only in G3, observing 90% seroconversion associated with high titers of Abs (log2=6.7) after the 2nd dose of vaccine (D42). Heifers from G1 and G3 responded to BoHV-1 after the first dose (D21): G1 (log2=2.5, seroconversion rate = 67%) and G3 (log2=0.7, seroconversion rate = 80%). In D42, a higher magnitude response was observed in the heifers from G3 (log2=6.1, 100%) compared with G1 (log2=4.3, 100%) and G2 (log2=2.7, 60%). Based on the data obtained, it can be concluded that the commercial vaccine contained aluminum hydroxide (G1) was most effective in the induction of antibodies against BVDV-1. On the other hand, this vaccine did not induce the production of neutralizing Abs against BVDV-2. Only the heifers from G3 (Quil A, amphigen and cholesterol) generated neutralizing Abs against BVDV-2. The animals that received commercial vaccine containing oil emulsion as adjuvant (G2) had a weak/undetectable response against BVDV-1 and BVDV-2. The best protective response against BoHV-1 was observed in heifers vaccinated with the live modified thermosensitive virus.(AU)

A vacinação é utilizada como estratégia para a prevenção e controle das doenças reprodutivas, causadas pelos vírus da diarreia viral bovina (BVDV) e herpesvírus bovino tipo 1 (BoHV-1), entretanto, as diversas composições de vacinas comerciais devem ser avaliadas quanto a sua eficiência protetiva mediada por anticorpos (Acs). O objetivo desta pesquisa foi avaliar a produção Acs neutralizantes específicos para cepas de BVDV-1 e 2, e BoHV-1 induzida por vacinas comerciais contendo diferentes tipos de adjuvantes. Para tal, novilhas Holandesas foram vacinadas e distribuídas em três grupos experimentais: Grupo I (G1) foi vacinado com uma vacina comercial composta por cepas inativadas de BVDV-1, BVDV-2 e BoHV-1 diluídas em hidróxido de alumínio como adjuvante (n=9); Grupo II (G2) foi vacinado com produto contendo as cepas inativadas de BVDV-1, BVDV-2, BoHV-1 e BoHV-5 em uma emulsão oleosa como adjuvante (n=10); O Grupo III (G3) foi vacinado com uma vacina comercial contendo BVDV-1 e BVDV-2 inativado, além do BoHV-1 vivo modificado e termosensivel, diluídos em adjuvante contendo Quil A, Amphigem e colesterol (n=10); O Grupo Controle não vacinado (n=6) foi inoculado com solução salina. As novilhas receberam duas doses das respectivas vacinas ou solução salina (5mL), com intervalo de 21 dias, por via subcutânea, na tábua do pescoço do lado direito. A resposta imune humoral foi avaliada pelo teste de vírus neutralização (VN) contra o BVDV-1 (cepas NADL e Singer), BVDV-2 (cepa SV253) e BoHV-1 (cepa Los Angeles) em amostras de soro coletadas nos dias (D) de vacinação zero (D0), 21 dias após 1ª dose (D21)e 42 (D42; 21 dias após A 2ª dose). Os anticorpos neutralizantes contra o BVDV-1 NADL foram detectados apenas em D42, independentemente da vacina utilizada. Os títulos médios geométricos (GMT) de anticorpos foram semelhantes entre G1 (log2=5,1) e G3 (log2=5,1). A taxa de soroconversão foi maior no G1 (78%) quando comparado ao G2 (10%) e G3 (40%). Para o BVDV-1 Singer, somente após D42 foi observada a produção de Acs no G1 (log2=5,8; taxa de soroconversão de 100%) e G3 (log2=3,5; taxa de soroconversão = 60%). Os anticorpos contra BVDV-2 (SV253) foram detectados apenas nas novilhas do G3, observando-se taxa de soroconversão de 90% com altos títulos de anticorpos neutralizantes (log2=6,7) em D42. Novilhas G1 e G3 responderam ao BoHV-1 após a primeira dose (D21): G1 (log2=2,5; taxa de seroconversão = 67%) e G3 (log2=0,7; taxa de seroconversão = 80%). Em contrapartida, foi observada uma maior magnitude de resposta para as novilhas G3 (log2=6,1; 100%) em D42, em relação aos animais G1 (log2=4,3; 100%) e G2 (log2=2,7; 60%). Com base nos dados obtidos, foi possível concluir que a vacina composta por hidróxido de alumínio (G1) foi mais eficaz na produção de anticorpos contra o BVDV-1, em contrapartida esse produto não induziu anticorpos contra o BVDV-2. Apenas as novilhas do G3 (Quil A, amphigen e colesterol) geraram Acs neutralizantes contra o BVDV-2. Os animais que receberam a vacina em emulsão oleosa (G2) como adjuvante apresentaram uma resposta fraca/indetectável contra o BVDV-1 e BVDV-2. A melhor resposta protetiva contra o BoHV-1 foi observada nas novilhas vacinadas com a vacina viva modificada termosensível.(AU)

Field-testing a single-dose immunocontraceptive in free-ranging male capybara (Hydrochoerus hydrochaeris): Evaluation of effects on reproductive physiology, secondary sexual characteristics, and agonistic behavior.

Controlling wildlife populations to mitigate human-wildlife conflicts and the spread of zoonotic diseases is an ever-growing necessity. The objective of this study was to evaluate a single-dose anti-gonadotropin-releasing hormone vaccine (GonaCon, USDA/NWRC, Fort Collins, CO, USA) as a non-lethal alternative for population control in free-ranging, synanthropic male capybara. In addition to infertility efficacy of this treatment, potential effects on the alpha male's secondary sexual characteristics and agonist behavior need to be assessed because any alterations in these factors could lead to population management failure. The treatment group (n = 3) received 1 mL of the anti-GnRH vaccine, intramuscularly, and the control group (n = 2) a 1 mL sham vaccine. Reproductive behavior and social group dynamics were monitored for 30 days prior to inoculation (June 2017) with continuous observations occurring during the study period. Antifertility effects were assessed by conducting exams of testicular morphology, semen characteristics, and histological analysis (after 270 days via hemi-gonadectomy). Compared to the control group, the testicles of the treated males had severe atrophy (P <  0.05), oligozoospermia and greater numbers of sperm cells in a static developmental phase. Courtship and agonistic alpha male behavior were not altered, and the group's social integrity was maintained. Results indicate there was 100% infertility in capybara males, observed throughout the study period of 18 months, and equally important, the male's alpha characteristics were not affected by the treatment, which is imperative for successful capybara population control efforts.

Antioxidant Effect of a Polyphenol-Rich Murtilla (Ugni molinae Turcz.) Extract and Its Effect on the Regulation of Metabolism in Refrigerated Boar Sperm.

The production of reactive oxygen species (ROS) in boar spermatozoa increases in refrigeration; this can have an impact on sperm quality and fertilization capacity. We evaluated the effect of polyphenol-rich aqueous extract of murtilla (Ugni molinae Turcz) on boar sperm stored at 17°C in order to reduce oxidative stress and improve sperm quality in the long term. Five experiments were performed: first, characterization of the polyphenol content from five genotypes of murtilla; second, determination of the genotype with the best antioxidant effect (MT-Ex); third, the antioxidant capacity on O2 - and lipid peroxidation; fourth, the influence of MT-Ex on motility, calcium movement, cAMP, and metabolic parameters; and fifth, analysis of long-term refrigeration. The average phenolic content was 344 ppm; gallic acid, catechin, quercetin, myricetin, and kaempferol were detected. All extracts evaluated presented a concentration-dependent antioxidant effect. MT-Ex reduces intracellular O2 -/peroxides but low lipid peroxidation. MT-Ex in nonstimulated ROS conditions reduces sperm motility, mitochondrial membrane potential, cAMP, and ATP, but the succinate dehydrogenase activity remained normal; also, we observed a reduction in calcium movement in in vitro sperm capacitation. The long-term analyses showed that MT-Ex improved sperm motility decay and reduced membrane damage and ROS at 168 h. Based on this study, we propose MT-Ex as a supplement in semen extenders.

Assessment of different sperm functional tests in golden-headed lion tamarins (Leontopithecus chrysomelas).

The golden-headed lion tamarin (Leontopithecus chrysomelas) is an endangered species endemic to Brazil's Atlantic Forest, a shrinking biodiversity hotspot. As in other Neotropical primates, its semen characteristics and freezability are poorly studied. Hence, reproductive technologies for callitrichids would greatly benefit from reliable methods of semen analysis. In a bid to promote reproductive research in tamarins, we validated simple and inexpensive sperm function tests that can be used to monitor sperm-egg binding, plasma membrane and acrosome integrity, mitochondrial activity, and DNA fragmentation. Ejaculates from adult males were individually diluted and divided into control and damage-induced aliquots, and then samples comprising assorted amounts of damaged spermatozoa were examined by organelle-specific tests. Our findings showed that sperm-binding in chicken egg perivitelline membrane (EPM) positively correlated with the number of spermatozoa injured by snap-freezing. Eosin-nigrosin (EN) and propidium iodide readings were correlated with each other, and both provided robust measurements of plasma membrane integrity. A high correlation between expected and measured amounts of acrosome-intact spermatozoa was found using Fast Green-Rose Bengal (FG-RB), Coomassie Blue (CB), and FITC-PSA stains, and all three methods exhibited comparable results. Likewise, different percentages of UV-irradiated spermatozoa were accurately assessed for DNA integrity by Toluidine Blue (TB) and sperm chromatin dispersion (SCD) tests. Comparisons between 3,3'-diaminobenzidine (DAB) and JC-1 stains also indicated the reliability of the former assay to ascertain gradual increases in spermatozoa with greater mitochondrial function. These data confirmed that different parts of the tamarin spermatozoa can be simply and consistently evaluated by EPM, EN, FG-RB, CB, TB, and DAB protocols.

Immunolocalisation and expression of oxytocin receptors and sex hormone-binding globulin in the testis and epididymis of dogs: correlation with sperm function.

The aim of this study was to confirm gene and protein expression of oxytocin receptor (OTR) and sex hormone-binding globulin (SHBG) in the testis and epididymis of dogs, correlating these data with sperm quality and production and testosterone concentrations. Positive correlations were found between OTR and SHBG expression in both the testis and epididymis. Testicular OTR expression was positively associated with plasma membrane and acrosome integrity in canine spermatozoa, whereas SHBG expression in the testis was positively correlated with various sperm characteristics, such as sperm concentration, total and progressive motility, plasma membrane integrity and acrosome integrity. Testicular expression of both OTR and SHBG was negatively correlated with low sperm mitochondrial activity. In the epididymis, SHBG expression was only positively correlated with plasma membrane integrity. Analysis of protein expression revealed that testicular OTR was positively correlated with testosterone concentrations and negatively correlated with the absence of sperm mitochondrial activity. In addition, SHBG expression in the testes was associated with epididymis SHBG expression and morphologically normal cells. Immunohistochemical (IHC) analysis revealed the presence of both OTR and SHBG in testicular smooth muscles and Leydig cells. However, in the epididymis, OTR was only located in smooth muscle cells, whereas neither IHC nor western blotting detected SHBG. Together, the results of this study suggest that OTR and SHBG play key roles in spermatogenesis and sperm maturation, being essential for male reproductive success.

Follicular environment as a predictive tool for embryo development and kinetics in cattle.

Follicular fluid composition and the transcription pattern of granulosa cells were analysed to better comprehend associations between embryo development and morphokinetics. Bovine follicles were punctured and their respective follicular fluid and granulosa cells were collected. Cumulus-oocyte complexes derived from these follicles were matured and fertilised invitro. Embryo morphology and kinetics were evaluated at 40h after insemination, when embryos were classified as fast (FCL, four or more cells), slow (SCL, 2-3 cells) or non-cleaved (NCL). Their development was followed until the blastocyst stage. Glucose, pyruvate, cholesterol and oestradiol were quantified in the follicular fluid and the transcription pattern of 96 target genes was evaluated in granulosa cells by large-scale quantitative reverse transcription polymerase chain reaction. Follicular fluid from the blastocyst group had increased levels of glucose, total cholesterol and pyruvate compared to the non-blastocyst group, whereas higher levels of oestradiol were observed in the follicular fluid of embryos and blastocysts with fast cleavage. The transcriptional pattern revealed altered metabolic pathways between groups, such as lipid metabolism, cellular stress and cell signalling. In conclusion, both follicular fluid and granulosa cells are associated with the possibility of identifying follicles that may generate embryos with high potential to properly develop to the blastocyst stage.

Sperm traits on in vitro production (IVP) of bovine embryos: Too much of anything is good for nothing.

Sperm samples used on fertilization strongly influence the in vitro production (IVP) rates. However, sperm traits behind this effect are not stated consistently until now. This study aimed to evaluate the isolated and combined effect of some sperm traits (MB: total motility before Percoll® gradient, MA: total motility after Percoll® gradient, AI: acrosome integrity, MI: membrane integrity, MP: mitochondrial membrane potential, and CR: chromatin resistance) on IVP rates. This is the first study focusing on the isolated effect of distinct traits. For this purpose, the experiment was divided in three steps. In first step, to study behavior of traits sperm samples (n = 63 batches) were analyzed and ranked based on each trait. In second step, samples ranked were selected from target ranks regions and allocated in groups of four to five batches, creating Higher and Lower groups, according to two different approaches. One aimed to form groups that differed to all sperm traits simultaneously (effect of combined traits). The other aimed to form groups that differed only to a single sperm trait while no differences were observed for the remaining traits (effect of each isolated trait). In third step, for each group successfully formed in step 2, sperm samples were individually and prospectively used for IVP. Cleavage, embryo development and blastocyst rates were recorded and compared between Higher and Lower of respective trait groups. Surprisingly, evaluation of isolated effects revealed that lower levels of MB, AI and MP resulted in higher embryo development and blastocyst rates (p<0.05), which was not observed on cleavage rate. We conclude that sperm traits strongly influence embryo development after in vitro fertilization (IVF), affecting the zygote competence to achieve blastocyst stage. Individually, levels of MB, AI or MP could be some of the key traits that may define IVP efficiency on current systems of embryo production.

Validation of simple and cost-effective stains to assess acrosomal status, DNA damage and mitochondrial activity in rooster spermatozoa.

Several methods have been developed to evaluate spermatozoa function in birds but many of these are sometimes complicated, costly and not applicable to field studies (i.e., performed within poultry breeding facilities). The objective was, therefore, to validate efficient, practical and inexpensive procedures to determine DNA fragmentation, acrosomal integrity, and mitochondrial activity in poultry spermatozoa. Initially, ejaculates were individually diluted and divided into control (4°C, 4h) and UV-irradiated aliquots (room temperature, 4h), and then samples containing different percentages of DNA-damaged spermatozoa (0%, 25%, 50%, 75% and 100%) were subjected to Toluidine Blue (TB) and Sperm Chromatin Dispersion assessments (SCD). Fast Green-Rose Bengal (FG-RB) and FITC-PSA staining protocols were subsequently used to assess acrosome status in aliquots comprising assorted amounts of acrosome-reacted spermatozoa. Furthermore, to validate 3,3'-diaminobenzidine (DAB) assay, ejaculates containing different gradients of spermatozoa with great amounts of mitochondrial activity were concurrently evaluated using DAB and JC-1 stains. The proportion of spermatozoa with abnormal DNA integrity when evaluated using the TB assessment correlated significantly with the expected percentages of UV-irradiated spermatozoa and with SCD results. A significant linear regression coefficient was also observed between expected amounts of acrosome-intact spermatozoa and FG-RB readings, and there was a significant correlation of the data when FG-RB and FITC-PSA were used. Likewise, the use of the DAB assay enabled for accurately ascertaining percentages of rooster spermatozoa with greater and lesser mitochondrial function, and results were highly correlated to results with staining with JC-1. Altogether, findings of the present study indicate acrosomal status, DNA integrity and mitochondrial activity in rooster spermatozoa can be easily and reliably determined using FG-RB, TB and DAB stains.

Antioxidant enzyme profile and lipid peroxidation products in semen samples of testicular germ cell tumor patients submitted to orchiectomy

ABSTRACT Purpose To determine enzymatic antioxidant and lipid peroxidation levels in seminal plasma of patients orchiectomized for testicular tumors. Materials and Methods The study included 52 patients: 26 control men and 26 orchiectomized patients for testicular tumor, of which 12 men had seminoma tumor and 14 men non-seminoma tumor. After semen analysis performed according to the WHO guidelines, an aliquot of semen was centrifuged and the seminal plasma was collected. Lipid peroxidation was performed by thiobarbituric acid reactive substances (TBARS) assay and antioxidant profile was assessed by analyzing catalase, glutathione peroxidase (GPx) and superoxide anion (SOD) activities using colorimetric assays with a standard spectrophotometer. Data were tested for normality and compared using one-way ANOVA (p<0.05). Results Seminoma and non-seminoma groups presented lower sperm concentration and morphology when compared to control group (p=0.0001). Both study groups (seminoma and non-seminoma) presented higher TBARS levels when compared to control group (p=0.0000013). No differences were observed for SOD (p=0.646) andGPx (p=0.328). It was not possible to access the enzymatic activity of catalase in any group. Conclusion Patients with testicular tumor present increased semen oxidative stress, but no differences were observed in antioxidant levels, even after orchiectomy. This indicates that most likely an increased generation of oxidative products takes place in these patients.

The Stimulated Glycolytic Pathway Is Able to Maintain ATP Levels and Kinetic Patterns of Bovine Epididymal Sperm Subjected to Mitochondrial Uncoupling.

Studies have reported the importance of mitochondria in sperm functionality. However, for some species, the glycolytic pathway appears to be as important as oxidative phosphorylation in ATP synthesis and sperm kinetics. These mechanisms have not been fully elucidated for bovine spermatozoa. Therefore, the aim of this study was to evaluate the role of mitochondria and the glycolytic pathway in ATP synthesis, sperm movement patterns, and oxidative homeostasis of epididymal spermatozoa in bovine specimens. We observed that mitochondrial uncoupling with protonophores significantly reduced ATP levels. However, these levels were reestablished after stimulation of the glycolytic pathway. We verified the same pattern of results for sperm kinetic variables and the production of reactive oxygen species (ROS). Thus, we suggest that, after its appropriate stimulation, the glycolytic pathway is capable of maintaining ATP levels, sperm kinetic patterns, and oxidative balance of bovine epididymal spermatozoa submitted to mitochondrial uncoupling.

Antioxidant enzyme profile and lipid peroxidation products in semen samples of testicular germ cell tumor patients submitted to orchiectomy.

PURPOSE: To determine enzymatic antioxidant and lipid peroxidation levels in seminal plasma of patients orchiectomized for testicular tumors. MATERIALS AND METHODS: The study included 52 patients: 26 control men and 26 orchiectomized patients for testicular tumor, of which 12 men had seminoma tumor and 14 men non-seminoma tumor. After semen analysis performed according to the WHO guidelines, an aliquot of semen was centrifuged and the seminal plasma was collected. Lipid peroxidation was performed by thiobarbituric acid reactive substances(TBARS) assay and antioxidant profile was assessed by analyzing catalase, glutathione per-oxidase (GPx) and superoxide anion (SOD) activities using colorimetric assays with a standard spectrophotometer. Data were tested for normality and compared using one-way ANOVA (p<0.05). RESULTS: Seminoma and non-seminoma groups presented lower sperm concentration and morphology when compared to control group (p=0.0001). Both study groups (seminoma and non-seminoma) presented higher TBARS levels when compared to control group (p=0.0000013). No differences were observed for SOD (p=0.646) and GPx (p=0.328). It was not possible to access the enzymatic activity of catalase in any group. CONCLUSION: Patients with testicular tumor present increased semen oxidative stress, but no differences were observed in antioxidant levels, even after orchiectomy. This indicates that most likely an increased generation of oxidative products takes place in these patients.

C-Peptides for diagnostics and therapy: a veterinary medicine point of view / Peptídeos-C para diagnóstico e terapia: um ponto de vista na medicina veterinária

Empirical studies proved that C-peptides are performing numerous intrinsic biological roles, and serve as a marker for pancreatic performance analysis. Since the last decade, C-peptide assays for differential diagnosis in veterinary diabetic patients are becoming more available, but still only for a very limited number of species. Studies on C-peptide as a diagnostic tool, therapy for associated complications, or as replacement therapies for C-peptide deficiency still showed not to be a common practice in veterinary medicine. This review was conducted to determine the potential importance of C-peptide in Veterinary Medicine, relevant in the diagnosis of diabetes and for other metabolic processes, as well as its proposed therapeutic benefits. Numerous articles were identified that reported positive results in their experimental studies, whether C-peptide as a biomarker for pancreatic performance in dogs, cats, and horses, as a non-invasive method to monitor nutritional status in primates, or to investigate its potential therapeutic benefits for diabetes-related illnesses.(AU)

Os estudos empíricos provaram que o peptídeo C realiza várias funções biológicas intrínsecas e serve também como um marcador para a análise de desempenho do pâncreas. Nesta última década os ensaios peptídeo C para o diagnóstico diferencial em doentes diabéticos veterinários estão mais disponíveis, contudo apenas em um limitado número de espécies. Estudos sobre C-peptídeo como ferramenta de diagnóstico, terapia de complicações associadas ou como terapias de reposição na sua deficiência ainda não é prática comum na Medicina Veterinária. Esta avaliação foi realizada para determinar a importância potencial de peptídeo C na Medicina Veterinária, relevante no diagnóstico de diabetes e também em outros processos metabólicos, assim como os benefícios terapêuticos propostos. Foram identificados numerosos artigos que reportaram resultados positivos nos seus estudos experimentais, quer o peptídeo C fosse utilizado como biomarcador para o desempenho do pâncreas em cães, gatos e cavalos, como um método não invasivo para monitorar o estado nutricional em primatas, quer para investigar o seu potencial terapêutico benéfico para doenças relacionadas ao diabetes.(AU)

Sperm-binding to the perivitelline membrane of chicken egg yolk as a functional test for sperm evaluation in dogs / Teste de ligação espermática à membrana perivitelínica da gema de ovo de galinha como avaliação funcional do sêmen de cães

During fertilization, spermatozoa interact with the zona pellucida (ZP) through the binding between the acrosome and proteins 2 and 3 (ZP2 and ZP3). The perivitelline membrane of chicken egg yolk is homologous to the mammalian ZP3, which allows the binding of sperm of several species. The aim of this study was to standardize and evaluate the efficiency of sperm-binding to the perivitelline membrane of chicken eggs as a functional method for canine semen evaluation. For this purpose, nine post-thaw sperm samples were used, which were divided into two aliquots: the first was kept in water bath at 37ºC (live sample) and the second was submitted to cold shock to induce cellular damage (dead sample). The two aliquots were mixed on five proportions, corresponding to 0%, 25%, 50%, 75%, and 100% of viable cells, and the binding test was performed by analyzing the number of spermatozoa bonded to the perivitelline membrane by means of computerized assessment of sperm motility (CASA) or conventional microscopy. Additionally, samples were submitted to sperm motility analysis, evaluation of plasmatic and acrosomal membrane integrity, and sperm mitochondrial activity. The sperm-binding test to the perivitelline membrane of chicken egg yolk was considered a feasible sperm analysis test for both fertilizing capacity and overall sperm attributes evaluation, mainly when the analysis is performed by a conventional microscope, which expands its practicality to the majority of canine reproduction laboratories.(AU)

Durante a fecundação, os espermatozoides interagem com a zona pelúcida (ZP) por meio da ligação entre o acrossomo e as proteínas 2 e 3 (ZP2 e ZP3). A membrana perivitelínica da gema de ovo de galinhas é homóloga à ZP3 de mamíferos, possibilitando a ligação espermática de diversas espécies. Este trabalho padronizou e avaliou a eficiência do teste de ligação espermática à membrana perivitelínica da gema de ovo de galinhas como avaliação funcional do sêmen de cães. Para tal, foram utilizadas nove amostras seminais previamente criopreservadas. Cada amostra foi dividida em duas alíquotas: a primeira foi mantida em banho-maria à 37ºC (vivos) e a segunda submetida a choque térmico com o intuito de induzir dano celular (mortos). As duas alíquotas foram misturadas, correspondendo a 0, 25, 50, 75 e 100% de células viáveis. As amostras foram avaliadas quanto ao número de espermatozoides ligados à membrana perivitelínica por meio da análise computadorizada da motilidade (CASA) ou microscopia convencional. Ademais, as amostras foram avaliadas quanto à motilidade espermática, integridade das membranas acrossomal e plasmática e atividade mitocondrial espermática. O teste de ligação espermática à membrana perivitelínica de ovos de galinha foi considerado um teste de análise seminal exequível tanto para avaliar a capacidade fecundante dos espermatozoides como atributos seminais gerais, especialmente quando realizado em microscopia convencional, expandindo sua praticidade para a maioria dos laboratórios de análise de sêmen canino.(AU)

Avaliação do tocoferol no congelamento do sêmen bovino e nas taxas de prenhez após inseminação artificial em tempo fixo / Tocopherol evaluations at the freezing of bovine semen on the pregnancy rates after fixed time artificial insemination

O presente estudo avaliou se a adição de tocoferol no diluidor para a criopreservação de sêmen bovino reduz os danos causados pelo estresse oxidativo e melhora a capacidade fertilizante do sêmen utilizado na IATF. No primeiro lote sincronizado foram utilizadas 84 fêmeas Nelore (Bostaurusindicus) e no segundo lote 44 fêmeas. Para a inseminação artificial as vacas foram divididas aleatoriamente em dois grupos: controle (inseminadas com sêmen criopreservado sem uso de aditivos) e tocoferol (inseminadas com sêmen criopreservado com adição de 10mmol/mLtocoferol). Foram utilizados sêmen de três reprodutores da raça Nelore (Bos taurus indicus), sendo as doses provenientes de uma única partida, distribuída aleatoriamente entre as fêmeas inseminadas. O diagnóstico de gestação foi realizado 35 dias após a IATF por exame ultrassonográfico retal. O experimento foi realizado em delineamento inteiramente casualizado, e a taxa de prenhez comparada pelo teste qui-quadrado. A adição de 10mmol/mL de tocoferol no diluidor do sêmen não influenciou (P>0,05) na taxa de prenhez após IATF em comparação ao grupo controle (sem aditivos) para as médias do primeiro lote sincronizado (n=84; 38,5% vs 40%), segundo lote sincronizado (n=44; 28% vs 31,6%) e para a média geral dos lotes (n=128; 34,4% vs 37,5%). Nas condições experimentais a adição de 10mmol de tocoferol ao meio crioprotetor do sêmen não melhorou a taxa de prenhez após a inseminação artificial em tempo fixo na espécie bovina.

The present study evaluated if the addition of tocopherol to the extender semen cryopreservation reduces the damage caused by oxidative stress and preserves the fertilizing capacity of semen used in FTAI. In the first lot we used 84 synchronized females Nelore (Bos taurus indicus) and the second lot 44 females.For the artificial insemination the cows were randomly divided into two groups: control (inseminated with semen cryopreserved without using additives) and treatment (inseminated with semen cryopreserved with added 10 mmol/ml tocoferol). We used three semen sires Nelore (Bos taurus indicus), with doses starting from a single, randomly distributed among females inseminated. The pregnancy diagnosis was done 35 days after FTAI by rectal ultrasonography. The experimental design was completely randomized and pregnancy rates compared by chi-square test. There were no effect (P> 0.05) in pregnancy rate using cryopreserved semen with added 10mmol/ml tocopherol in the bovine semen extender compared to the control group (no additives) in the first synchronized group (n = 84, 38.5% vs 40%), in the second synchronized group (n = 44, 28% vs 31.6%) and the all animals (n = 128, 34.4% vs 37.5%). We conclude that, under the experimentalconditions, the addition of 10 mmol of tocopherol in the semen extender did not improve the pregnancy rate after FTAI in the bovine species.

Seasonal variation in urinary and salivary reproductive hormone levels in Amazonian manatees (Trichechus inunguis).

The Amazonian manatee (Trichechus inunguis) is a threatened aquatic mammal endemic to the Amazon basin. The aim of this study was to evaluate the urinary and salivary reproductive hormone levels of captive Amazonian manatees collected during two seasons of the year. Salivary samples from four males and urinary and salivary samples from three females were collected during two seasons (March-June and September-November) over two consecutive years. Salivary testosterone in males was measured by radioimmunoassay and reproductive hormones in females (salivary progesterone and oestradiol and urinary progestogens, oestrogens and luteinising hormone) were measured by enzyme immunoassay. The data were analysed in a 2×2 factorial design, where the factors were year and season. There was no effect of year or season for salivary testosterone. All female hormones showed a seasonal effect (higher hormone levels during March-June than September-November) or an interaction between year and season (P<0.05). These results strongly indicate the existence of reproductive seasonality in Amazonian manatees; however, apparently only females exhibit reproductive quiescence during the non-breeding season. Further long-term studies are necessary to elucidate which environmental parameters are related to reproductive seasonality in T. inunguis and how this species responds physiologically to those stimuli.

Oxidative and functional status of bovine semen cryopreserved in different seasons / Perfil oxidativo e funcional de sêmen bovino criopreservado em diferentes estações do ano

In general, Taurus bulls under tropical conditions demonstrate reduced fertility due to heat and oxidative stress on testicular tissue. This high incidence of sperm damage is generally enhanced by the large amount of polyunsaturated fatty acids (PUFA) naturally present in bull sperm. Despite PUFA increase cellular sensitivity to lipid peroxidation, they are essential for membrane fluidity and also promote high cellular protection during cryopreservation process. Some reports related that animals and plants provided from cold weather present higher cellular concentration of PUFA, so the present study aims to compare the effect of the season on sperm quality of Taurus and Zebu bulls. Cryoprotected semen samples of 10 Nelore (Bos taurus indicus) and 10 Simmental (Bos taurus taurus) bulls were analyzed during winter and summer seasons. After freezing-thawing process, semen samples were submitted to sperm motility and vigor analysis, to tests of plasma membrane integrity (MPIEosin/Nigrosin), acrosomal integrity (MAI-POPE), DNA fragmentation degree (DNAf-Comet Assay) and high mitochondrial activity (ACM-DAB). Moreover, frozen-thawed semen samples were induced to lipid peroxidation for measurement of thiobarbituric acid reactive substances (TBARS) and malondialdehyde (MDA) concentration. Higher post-thawing sperm motility (26.50% winter; 13.30% summer) and ACM (22.34% winter; 13.30% summer) were observed during the winter in Nelore group. In Simmental group, no differences were observed for the studied variables. It was concluded that, despite the heat stress, no seasonal effect on sperm quality was observed in Taurus cattle, which may be related to higher concentration of PUFA in seminal composition.

Bovinos taurinos, em clima tropical, mostram menores índices de fertilidade devido ao estresse térmico e oxidativo testicular. Essa alta incidência de alterações espermáticas é potencializada pela grande quantidade de ácidos graxos poli-insaturados (PUFA) presentes nos espermatozoides taurinos. Embora os PUFA aumentem a sensibilidade celular à peroxidação lipídica, são essenciais para a maleabilidade de membrana e promovem maior proteção celular durante a criopreservação. Devido aos relatos de que animais e vegetais de clima frio possuem maior concentração de PUFA nas células, o presente estudo teve como objetivo comparar o efeito da estação do ano sobre a qualidade espermática de taurinos e zebuínos. Foram avaliadas amostras criopreservadas de 10 touros da raça Nelore (Bos taurus indicus) e 10 da raça Simental (Bos taurus taurus), coletadas durante inverno e verão. Após a descongelação, foram realizados testes espermáticos de motilidade e vigor, testes de integridade de membrana plasmática (MPIEosina/Nigrosina), integridade acrossomal (MAI-POPE), grau de fragmentação de DNA (DNAf-Ensaio Cometa) e atividade citoquímica mitocondrial (ACM-DAB). Adicionalmente, avaliou-se a suscetibilidade das células espermáticas à peroxidação lipídica induzida, pela mensuração das substâncias reativas ao ácido tiobarbitúrico (TBARS) e malondialdeído (MDA). No grupo Nelore, foi observada maior motilidade espermática pós-descongelação (26,50% inverno; 13,30% verão) e maior ACM (22,34% inverno; 13,30% verão) durante o inverno. No grupo Simental, não houve diferença de época do ano nas variáveis. Concluiu-se que, apesar de sofrerem maior estresse térmico, nos animais taurinos não foi observado efeito da estação do ano sobre a qualidade espermática, o que pode estar relacionado a uma maior concentração de PUFA em sua composição seminal.

Endocrine monitoring of the ovarian cycle in captive female Amazonian manatees (Trichechus inunguis).

The Amazonian manatee (Trichechus inunguis; Mammalia: Sirenia), a threatened aquatic mammal endemic to the Amazon basin, is the only sirenian that lives exclusively in fresh water. Information about the reproductive endocrinology of the Amazonian manatee is scarce; therefore, the aim of this study was to monitor salivary progesterone and estradiol patterns during the ovarian cycle in T. inunguis. Salivary samples were collected daily during a 12-week period of two consecutive years from two captive adult females. The salivary estradiol and progesterone were measured by enzyme immunoassay. The results were analyzed in an iterative process of excluding values that were higher than the mean plus 2 standard deviations until the basal values were determined. The interval between two peaks of salivary estradiol followed by a rise of progesterone was considered as one complete cycle for the calculation of the cycle length. We observed only three complete cycles in all samples analyzed. The cycle length ranged from 42 to 48 days (mean of 44.67 days). We also observed two distinct salivary estradiol peaks during all cycles analyzed, with the first peak occurring before the rise in salivary progesterone and the second occurred followed by a return to basal progesterone levels. This is the first in-depth study of the ovarian cycle in Amazonian manatees. Our results demonstrate that salivary samples can be a useful tool in the endocrine monitoring of this species and suggest that T. inunguis shows a peculiar hormonal pattern during the ovarian cycle, a finding that may have physiological and ecological significance in the reproductive strategy of these animals.