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The traffic in international animal products can become a public health hazard when legal import sanitary procedures are not followed. In Brazil, due to its extensive border area, the importation of animal products is a common practice in many areas, especially in Rio Grande do Sul, a state that borders Argentina and Uruguay. The objective of this study was to evaluate the presence of veterinary drug residues (antibiotics and antiparasitics) in animal products consumed in Rio Grande do Sul. The presence of residues of veterinary antibiotics and antiparasitics was assessed in 189 meat (beef, pork, and chicken), processed dairy, and meat product samples bought in Argentina (n = 90) and Uruguay (n = 99). Residues of these veterinary drugs were detected in 50 (26.45%) of the samples; 28 samples (14.81%) had antibiotic residues, and 22 samples (11.64%) had antiparasitic residues. Of the 50 positive samples, 40% (15 from Argentina and 5 from Uruguay) had residues above the maximum residue limits (MRLs). Of these 20 samples, 12 had antiparasitic residues above the MRLs (11 beef samples had ivermectin and 1 pork sample had ivermectin and doramectin) and 8 had antibiotic residues above the MRLs (2 pork and 2 sausage samples had doxycycline, 2 cheese samples had doxycycline and chlortetracycline, 1 poultry meat sample had chloramphenicol, and 1 cheese sample had monensin). Because of the potential toxic effects on humans and the potential for pathogens to develop antibiotic resistance, the presence of these residues above the MRLs is a potential risk to public health. The negative impact of consumption of imported animal products can be reduced by implementation of an effective surveillance system and educational campaigns for the general population.

Embryo cryopreservation methods have been used for commercialization and formation of genetic banks. Cryopreservation of equine embryos 300 μm in diameter, needing blastocoel collapse for satisfactory resistance to cryopreservation by vitrification. To evaluate the viability of simplified blastocoel collapse by embryo puncture with low technology and low-cost equipment, 22 embryos, collected at day 8 post-ovulation (D8), were allocated to the following groups: (1) micropuncture with a 30 G needle, assisted by a mechanical micromanipulator, before vitrification (n=4); (2) manual blade microsection before vitrification (n=6); (3) no manipulation prior to vitrification (n=8); and (4) freshly inovulated embryos (n=4). Despite the high re-expansion rates observed after vitrification, embryos manipulated prior to vitrification (groups MP and MS) did not result in pregnancy 25 days after transfer. On the other hand, embryos from groups NM (non-micromanipulated) and FR (freshly inovulated) resulted in pregnancies at 25 days. Under the conditions of the present study, manual blastocoel collapse was not efficient in increasing cryotolerance to vitrification among large embryos, requiring improvements to obtain pregnancies.

The high lipid content in porcine oocytes impairs in vitro embryo production (IVP). Here, we evaluated the influence of two different lipid modulators during in vitro maturation (IVM) on the embryo development and the lipid content of oocytes and embryos. In Experiment I, oocytes were exposed to 50 μM docosahexaenoic acid (DHA) with (+) or without (-) the presence of porcine follicular fluid (pFF). In Experiment II, phenazine ethosulfate (PES) was added during IVM at two concentrations (0.5 and 0.05 μM). The pFF- with 50 μM DHA treatment impaired nuclear maturation, cleavage and blastocyst rates (p 0.05). Oocytes in pFF- media accumulated less lipids (p 0.05). The addition of 0.5 μ M PES reduced all development rates (p 0.05) and resulted in higher lipid content for oocytes and embryos. Only 0.05 μM PES oocytes matured similarly to the control (p 0.05), although embryo development and embryo lipid content was similar to 0.5 μM PES oocytes (p 0.05). Thus, 50 μM DHA supplementation in the IVM medium without pFF impaired oocyte maturation and embryo development rates without interfering in oocyte lipid content even in the presence of pFF. Maturation with PES neither favored porcine embryo development nor reduced their lipid content.

Conditions of impaired energy and nutrient homeostasis, such as diabetes and obesity, are associated with infertility. Hyperglycemia increases endoplasmic reticulum stress as well as oxidative stress and reduces embryo development and quality. Oxidative stress also causes deoxyribonucleic acid damage, which impairs embryo quality and development. The natural bile acid tauroursodeoxycholic acid reduces endoplasmic reticulum stress and rescues developmentally incompetent late-cleaving embryos, as well as embryos subjected to nuclear stress, suggesting the endoplasmic reticulum stress response, or unfolded protein response, and the genome damage response are linked. Tauroursodeoxycholic acid acts via the Takeda-G-protein-receptor-5 to alleviate nuclear stress in embryos. To evaluate the role of tauroursodeoxycholic acid/Takeda-G-protein-receptor-5 signaling in embryo unfolded protein response, we used a model of glucose-induced endoplasmic reticulum stress. Embryo development was impaired by direct injection of tauroursodeoxycholic acid into parthenogenetically activated oocytes, whereas it was improved when tauroursodeoxycholic acid was added to the culture medium. Attenuation of the Takeda-G-protein-receptor-5 precluded the positive effect of tauroursodeoxycholic acid supplementation on development of parthenogenetically activated and fertilized embryos cultured under standard conditions and parthenogenetically activated embryos cultured with excess glucose. Moreover, attenuation of tauroursodeoxycholic acid/Takeda-G-protein-receptor-5 signaling induced endoplasmic reticulum stress, oxidative stress and cell survival genes, but decreased expression of pluripotency genes in parthenogenetically activated embryos cultured under excess glucose conditions. These data suggest that Takeda-G-protein-receptor-5 signaling pathways link the unfolded protein response and genome damage response. Furthermore, this study identifies Takeda-G-protein-receptor-5 signaling as a potential target for mitigating fertility issues caused by nutrient excess-associated blastomere stress and embryo death.

Estradiol cypionate (EC) or GnRH have been widely used for ovulation induction in timed embryo transfer (TET). EC administration increases the proportion of cows that show estrus, whereas GnRH promotes more synchronized ovulations. The aim of the present study was to evaluate the potential beneficial effects of combining EC and GnRH in TET. In experiment 1, no difference was observed on serum progesterone concentrations on Day 6 and 13 after GnRH treatment between GnRH and EC+GnRH groups. In experiment 2, pregnancy per embryo transfer (P/ET) did not differ (

Oocyte-derived bone morphogenetic protein 15 (BMP15) is one of the main local regulators of ovarian physiology, but its role in the regulation of preovulatory follicles and ovulation is not well established. Therefore, this study was conceived to determine the effect of intrafollicular injection (IFI) of BMP15 on final follicular growth, ovulation and luteinization in cattle. Initially, it was observed that relative mRNA abundance of the BMP15 receptor BMPR1B in granulosa cells was regulated by GnRH treatment, and it was negatively correlated (R

SummaryRegulation of the transforming growth factor beta (TGFβ) superfamily by gonadotrophins in swine follicular cells is not fully understood. This study evaluated the expression of steroidogenic enzymes and members of the TGFβ superfamily in prepubertal gilts allocated to three treatments: 1200 IU eCG at D −3 (eCG); 1200 IU eCG at D −6 plus 500 IU hCG at D −3 (eCG + hCG); and the control, composed of untreated gilts. Blood samples and ovaries were collected at slaughter (D0) and follicular cells were recovered thereafter. Relative gene expression was determined by real-time PCR. Serum progesterone levels were greater in the eCG + hCG group compared with the other groups (P < 0.01). No differences were observed in the expression of BMP15, BMPR1A, BMPR2, FSHR, GDF9, LHCGR and TGFBR1 (P > 0.05). Gilts from the eCG group presented numerically greater mean expression of CYP11A1 mRNA than in the control group that approached statistical significance (P = 0.08) and greater expression of CYP19A1 than in both the eCG and the control groups (P < 0.05). Expression of BMPR1B was lower in the eCG + hCG treatment group compared with the control (P < 0.05). In conclusion, eCG treatment increased the relative expression of steroidogenic enzymes, whereas treatment with eCG + hCG increased serum progesterone levels. Although most of the evaluated TGFβ members were not regulated after gonadotrophin treatment, the downregulation of BMPR1B observed after treatment with eCG + hCG and suggests a role in luteinization regulation.

Caloric restriction (CR) increases the preservation of the ovarian primordial follicular reserve, which can potentially delay menopause. Rapamycin also increases preservation on the ovarian reserve, with similar mechanism to CR. Therefore, the aim of our study was to evaluate the effects of rapamycin and CR on metabolism, ovarian reserve, and gene expression in mice. Thirty-six female mice were allocated into three groups: control, rapamycin-treated (4 mg/kg body weight every other day), and 30% CR. Caloric restricted females had lower body weight (P 0.05) and induced insulin resistance (P

Bacteriocins are ribosomal-synthesized antimicrobial peptides that inhibit the growing of pathogenic and/or deteriorating bacteria. The most studied bacteriocin-producing microorganisms are lactic acid bacteria (LAB), as they have great potential application in food biopreservation, since the majority have GRAS (Generally Recognized as Safe) status. The LAB-producing bacteriocins and/or bacteriocins produced by these bacteria have been widely studied, with the emphasis on those derived from milk and dairy products. On the other hand, isolates from meat and meat products are less studied. The objective of this review is to address the main characteristics, classification, and mechanism of action of bacteriocins and their use in food, to highlight studies on the isolation of LAB with bacteriocinogenic potential from meat and meat products and also to characterize, purify, and apply these bacteriocins in meat products. In summary, most of the microorganisms studied areLactococcus,Enterococcus,Pediococcus, andLactobacillus, which produce bacteriocins such as nisin, enterocin, pediocin, pentocin, and sakacin, many with the potential for use in food biopreservation.

Evidence points to an important role of the growth hormone (GH) in the aging process and longevity. GH-deficient mice are smaller, live longer than normal littermates, and females have an increased ovarian reserve. The aim of the study was to evaluate the role of GH in the ovarian reserve by evaluating DNA damage, macrophage infiltration, and granulosa cell number in primordial and primary follicles. Experiment 1 used GH-deficient Ames dwarf mice (df/df, n = 12) and their normal littermates (N/df, n = 12), receiving GH or saline injections. Experiment 2 included transgenic mice overexpressing bovine GH (bGH) (n = 6) and normal mice (N, n = 6). DNA damage (anti-γH2AX) and macrophage counting (anti-CD68) were evaluated by immunofluorescence. Female df/df mice had lower γH2AX foci intensity in both oocytes and granulosa cells of primordial and primary follicles (p

Germ cells are physically coupled to somatic support cells of the gonad during differentiation, but this coupling must be disrupted when they are mature, freeing them to participate in fertilization. In mammalian females, coupling occurs via specialized filopodia that project from the ovarian follicular granulosa cells to the oocyte. Here, we show that signaling through the epidermal growth factor receptor (EGFR) in the granulosa, which becomes activated at ovulation, uncouples the germ and somatic cells by triggering a massive and temporally synchronized retraction of the filopodia. Although EGFR signaling triggers meiotic maturation of the oocyte, filopodial retraction is independent of the germ cell state, being regulated solely within the somatic compartment, where it requires ERK-dependent calpain-mediated loss of filopodia-oocyte adhesion followed by Arp2/3-mediated filopodial shortening. By uncovering the mechanism regulating germ-soma uncoupling at ovulation, our results open a path to improving oocyte quality in human and animal reproduction., Uncoupling of mature oocytes from somatic granulosa cells is required for their fertilization. Here the authors show that activation of EGFR signalling in granulosa cells during ovulation triggers ERK-dependent loss of filopodia oocyte adhesion, and Arp2/3 mediated retraction of granulosa cell filopodia.

The aim of this study was to investigate the effect of calorie restriction (CR) during pregnancy in mice on metabolism and ovarian function in the offspring. Pregnant female mice were divided into two groups, a control group and a CR group (n=7 in each). Mice in the CR group were fed 50% of the amount consumed by control females from Day 10 of gestation until delivery. After weaning, the offspring received diet ad libitum until 3 months of age, when ovaries were collected. Ovaries were serially cut and every sixth section was used for follicle counting. Female offspring from CR dams tended to have increased bodyweight compared with offspring from control females (P=0.08). Interestingly, fewer primordial follicles (60% reduction; P=0.001), transitional follicles (P=0.0006) and total follicles (P=0.006) were observed in offspring from CR mothers. The number of primary, secondary and tertiary follicles did not differ between the groups (P>0.05). The CR offspring had fewer DNA double-strand breaks in primary follicle oocytes (P=0.03). In summary, CR during the second half of gestation decreased primordial ovarian follicle reserve in female offspring. These findings suggest that undernutrition during the second half of gestation may decrease the reproductive lifespan of female offspring.

The aim of this study was to evaluate the effect of growth hormone (GH) deficiency in primordial follicle reserve, DNA damage and macrophage infiltration in the ovaries of young mice. Ovaries from six-month-old GH-deficient Ames Dwarf (df/df) and Normal (N/df) mice were used. The number of primordial follicles was higher in df/df mice (p = 0.0026). Also, df/df mice had a lower number of primary (p = 0.023), secondary (p = 0.0052) and tertiary (p = 0.019) follicles. These findings indicate a slower rate of primordial follicle activation in df/df mice. Female df/df mice had decreased γH2AX foci intensity in oocytes of primordial (p = 0.015) and primary (p = 0.0004) follicles compared to N/df mice. Also, df/df mice had reduced γH2AX intensity in granulosa cells of primordial (p = 0.0002) and primary (p < 0.0001) follicles. Overall, this indicate to us that df/df mice accumulate less DNA damage in the ovarian reserve compared to N/df mice. Additionally, macrophage infiltration was also reduced in ovaries of df/df mice compared to N/df mice (p = 0.033). Interestingly, df/df mice had a reduced number of granulosa cells around primordial (p = 0.0024) and primary (p = 0.007) follicles compared to N/df mice. Also, df/df mice had a small diameter of primordial follicle nuclei (p = 0.0093), secondary follicle oocyte (p = 0.046) and tertiary follicle (p = 0.012). This points to the role of granulosa cell proliferation and oocyte growth for primordial follicle activation. The current study points to the role of the GH/IGF-I axis in extending lifespan of reproductive health, along with maintenance of oocyte DNA integrity and reduced ovarian inflammation.

ObjectiveThe aim of the study was to evaluate the role of growth hormone (GH) in DNA damage, macrophage infiltration and the granulosa cells number of primordial and primary follicles.MethodsFor these experiments six groups of female mice were used. Four groups consisted of Ames dwarf (Prop-1df, df/df, n=12) and their normal littermates (N/df, n=12) mice, between sixteen and eighteen month-old, receiving GH (n=6 for df/df, and n=6 for N/df mice) or saline injections (n=6 for df/df, and n=6 for N/df mice). The other two groups consisted of ten to twelve-month-old bGH (n=6) and normal mice (N, n=6). Immunofluorescence for DNA damage (anti-γH2AX) and macrophage counting (anti-CD68) were performed. Granulosa cells of primordial and primary follicles were counted.ResultsFemale df/df mice had lower γH2AX foci intensity in in both oocytes and granulosa cells of primordial and primary follicles (p0.05). However, bGH mice had an increased number of granulosa cells on primordial and primary follicles compared to normal mice (pConclusionThe current study points to the role of the GH/IGF-I axis in maintenance of oocyte DNA integrity and macrophage ovarian infiltration in mice.

Many microorganisms from various sources may be present in ejaculates of bulls. This study identified and isolated bacteria from bull sperm samples in a commercial stud and evaluated their resistance to antibiotics. The number of colony-forming units was determined in semen samples collected at distinct steps during freezing and thawing. The minimum inhibitory concentration and the minimum bactericidal concentration were determined for four antibiotics commonly used in commercial studs. A total of 135 microorganisms from 25 genera were isolated. After a sensitivity test, all evaluated microorganisms (n = 55) were resistant to penicillin and most of them were resistant to tylosin and lincomycin (n = 54). Resistance to all tested antibiotics was observed in 22% of all isolates, whereas only 3.9% of the isolates were inhibited by the tested antibiotics at the concentrations recommended by the international legislation. As the isolated microorganisms presented high resistance to frequently used antibiotics, sensitivity tests should be periodically conducted in commercial bull semen studs to prevent the use of contaminated semen in artificial insemination.

Bull Semen Collection and Processing Centers (SCPC) have satisfactory control of sperm quality, but commonly lack standardized quality control of hygiene procedures. This study assessed the impact of implementing a Hazard Analysis and Critical Control Points (HACCP) system in a bull SCPC, comparing microbial counts on various steps of semen processing, semen quality and costs across two periods (before and after the HACCP implementation). After surveying all routine activities of the SCPC, control points were identified, preventive measures were designed and corrective actions were employed, whenever necessary. Six months after HACCP implementation, the system was audited and production data covering two similar periods of two consecutive years were compared. Counts of colony forming units in samples collected from artificial vaginas, flexible tubes from the straw filling machine and from fresh and frozen semen after HACCP implementation were lower than during the previous period (P < 0.05). Improved post-thawing sperm motility, membrane integrity and acrosome integrity (P < 0.0001) and reduced rejection of semen batches and frozen doses were observed after HACCP implementation (P < 0.01), resulting in reduced opportunity costs. Thus, the implementation of a HACCP system in a bull SCPC allowed low-cost production of high-quality semen doses with reduced microbial contamination.

The techniques currently available for embryo collection in sheep include surgical (laparotomy; LP) and transcervical (TC) methods Although both methods are well established, there is no data about the levels of inflammatory markers in ewes after using such procedures. The objective of this study was to compare the systemic reaction to inflammation in ewes submitted to superovulation and embryo collection through LP or TC methods Blood samples were collected before each procedure (at D0) and at 3, 6 and 9 days after procedures to determine the levels of total protein, haptoglobin, fibrinogen and paraoxonase 1. The levels of inflammatory markers did not differ (P > 0.05) between methods Serum levels of haptoglobin and total protein and paraoxonase 1 activity increased after D0 (P

The aim of this study was to evaluate the effect of growth hormone (GH) in the maintenance of the ovarian primordial follicle reserve. Ovaries from 16 mo old GH-deficient Ames Dwarf (df/df) and Normal (N/df) mice were used. A subgroup of df/df and N mice received GH or saline injections for six weeks starting at 14 mo of age. In addition, ovaries from 12 mo old mice overexpressing bovine GH (bGH) and controls were used. df/df mice had higher number of primordial and total follicles than N/df mice (p < 0.05), while GH treatment decreased follicle counts in both genotypes (p < 0.05). In addition, bGH mice had lower number of primordial and total follicles than the controls (p < 0.05). pFoxO3a levels were higher in mice treated with GH and in bGH mice (p < 0.05) when comparing with age match controls. These results indicate that increased circulating GH is associated with a reduced ovarian primordial follicle reserve and increased pFoxO3a content in oocytes.

In Experiment I, during the non-breeding season, after intravaginal devices containing progesterone (P4) were withdrawn (n = 28), estrous rates were greater with treatment with 400 IU eCG (P 0.05). In Experiment II (non-breeding season), ewes of one group were treated with 300 IU eCG (n = 8) and of two groups were treated with 10 mg FSH. In one FSH group, 250 μg estradiol benzoate (EB) were administered after 24 h (n = 9); in the other, 4 μg GnRH were administered after 36 h (n = 10). Serum P4 concentrations were greater in eCG-treated ewes (P 0.05). In Experiment III (breeding season), the treatments were: 300 IU eCG; 250 μg estradiol cypionate; 250 μg EB; or control (n = 22). Follicular growth was greater for eCG-treated ewes within 0−24 h and for control ewes within 48−72 h (P = 0.001). Although estrous and ovulation rates did not differ (P > 0.05), all eCG-treated ewes had ovulations. During the non-breeding season, FSH treatment promoted follicular growth but did not induce ovulations. For FTAI regimens, eCG was more effective than FSH plus GnRH and estradiol esters in inducing estrus and ovulation.

Addition of seminal plasma (SP) to freezing extenders may help reducing post-thawing sperm cryoinjuries. This study evaluated the effects of adding heterologous (boar) SP to the freezing extender on ram sperm quality after thawing. Protein content was quantified in pooled SP samples from rams and boars. Five ejaculates from four rams were frozen in a Tris-egg yolk-glycerol extender including: no (control), ram and boar SP (both at 20% of the volume, with 48 μg protein/mL). After thawing, total and progressive motility for sperm treated with SP were lower than that of the control (P 0.05). Apoptotic-like changes were similar among treatments (P > 0.05), but there were fewer intact spermatozoa extended with boar SP than in the control (P

The aim of this study was to evaluate the effect of exposing bovine oocytes to lipopolysaccharides (LPS) in vivo and in vitro on early embryo development. In experiment 1, cumulus oocyte complexes (COCs, n = 700/group) were challenged with 0, 0.1, 1.0 or 5.0 μg/mL of LPS during in vitro maturation (IVM). Later, in vitro fertilization (IVF) and in vitro culture (IVC) were performed. In experiment 2, COCs (n = 200/group) matured and in vitro fertilized without LPS were subjected to IVC with the same doses of LPS from experiment 1. In experiment 3, heifers received two injections of saline solution (n = 8) or 0.5 μg/kg of LPS (n = 8) 24 h apart, and 3 days later, COCs were recovered and submitted to IVM, IVF, and IVC. In experiments 1 and 3, the expression of TLR4, TNF, AREG and EREG genes in cumulus cells was evaluated. Exposure to 1 and 5 μg/mL of LPS during IVM decreased nuclear maturation (39.4 and 39.6%, respectively) compared with control (63.6%, P In vivo exposure to LPS decreased the in vitro cleavage rate (54.3 vs 70.2%, P = 0.032), but cleaved embryos developed normally. Number of cells per embryo and gene expression were not affected by the LPS challenge in any experiment. In conclusion, although in vitro exposure to LPS did not affect early embryo development, in vivo LPS exposure reduced cleavage rate.

As oocytes and embryos of pigs have greater lipid content in the cytoplasm than those of other species, supplementation of the medium for in vitro maturation (IVM) of oocytes with omega-3 polyunsaturated fatty acids (PUFA) may help to improve embryo development. This study was conducted to evaluate effects of the inclusion of the docosaexaenoic (DHA) and of the eicosapentaenoic acids (EPA) in the IVM medium on the development of pig oocytes and on the lipid content of oocytes and embryos. In all experiments, control media consisted of porcine follicular fluid and oocytes were activated through parthenogenesis. In Experiment 1, there were four treatments for each PUFA: one control; and three treatments including EPA or DHA in the IVM medium at 12.5 μM, 25.0 μM and 50.0 μM). In Experiment 2, inclusion of 50 μM DHA was compared against the control. Cleavage rates in the IVM medium including 12.5 μM EPA and blastocyst development rates in media at any EPA concentration were less than for the control in Experiment 1 (P 0.05). Compared to the control, inclusion of 50 μM DHA in the IVM medium was related to greater cleavage rates and greater number of embryo cells, in Experiment 1, and lesser lipid content in oocytes after 22 and 44 h and in embryos after 7 days, in Experiment 2 (both P 0.05). Addition of DHA in the IVM medium may benefit the development of pig oocytes, but EPA appears to be cytotoxic.

Considering the specific biochemical composition of buffalo (Bubalus bubalis) meat (high iron content, high biological value proteins and essential fatty acids, low amounts of fat and cholesterol), we evaluated the influence of cutting and deboning operations on the microbiological quality and shelf-life of vacuum-packed buffalo meat stored under refrigeration. On the processing day, samples were collected from carcass, deboning room surfaces and meat cuts. Samples from meat cuts were evaluated weekly for two months. On the processing day, higher counts of Pseudomonas spp. were observed in samples from meat cuts compared with the hindquarters and the processing surfaces. For thermotolerant coliform scores, the averages were -0.5 log MPN·cm(-2), -0.4 log MPN·cm(-2) and 0.9 log MPN·g(-1), respectively. Higher counts of Pseudomonas spp. and LAB in meat cuts were observed on the processing day and after the first week of storage, respectively, remaining constant during shelf life. Listeria grayi was identified in two samples of hindquarters and meat cuts during storage. Listeria innocua was identified in one meat cut. In conclusion, cutting and deboning operations influence the microbiological quality and shelf life of vacuum-packed buffalo meat stored under refrigeration.

The ovarian follicular reserve of primordial follicle declines with aging in female mammals. Caloric restriction (CR) has been shown to increase the preservation of the ovarian follicular reserve. Likewise, rapamycin has similar effects to CR on the ovarian reserve. Therefore, the aim of our study was to evaluate the effects of rapamycin and CR on the metabolism and ovarian follicular reserve and gene expression in mice. Thirty-six female mice were used, and allocated into 3 groups: control, rapamycin (4mg/kg body weight every other day) and 30% CR. At 85 days of treatment, an insulin tolerance test (ITT) and glucose tolerance test (GTT) was performed. At 93 days ovaries were collected for analysis. CR females had lower body weight (P0.05) and were more resistant to insulin (P

Oocytes collected from prepubertal animals are known to be less developmentally competent than those from adult animals. There is evidence suggesting that acquisition of developmental competence in bovine oocytes may be linked to the expression profile of genes in the granulosa cells (GCs). Cumulus-oocyte complexes (COC) and GCs were collected from 12 Holstein heifers between 2 and 6 months of age (nine follicle-stimulating hormone [FSH] treated and three untreated) and eight FSH-treated cows. The COCs from prepubertal animals were matured, fertilized, and cultured in vitro to assess development to the blastocyst stage. The relative messenger RNA (mRNA) abundance of FSHR, StAR, CYP19A1, HSD3B1, CX43, FOXO1, and XIAP in GCs were quantified by real-time quantitative polymerase chain reaction. Results from this study revealed that GCs of prepubertal animals respond to FSH treatment by increasing mRNA levels of genes promoting estradiol synthesis and follicular growth ( FSHR and CYP19A1), and preventing cell apoptosis ( XIAP), and by decreasing mRNA levels of genes promoting progesterone production ( StAR and HSD3B1). This study also revealed that the relative mRNA abundance of FOXO1 in GCs is associated with oocyte competence to support embryo development to the blastocyst stage in prepubertal Holstein heifers.

Bull semen production centres (SPC) generally present satisfactory quality control for sperm processing, but non-standardized hygiene procedures. This study describes a Hazard Analysis and Critical Control Points (HACCP) system developed for bull SPC and subsequently implemented in a commercial SPC. After the identification of hazards at each step of semen processing and the determination of their risk and severity, monitoring and corrective procedures were designed to assess the system's efficiency. The HACCP system identified six microbiological hazards, 10 physical hazards, four chemical hazards and three critical control points. After the establishment of Good Processing Practices, Standard Operating Procedures and Standard Sanitizing Operating Procedures, the system was validated through an audit, to identify eventual failures and to define measures to correct them.

Leptin acts on energy metabolism, affecting reproductive functions through activation of its receptors in the brain and in reproductive organs. This study compared the presence of leptin and its receptor (ObR-b) in hypothalamus neurons, endometrial glands and oocytes of culled swine females across ovarian statuses and parities. Immunohistochemistry was done in samples of uterus, ovaries and hypothalamus from 28 culled females, using polyclonal antibodies antileptin and ObR-b. Immunolabelling was compared for sows categorized by parity at culling (0, 1, 2-4 and4) and ovarian status (luteal and follicular phases of the oestrous cycle and with cysts). Immunolabelling for leptin and ObR-b in neurons and oocytes was weaker in females with cysts (p 0.05) than in those at the follicular phase. In endometrial glands, leptin immunolabelling was less intense in females with cysts (p 0.05), but immunolabelling for ObR-b was similar across ovarian statuses (p 0.05). In sows culled with 2-4 parities, leptin immunolabelling in neurons and endometrial glands was more intense than in nulliparous females (p 0.05). In comparison with sows culled at greater parities, ObR-b immunolabelling for nulliparous females was less intense in endometrial glands and in oocytes (p 0.05), but more intense in neurons (p 0.05). Thus, in swine, the presence of leptin and ObR-b varies across parities and is more intense in the uterus, ovaries and hypothalamus of females that were cycling before culling than in those having cystic ovaries.

The massive agricultural expansion converted the Cerdocyon thous, a South American native predator, in vulnerable specie. Basic data, such as histological description, are important to raise awareness on animal species, helping on preservation strategies. Considering the difficult in obtain samples, as the euthanasia of wild animals for this purpose is not allowed, data on histology are very scarce or inexistent. The objective of this paper was to provide a detailed histological description of the trachea and bronchial tree of the crab-eating fox Cerdocyon thous (Linnaeus, 1766). The specimens (one adult male and one adult female) used were provided by the Federal University of Pelotas (Pelotas, RS, Brazil) Rehabilitation Center of Wild Fauna (NURFS). Tissue samples were fixed in 10% formalin and included in paraffin. After slicing, samples were stained with HE (hematoxylin and eosin), PAS (periodic acid-Schiff) and resorcin fuchsin. Trachea had an average diameter of 7.87mm, and approximately 57% of the mucosa ciliated pseudo-stratified columnar epithelium was composed of goblet cells, mostly in the dorsal region. Bronchia and bronchioles had a mucosal fold with higher number of goblet cells. Using all these techniques there is no great remarkable differences from C. thous trachea and lung, when compared with the previous described structures for carnivores and most mammals, except for the goblet cells “regionalization”. Described results are important to understand the animal physiological and behavioral habits, allowing the development of preservation and protection strategies. RESUMO: A expansão agrícola maciça tornou o Cerdocyon thous, um predador nativo sul-americano, vulnerável. Dados básicos, tais como descrição histológica, são importantes para aumentar o conhecimento sobre as espécies, ajudando nas estratégias de preservação. A eutanásia de animais selvagens para a coleta de amostras não é permitida, por isso os dados sobre a histologia são muito escassos ou inexistentes. O objetivo deste trabalho foi de fornecer uma descrição histológica detalhada da traqueia e árvore brônquica do cachorro do mato Cerdocyon thous (Linnaeus 1766). Os espécimes (um macho e uma fêmea adultos) utilizados foram fornecidos pela Universidade Federal de Pelotas (Pelotas, RS, Brasil), Centro de Reabilitação da Fauna (NURFS). As amostras de tecido foram fixadas em formalina a 10% e incluídas em parafina. Após o corte, as amostras foram coradas com HE (hematoxilina e eosina), PAS (ácido periódico de Schiff) e resorcina fucsina. A traqueia tinha um diâmetro médio de 7,87 milímetros e aproximadamente 57% do diâmetro do epitélio colunar pseudo-estratificado ciliado da mucosa composto por células caliciformes, principalmente na região dorsal do órgão. Os brônquios e bronquíolos apresentaram cararísticas similares aos outros animais, contudo aparenta ter maior número de células caliciformes. Usando distintas técnicas de coloração, observou-se que não há diferenças notáveis da traqueia e do pulmão de C. thous quando comparados com os dados para carnívoros e para a maioria dos mamíferos, exceto a regionalização de células caliciformes. Os resultados descritos são importantes para compreender a fisiologia dos animais e hábitos comportamentais, permitindo o desenvolvimento de estratégias de preservação e proteção.

Antibiotics are used in extenders for sperm cryopreservation, however no specific regulation has been established for ram sperm. This study evaluates the effects of antibiotics in ram sperm extenders for bacterial control and its effect on sperm viability. Sperm from five rams was collected, cooled and frozen using extenders with the following antibiotics: 100,000 IU/mL penicillin and 100 µg/mL streptomycin (PES); 500 µg/mL gentamycin, 100 µg/mL tylosin, 300 µg/mL lincomycin and 600 µg/mL spectinomycin (GTLS); 50 µg/mL ceftiofur sodium (CEF); and 1,000 µg/mL enrofloxacin (ENR). Bacillus sp., Corynebacterium sp., Klebsiella sp. and Staphylococcus sp. were isolated from fresh sperm and perpetual area. For cooled sperm, the antibiotic PES, GTLS and ENR were able to reduce the number of colony forming units per mL (CFU/mL), however, CEF was inefficient in all tested concentrations. Motility of cooled sperm was reduced when GTLS and ENR were used (P 0.05), however, motility was reduced with ENR treatment at doses greater than 50% (P < 0.05). The antibiogram revealed resistance of Staphylococcus sp . and Klebsiella sp. to the penicillin/streptomycin association and to tylosin. Both PES and GTLS were able to control bacterial growth on cooled sperm. However, attention should be given to the antibiotics added to the extenders, which may impair sperm motility.

Intense manipulation during beef jerky production increases the possibility of contamination with pathogenic microorganisms. This study evaluated the contamination by thermotolerant coliforms, Escherichia coli and Salmonella spp., on processing surfaces and raw materials during beef jerky production, as well as in the final product. Thermotolerant coliforms were found on all surfaces tested and in the raw material. Escherichia coli was identified in 6.7% of the surface samples, while Salmonella spp. was found in 3.3% of the surface samples and 8.6% of raw material samples. Virulence genes were detected in Salmonella spp. isolates. One Salmonella spp. isolate was resistant to sulfonamide, while one E. coli isolate was multiresistant, including the presence of resistance genes sul2, strA, strB, tetA and tetB. The presence of coliforms demonstrates failings in hygienic-sanitary procedures. The presence of pathogenic microorganisms causing foodborne diseases in the production line indicates persistent contamination in the production plant. Although the drying process applied to beef jerky should guarantee the safety of the final product, the presence of multiresistant pathogenic microorganisms, presenting virulence genes, should be a matter of concern. Because beef jerky is a ready-to-eat product, a failure in the production process may cause such microorganisms to pose a public health risk.

SummaryStaining with brilliant cresyl blue (BCB) may be used for oocyte selection, but BCB staining itself and the most commonly used selection medium (DMPBS) may compromise the development of porcine oocytesin vitro.This study evaluated DNA fragmentation, nuclear maturation, the area of migration of cortical granules (CG) and embryo development for stained (BCB+) and unstained (BCB−) oocytes incubated in DMPBS and in a modified medium (ReproPel) tested for the first time. Unexposed (UN), BCB+ and BCB− oocytes were incubated composing six groups: DMPBS/UN; DMPBS/BCB+; DMPBS/BCB−; ReproPel/UN; ReproPel/BCB+; and ReproPel/BCB−. There were more BCB+ oocytes in ReproPel than in DMPBS (P< 0.05). The DNA fragmentation was evaluated for oocytes in DMPBS/BCB+, DMPBS/BCB−, ReproPel/BCB+, ReproPel/BCB− and in porcine follicular fluid (control). The frequency of oocytes with no DNA fragmentation was greatest (64.6%) in DMPBS/BCB+ and lowest in ReproPel/BCB+ and ReproPel/BCB− (26.8 and 34.1%, respectively) (P< 0.05). Nuclear maturation rates were greater (P< 0.05) for DMPBS/BCB+ (63.1%), ReproPel/UN (55.1%) and ReproPel/BCB+ (50.2%) than for DMPBS/UN (40.8%) and ReproPel/BCB− (35.5%). The area of CG was greater (P< 0.05) for ReproPel/BCB− (80.7%) and DMPBS/UN (77.6%) than for ReproPel/UN (34.7%). Cleavage rates for DMPBS/BCB+ and ReproPel/BCB+ were greater than for DMPBS/UN (P< 0.05). Blastocyst development rates were greatest (P< 0.05) for ReproPel/UN and ReproPel/BCB+. In both media, BCB staining was apparently unable to select competent oocytes, which likely occurred due to toxicity. Despite the similar nuclear maturation and area of CG compared with DMPBS, oocytes selected in ReproPel presented impaired DNA integrity.

Leptin is a modulator of oocyte maturation and follicular development in swine. The MAPK are serine/threonine kinases that act as signal transduction pathways in swine ovaries. This study evaluated the presence of leptin, activated MAPK ERK 1/2 and p38 in oocytes of primordial/primary, secondary and tertiary follicles of gilts and sows. Ovaries from ten gilts and ten sows were collected in an abattoir, fixed in 10% formalin and prepared with classical histology methods. For immunohistochemistry, slides were incubated with polyclonal antibodies anti-leptin, anti-phospho ERK1/2 MAPK and anti-phospho p38 MAPK. Leptin immuno-labeling and the presence of activated ERK 1/2 MAPK were more intense for oocytes of sows (P0.05), whereas p38 MAPK was more active for oocytes of gilts (P0.05). Although no differences in immunolabeling for leptin and p38 MAPK were observed for oocytes of gilts at distinct follicle developmental stages (P0.05), immunolabeling was intense for oocytes of sows included in primordial/primary follicles (P0.05). Thus, leptin and p38 MAPK may be important to start oocyte development.

The effects of β-mercaptoethanol (BME) and cysteine on the viability and oxidative activity of ram sperm after thawing and on development in vitro and viability of vitrified sheep embryos were evaluated. Ejaculates from four rams were pooled and extended, composing six treatments: no antioxidants; 2mM BME; 5mM BME; 2mM BME and 5mM cysteine; 5mM BME and 5mM cysteine; and 5mM cysteine. Sperm motility, membrane and acrosome integrity, mitochondrial functionality, production of reactive oxygen species and total antioxidant capacity were similar across treatments (P>0.05). A medium with no antioxidant presented cleavage and blastocyst development rates (60.3% and 33.6%, respectively) similar (P>0.05) to those of a medium with 50μM BME and 600μM cysteine (64.3% and 36.6%, respectively). Post-thawing viability of vitrified embryos was similar between media (P>0.05). Cysteine and BME had no influence on the post-thawing viability and oxidative activity of ram sperm and on the viability of vitrified sheep embryos. RESUMO Foram avaliados os efeitos do β-mercaptoetanol (BME) e da cisteína sobre a viabilidade e a atividade oxidativa após o descongelamento do sêmen ovino e sobre o desenvolvimento in vitro e a viabilidade de embriões ovinos vitrificados. Ejaculados de quatro carneiros foram agrupados e diluídos, compondo seis tratamentos: sem antioxidantes; com BME 2mM; com BME 5mM; com BME 2mM e cisteína 5mM; com BME 5mM e cisteína 5mM; e com cisteína 5mM. Motilidade, integridade da membrana e do acrossoma, função mitocondrial, produção de espécies reativas de oxigênio e capacidade antioxidante total foram semelhantes entre os tratamentos (P>0,05). Em um meio sem antioxidantes, as taxas de clivagem e de desenvolvimento embrionário até blastocisto (60,3%, e 33,6%, respectivamente) foram semelhantes (P>0,05) às obtidas em um meio comBME 50μM e cisteína 600μM (64,3% e 36,6%, respectivamente). A viabilidade pós-descongelamento dos embriões vitrificados não diferiu entre os meios (P>0,05). O BME e a cisteína não influenciaram a viabilidade e a atividade oxidativa do sêmen ovino após o descongelamento e a viabilidade de embriões ovinos vitrificados.

Intratesticular injection (ITI) of sodium chloride (NaCl) is efficient for chemical castration of young calves, but its effects on calves welfare are unknown. Two experiments were conducted to evaluate the effects of ITI of 20% NaCl on stress and inflammatory markers in calves less than 20 days old and to assess the efficiency of ITI of 30% NaCl in 5 months old calves. In Experiment 1, control calves were only restrained and compared to calves submitted to castration through surgery (SC) and ITI with 20% NaCl (n = 9/group). No differences were observed for the eye corner temperature measured by thermography from 60 s before to 60 s after the procedures (P > 0.05). In the SC group, acute serum cortisol levels increased at 30 and 60 min after the procedure, but increased levels in the ITI group occurred only at 30 min (P 0.05). Scrotal temperature was higher at D1 in the SC group than for the other groups, but lowest at D4 compared to the control (both P < 0.05). In Experiment 2, histological sections of testes were compared after ITI with either 30% NaCl or 30% calcium chloride (CaCl2), to intact calves (control). After 60 days, intact seminiferous tubules and mediastinum were observed after ITI with 30% NaCl, whereas coagulative necrosis, inflammatory infiltration and calcification occurred after ITI with 30% CaCl2. Efficient chemical castration through ITI of 20% NaCl in young calves was followed by slight stress and inflammatory responses compared to surgical castration. However, ITI of 30% NaCl was ineffective for chemical castration of 5 months old calves.

The aim of this study was to describe the ultrastructure of sheep oocytes during 24 h of in vitro maturation (IVM). Ovaries were collected from a slaughterhouse and the follicles aspirated. The selected cumulus oocyte complexes (COC's) were divided into groups and transferred to maturation medium, where they were maintained for 6, 12, 18 or 24 h. Control immature oocytes were also collected (0 h). After different maturation intervals the structures were removed and processed for transmission electron microscopy. Immature oocytes (0 h) demonstrated pleomorphic mitochondria and presumptive lipid droplets as normally expected, and a moderate number of vesicles scattered throughout the cytoplasm. After 6 h of IVM, there was a migration of the mitochondria and presumptive lipid droplets to the cytoplasm periphery and the onset of cumulus cell expansion. Between 12 and 18 h of IVM, cytoplasmic rearrangements of the organelles were also observed e.g. aligning and increasing of cortical granules under the plasma membrane. Hooded mitochondria were observed during oocyte maturation. After 24 h of IVM, the oocytes showed signs of degeneration, which can be related to cellular aging – because of the excessive time in the maturation medium, after acquisition of developmental competence. This work described the ultrastructural aspects of sheep oocytes during IVM and provided insight into oocyte maturation in the ovine species.

The aim of the present work was to compare the efficiency of methyl-formamide (MF), dimethyl-formamide (DF) and glycerol (GL) as cryoprotectants in canine semen cryopreservation. For the experiment, pooled semen was submitted to one of the three cryoprotectants, with a final concentration of 3% in egg yolk-TRIS extender. Semen was subjectively evaluated for total and progressive motility, vigour and morphology. Sperm membrane functional integrity was assessed by hypo-osmotic swelling test (HOST), and longevity was assessed using the thermoresistance test (TRT). Fresh semen showed normal physical and morphological characteristics. After thawing, differences were observed between semen frozen using GL and DF, regarding total and progressive motility and vigour (p < 0.05), but not between MF and GL or MF and DF. Means for total motility, progressive motility, vigour and morphologically normal spermatozoa were, respectively, 69.0 +/- 5.4%, 61.0 +/- 7.4%, 2.9 +/- 0.5 and 57.1 +/- 5.0% for GL; 59.0 +/- 8.9%, 50.0 +/- 10.0%, 2.5 +/- 0.7 and 66.9 +/- 7.7% for MF; and 44.0 +/- 21.0%, 37.0 +/- 19.8%, 2.1 +/- 0.6 and 61.1 +/- 5.5% for DF. On HOST, GL was superior (p < 0.05) to MF and DF (57.8 +/- 12.4%, 35.8 +/- 18.4% and 34.4 +/- 9.4%, respectively). During the TRT, both GL and MF were superior to DF, with no differences between GL and MF. In conclusion, the use of MF as cryoprotectant showed results similar to GL, and can be considered as an alternative in canine semen cryopreservation. Further studies testing different concentrations of MF may improve its effects on cryopreservation of canine semen.

The aims of the present study were to determine the role of protein kinase C (PKC) on meiotic resumption and its effects on pronuclear formation and cleavage in the bovine. Oocytes were matured in the presence of 0, 1, 10 and 100 nM of phorbol 12-myristate 13-acetate (PMA), to evaluate the percentage of germinal vesicle breakdown. To study pronuclear formation and cleavage, oocytes were randomly distributed in four groups and matured in modified TCM-199 with LH and FSH (negative control); 10% of estrous cow serum (positive control); 100 nM of PMA (treatment); 100 nM of 4alpha-PDD (phorbol ester control). Oocytes were also matured in positive control medium, fertilized and transferred to KSOM with increasing concentrations of a PKC inhibitor. The protein profile and the presence of PKC at the end of maturation period were determined by SDS-PAGE followed by Silver Stain and Western blot, respectively. PMA stimulated meiotic resumption in a concentration-dependent manner. PKC stimulation during oocyte maturation caused an increase in pronuclear formation and did not cause parthenogenetic activation. Inhibitor of PKC (MyrPKC) inhibited cleavage in a dose-dependent and irreversible manner. A protein band around 74 kDa was not detected in PMA-treated oocytes and PKC was not detected by Western blot at the end of the maturation period. In conclusion, meiotic resumption was accelerated and the rate of oocytes with two pronuclei was increased when PKC was activated during oocyte maturation. Moreover, cleavage was inhibited in the presence of PMA.

Castration of male calves is necessary for trading to facilitate handling and prevent reproduction. However, some methods of castration are traumatic and lead to economic losses because of infection and myiasis. The objective of the present study was to evaluate the efficiency of intratesticular injection (ITI) of hypertonic sodium chloride (NaCl; 20%) solution in male calf castration during the first weeks of life. Forty male calves were allocated to one of the following experimental groups: negative control—surgically castrated immediately after birth; positive control —intact males; G1—ITI from 1- to 5-day old; G2—ITI from 15- to 20-day old; and G3—ITI from 25- to 30-day old. Intratesticular injection induced coagulative necrosis of Leydig cells and seminiferous tubules leading to extensive fibrosis. Testosterone secretion and testicular development were severely impaired in 12-month-old animals from G1 and G2 groups (P

Objetivou-se avaliar a eficácia da metil-formamida na criopreservação do sêmen ovino. O pool de sêmen utilizado no experimento foi obtido a partir da coleta, com vagina artificial, do sêmen de quatro carneiros mestiços Santa Inês, com idade aproximada de quatro anos. As coletas foram realizadas uma vez por semana, por seis semanas consecutivas, correspondendo, cada semana, a uma repetição do experimento. As frações do pool foram diluídas em cinco diferentes meios de congelação: (1) tris-gema com 5,3% de glicerol (TG5,3G); (2) tris-gema com 3% de metil-formamida (TG3MF); (3) tris-gema com 5% de metilformamida (TG5MF); (4) tris-gema com 7% de metil-formamida (TG7MF); (5) tris-gema com 9% de metil-formamida (TG9MF). Foram avaliadas a motilidade progressiva e o vigor das células espermáticas e realizado o teste de termorresistência pós-descongelação. O tratamento que obteve maior motilidade foi o TG5,3G (50%), seguido do TG3MF (38%) e os tratamentos que apresentaram menor motilidade progressiva foram TG5MF (29%), TG7MF (1,0%), TG9MF (6,0%). Os meios contendo metil-formamida apresentaram resultados inferiores ao meio controle para preservar a integridade morfológica dos espermatozoides, sendo que nos meios TG7MF e TG9MF menos de 60% de espermatozóides apresentaram-se morfologicamente normais. Os espermatozoides do meio TG5,3G apresentaram motilidade (15%) e vigor (2,8) similares aos do meio TG3MF (15% e 2,6, respectivamente) no teste de termorresistência, mas o meio TG5,3G preservou melhor a integridade funcional da membrana plasmática. O glicerol foi mais eficiente como crioprotetor do que a metil-formamida na criopreservação de sêmen ovino. The aim of this study was to evaluate the efficacy of methyl-formamide in ram semen cryopreservation. The semen pool used in this experiment was obtained by artificial vagina collection from four mixed breed Santa Inês rams, around four years of age. Semen collection was performed once a week, during six weeks. Each week corresponded to one experiment replication. The semen pool was divided in five fractions in order to be diluted in one of the following freezing media: (1) tris-egg yolk with 5.3% of glycerol (TG5.3G); (2) tris-egg yolk with 3% of methyl-formamide (TG3MF); tris-egg yolk with 5% of methyl-formamide (TG5MF); tris-egg yolk with 7% of methyl-formamide (TG7MF); tris-egg yolk with 9% of methyl-formamide (TG9MF). Semen progressive motility, vigor and thermoresistance were evaluated. The treatments TG5.3G (50%) and TG3MF (38%) showed higher progressive motility after thawing, while TG5MF (29%), TG7MF (1%) and TG9MF (6%) showerd lower motility. Freezing media containing methyl-formamide were less effective in preserving spermatozoa membrane integrity and morphology than control media. In TG7MF and TG9MF extenders, less than 60% spermatozoa showed normal morphology. After thermoresistance test, semen cryopreserved in TG3MF showed vigor (2.6) and motility (15%) statistically similar to TG5.3G media (15% and 2.8, respectively); however, the extender TG5.3G was more effective in preserving plasma membrane functional integrity. In conclusion, in the experimental conditions used, glycerol showed more cryoprotectant potential than methyl-formamide.