Ivan Cunha Bustamante-Filho

Criopreservação de espermatozóides eqüinos comparando duas curvas de congelamento combinadas com diluentes comerciais: uma análise laboratorial

During semen cryopreservation, sperm cells were submitted to several deleterious events leading to membrane damage which result in fertility decrease. This study was designed to compare the effects of two freezing techniques (conventional and automated), and the use of two commercial extenders as cryoprotectants (FR-5® and Botu-Crio®) on total and progressive motility, integrity and functionality of spermatic membranes during the cryopreservation of equine semen. Twenty ejaculates from two stallions were analyzed. The total and progressive motility of fresh and post-thawing semen samples were evaluated by patterns assays. Function of plasmatic membrane was measured by the hipoosmotic swelling test. Integrity of plasmatic membrane was evaluated using carboxifluorescein diacatate and iodidium propide fluorescent probes. There were significant differences between the two freezing techniques and/or between cryoprotectants for all assessed parameters. The combination of Botu-Crio® and automated curves showed better results on total and progressive post-thawing motility. The extender Botu-Crio®, alone, showed to better preserve the membrane integrity and function.

Enzymatic scavengers in the epididymal fluid: Comparison between pony and miniature breed stallions

The use of stallion semen collected from cauda epididymis for AI has increased due to the new protocols available for cryopreservation. Preserving the genetic material from valuable males that suffer sudden death or other events that prematurely end the stallions reproductive life is an important strategy for Stud breeding management. While protecting spermatozoa from oxidative stress and infectious agents, the epididymis promotes the enhancement of sperm cell morphology and changes in membrane protein profile, increasing its fertility potential. The epididymal fluid must be a balanced redox environment to allow sperm preservation and protein-protein and protein-lipids interactions to quantify. The aim of this study was quantify the enzymatic ROS scavengers in epididymal fluid of pony and miniature breed stallions. Epididymides from 8 pony stallions and 12 miniature breed stallions were dissected and fluid from caput, corpus and cauda epididymis collected. Spermatozoa were separated of epididymal fluid by 2-step centrifugation. The activities of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured and compared between stallion groups and epididymal regions. The three enzymes were present in all epididymal regions tested, with higher activities of catalase and SOD in cauda epididymis in miniature breed stallions (P<0.05). GPx activity was higher in caput epididymis in pony stallions (P<0.05), however with no difference to fluid from cauda epididymis of both breeds. These results show a difference in antioxidant enzymatic scavengers between pony and miniature breed stallions. Also, our data confirm the protective role of cauda epididymis, preserving spermatozoa integrity from oxidative damage. As glutathione peroxidase is involved in several signaling pathways, its constant activity during epididymal transit corroborates the importance of this enzyme for spermatozoa maturation.

GnRH immunization alters the expression and distribution of protein disulfide isomerases in the epididymis.

Hypogonadism is defined as the inadequate gonadal production of testosterone. Low serum testosterone leads to infertility by impairing spermatogenesis and reducing sperm count, however, the impact of hypogonadism in epididymal sperm maturation is poorly understood. From the testis, spermatozoa are transported into the epididymis where they find a specific microenvironment composed of a complex mixture of proteins that facilitate sperm storage and maturation. Inside the epididymal ductule, spermatozoa undergo several changes, resulting in their becoming capable of fertilizing eggs. Protein disulfide isomerases (PDIs) are known to participate in the folding and assembly of secreted proteins in the endoplasmic reticulum. However, little is known about the control and function of PDIs in the testis and epididymis, particularly during male development. The aim of this work was to compare the expression and distribution of PDI and PDIA3 (ERp57) in the testis and epididymis of healthy and GnRH‐immunized boars. We detected higher amounts of PDIA3 and PDI in sperm preparations and fluid from the proximal regions of the epididymis of healthy boars. However, we observed an increase in PDIA3 expression in the testis and cauda epididymis in the immunocastrated group. GnRH‐immunized boars showed a marked increase in PDI content in cauda spermatozoa and fluid, indicating a possible endocrine dysregulation of PDI. The results of our study suggest that PDIs are associated with epididymal sperm maturation and may be attractive candidates for monitoring male fertility.

Nutritional supplementation in boars with low production of semen doses

The boar in semen collection routine has a different nutritional demand in comparison to swine in weight gain. The maintenance of spermatogenesis needs a higher energy and protein intake, as well macro and micro elements have limiting effects. Despite all nutritional supplementation products for boars, there are few data available about its effects on seminal parameters of boars with low productivity of doses for artificial insemination. The aim of this work was to evaluate the effect of a nutritional supplementation on sperm quality of boars with low productivity of doses. Twenty eight breeding Landrace boars with average age of 25.28 (4.04) years and a mean weight of 300 kg were used. Animals were fed with 500g/day of a commercial nutritional supplementation, together with ration. A total of 199 ejaculates were analyzed in pre-supplementation period, and 215 ejaculates were accessed after 1 month of supplementation. The seminal parameters evaluated were: volume, sperm concentration, motility, vigor and agglutination. t-test was used to compare the means in pre and post-supplementation periods. It was observed a significant increase in ejaculate volume (p = 0.04), concentration (p = 0.03), agglutination (p = 0.002) and motility (p = 0.007). Vigor had no significant variation (p = 0.38). The average doses produced before supplementation was 23.58 (5.50), rising to 28.40 (6.27) after supplementation, showing the increased profitability with the inclusion of nutritional supplements in the diet.

Structural modeling and mRNA expression of epididymal β-defensins in GnRH immunized boars: a model for secondary hypogonadism in man: WEBER et al.

Human secondary hypogonadism is associated with impaired testicular function, however, little is known about its impact on sperm epididymal maturation. Endocrine disruption in the epididymis could impair the secretion of key proteins, such as β‐defensins, responsible for spermatozoa maturation during epididymal transit. This study evaluated the sequence and structural similarities between porcine epididymal β‐defensins porcine β‐defensins (pBD3), pBD4, pBD125, and pEP2C and their human homologs using bioinformatics integrated with information derived from protein databanks. We then verified whether the expression of pBD3, pBD4, pBD125, and pEP2C genes in the testis and epididymis are influenced by disruption of the hypothalamic‐pituitary‐testicular (HPT) axis in a pig model for male human secondary hypogonadism. Upon modeling porcine β‐defensins, structural and functional analysis confirmed the presence of motifs associated with β‐defensin function, validating the models generated in silico. pBD3 and pBD4 showed acceptable structural alignments with human β‐defensins BDEF103 and BDEF110, respectively. In addition, evaluation of hormonal regulation of β‐defensins was assessed by analyzing the expression of these four β‐defensins in adult boars immunized against gonadotropin‐releasing hormone (GnRH). Our results indicate that HPT axis disruption modifies the expression of pBD3, pBD4, pBD125, and pEP2C in boar testis and epididymis, suggesting an endocrine‐dependent regulation of β‐defensins in swine epididymis. In conclusion, sequence and structural homology between pBD3 and pBD4 and their human homologs provide a basis for using the pig as a model for the study of human secondary hypogonadism. Further investigation of the human homologs in hypogonadal men could elucidate the connections between fertility and epididymal expression of β‐defensins.

Essential oils rich in monoterpenes are unsuitable as additives to boar semen extender

Despite the development of efficient boar semen extenders, there is still room for improvement of new formulas using new molecules that could increase fertilisation outcomes and substitute cryoprotectants and antibiotics. The goal of this work was to evaluate if the essential oils from the leaves of Myrrhinium atropurpureum and Cymbopogon citratus are suitable as additives in boar semen extender. The major compounds found in the essential oils from M. atropurpureum were 1,8‐cineole (37.37%) and terpinolene (19.18%); and geranial (49.8%) and neral (33.24%) in essential oil of C. citratus. The addition of 1% and 0.1% of both essential oils to extended semen had immediate spermicidal effects (p < 0.05). Lower concentrations were tested and no cytotoxic effect was observed when M. atropurpureum essential oil was added at 0.001%. Differently, essential oil from C. citratus reduced sperm motility, membrane functionality and integrity and mitochondrial membrane potential even in concentrations as low as 0.001%. Also, addition of essential oils in low concentrations had no inhibitory effect against Escherichia coli and Pseudomonas aeruginosa growth. We conclude that the essential oils from C. citratus and M. atropurpureum, rich in monoterpenes, are cytotoxic to swine spermatozoa, therefore unsuitable as semen extender additives.

Uterine nitric oxide levels and isofluopredone treatment effect in mares susceptible to persistent post-breeding endometritis

Transient endometritis is a normal consequence of breeding and results from uterine contamination with both semen and bacteria. The modulation of the inflammatory response with the use of isoflupredone has been proposed as efficient for the treatment of endometritis by increasing pregnancy rates. The aim of the current study was to determine the effects of isoflupredone on nitric oxide (NO) levels in uterine samples from mares susceptible to persistent postbreeding endometritis, presenting or not the infectious process. Seven consecutive estrous cycles were induced in 11 mares, being the first one used as control (no treatment). All mares were submitted to the following four treatments: treatment 1: control, treatment 2: glucocorticoid (GC) treatment (20 mg isoflupredone acetate) every 12 h, for three consecutive days, treatment 3: infected treatment (intrauterine infusion of 1x109 CFU/ml Streptococcus equi subsp. zooepidemicus), treatment 4: combination of GC + infected treatment (infusion of bacteria 24 h after the first GC treatment). At 12 h after the end of each treatment, uterine samples were collected by flushing and NO was determined. After nitrate reduction, total nitrite was determined by spectrophotometer. No significant differences on nitric oxide concentration were verified by analysis of variance in the different experimental groups. It is concluded that the use of isoflupredone did not alter the nitric oxide concentration in uterine flushing’s from susceptible mares 12 h after treatment.

Prevalence and genotyping of pathogenic Escherichia coli on carcasses of pigs slaughtered in commercial slaughterhouses in southern region of Brazil

The pork is an important source of animal protein for the world, however it’s associated to food poisoning outbreaks. One of the causes of such episodes is the contamination by Escherichia coli, that can be found in the intestinal tract and environment of pigs slaughtered for production of “in natura” and industrialized meat. In the context of food safety, outbreaks of Escherichia coli that producing Shiga toxin (STEC) are the best documented. In the context of food security, outbreaks of Escherichia coli Shiga toxin-producing (STEC) are well documented. However, there are few data about the occurrence of this pathogen in swine and pork meat in Brazil. The aim of this study was to quantify contamination by E. coli of swine carcass slaughtered in abattoirs located in the Southern region of Brazil and to identify by PCR the presence of shiga toxin and intimin producing E. coli. Swabs of 272 swine carcasses were performed in slaughterhouses of RS, SC and PR States. A total of 25 carcasses were contaminated, 20 at RS and 5 at PR. DNA was extracted of positive samples for genotyping by PCR. None of the samples were positive for stx1 gene, however 13 samples were positive for the eaeA gene in different parts of the carcass. The PCR technique can be a useful tool for screening microbiological contamination through slaughter, helping to reduce the occurrence of foodborne infections outbreaks caused by E. coli and other microorganisms.

Polymorphisms in SNP CGIL4: association stuty on the phenotype of clinical mastitis resistance in Holstein cows

Bovine mastitis is the most important udder pathology and have a significant economic impact in the dairy industry. Its etiology is associated to problems with health and milking management. However, there are animals with resistance or susceptibility to mastitis, even when environmental factors are controlled. Recently, some molecular markers were associated to the phenotype of resistance to mastitis. The present study aims to verify if, based on clinical history of cows of second and third lactation, the SNP CGIL4 is associated to the phenotype of resistance to mastitis in Holstein cows in a herd in southern Brazil. Genomic DNA was obtained from blood samples of 160 Holstein cows (second and third lactation) from dairy herds from Rio Grande do Sul state, Brazil. Phenotype of resistance or susceptibility to clinical mastitis was defined based on animal’s clinical history, and the animal was classified as susceptible if presented at least 3 episodes of clinical mastitis during the last lactation. The identification of the CGIL4 SNP was performed using a PCR-RFLP. The association between genotipes and phenotipes was acessed by the χ 2 test. To detect polymorphism, Polymerase Chain Reaction (PCR) was performed using a touch-down PCR reaction producing an amplicon of 399 bp. The Restriction Fragment Length

DETECÇÃO DE LISTERIA MONOCYTOGENES PELA TÉCNICA DA REAÇÃO EM CADEIA DA POLIMERASE (PCR) EM AMOSTRAS DE LEITE BOVINO IN NATURA

Considering the possible incidence of Listeria monocytogenes in raw foods and their pathogenicity and health risk, this study aimed to compare techniques for extraction of bacterial DNA from milk samples and investigate the presence of L. monocytogenes by Polymerase Chain Reaction (PCR) in raw milk. We tested four different extraction protocols (generally identified: A, B, C, and D) for isolation of bacterial DNA directly from milk. In all of them was obtained identifying the product of 702 bp (base pairs) corresponding to the listeriolysin gene from L. monocytogenes . The protocol B containing proteinase K and phenol buffered, was chosen for the extraction of DNA from milk samples from eight dairy farms within the RS. The subsequent PCR amplification with DNA obtained by the protocol B allowed the identification of L. monocytogenes from 10 3 CFU/mL. None of the samples was positive for the producer L. monocytogenes by PCR or by conventional microbiological analysis. With this study it is concluded that the tested protocols, the protocol B was more effective for the detection of L. monocytogenes by PCR. Moreover, for the samples of the producers, the result PCR technique was obtained in a shorter time than conventional analysis of L. monocytogenes , which may allow earlier treatment of infected animals and thus avoid losses to the producer.