Histopatology of the reproductive tract of Nellore pubertal heifers with genital ureaplasmosis.

In order to study and characterize the lesions in the reproductive tract of Nellore heifers naturally infected with Ureaplasma diversum and presenting granular vulvovaginitis syndrome (GVS), fragments of uterine tube, uterus, cervix, vagina and vulva of 20 animals were evaluated. The macroscopic lesions of the vulvovaginal mucosa were classified in scores of "1" mild, until "4", severe inflammation and pustular or necrotic lesions. The histopathological evaluation was performed using scores of "1" to "4", according to the inflammatory alterations. The fragments with severe microscopic lesions (3 and 4) were from the uterine tubes and uterus, which showed leukocytes infiltration and destruction and/or necrosis of epithelium. Alterations in the lower reproductive tract fragments were mild, but characteristics of acute inflammatory processes. The histopathological findings of the reproductive tract of females naturally infected with Ureaplasma diversum are consistent with injuries that compromise the environment from the local where spermatozoa acquires ability to fertilize an oocyte until those where the oocyte is fertilized. Therefore, animals with GVS should be identified early in the herd, because, besides the reduction in the fertility rates caused by tissue damages, they can contribute to disseminate the microorganism. Key words: bovine, tissue evaluation, reproduction, Ureaplasma diversum.

Assessment of cow and farm level risk factors associated with Ureaplasma diversum in pasture-based dairy systems - A field study.

Potential risk factors for Ureaplasma diversum in the vaginal mucus of 1,238 dairy cows were included in a multivariate logistic regression model, based on the cow level (i.e., granular vulvovaginitis [+GVV], yearly milk production [4500 kg or more], pregnancy, predominance of Bos taurus [+Bos Taurus], score of corporal condition [at least 2.5], concomitant positivity for Escherichia coli [+E.coli]), and farm level i.e., milking room hygiene (-Milking room), dunghill location, and replacement female). Ureaplasma diversum was present in 41.1% of the samples. Independent risk factors for U. diversum were +GVV (odds ratio [OR], 1.31); +Mycoplasma spp (OR, 5.67); yearly milk production (4500 kg or more) (OR, 1.99); +Bos taurus (OR, 1.68); +E. coli (OR, 4.96); -milking room (OR, 2.31); and replacement females (OR, 1.89). Ureaplasma diversum vaginal colonization was strongly associated with Mycoplasma spp., E. coli, and number of pregnant cows.

Effects of calving season on the voluntary waiting period and reproductive performance of Holstein cows in the tropical savannah.

The effects of calving season [rainy (RS) and dry (DS)] on the voluntary waiting period (VWP) of 58 Holstein cows raised in the tropical savannah were investigated using data of temperature humidity index (THI), total antioxidant status (TAS), respiratory rate (RR), rectal temperature (RT), glucose, total cholesterol (TC), triglycerides (TG), velocity of uterine regression, and subsequent reproductive performance. Blood samples and clinical data were taken once every week, from calving until the sixth postpartum week. Reproductive data were collected until 180 days postpartum. THI differed between seasons (P < 0.05], as well as TAS (P < 0.001), RR (P < 0.001), RT (P < 0.01), glucose (P < 0.001), TC, and TG (P < 0.05), with higher values in RS. Although the velocity of uterine regression showed to be slower (P < 0.001) during RS, no differences were present regarding uterine health. Days open increased in RS (P < 0.001), but the number of services/conception was similar (P = 0.33). The results suggested cows under heat stress during the rainy season in the tropical savannah are more susceptible to a decline in the reproductive performance due to oxidative, metabolic, and uterine health problems.

Photodisinfection of Alphaherpesvirus 1 in bovine semen.

Reproductive biotechnologies are widely consolidated as a methodology in cattle breeding and have an important impact on the genetic improvement of cattle herds. Semen is an important source of dissemination of pathogenic microorganisms during reproductive procedures. To ensure the sanitary quality of the semen, it is essential to consider the presence of various microorganisms including viruses. One of the main viral agents of reproductive interest is Bovine Alphaherpesvirus 1 (BoHV-1), the etiological agent responsible for bovine rhinotracheitis and vulvovaginitis and frequently associated with reproductive efficiency of matrices and bulls. In artificial insemination centers, semen treatment is generally based only on the use of antibiotics, ignoring the possibility of inactivating other non-bacterial infectious agents. In this context, photodisinfection emerges as a promising alternative to inactivate a wide range of microorganisms, offering a complementary or substitution approach to those conventional semen treatment methods. In this work, we evaluated the use of four halogenated sulfonated porphyrins as potential photosensitizers (PSs) for photodynamic inactivation of Bovine Alphaherpesvirus I (BoHV-1) for bovine semen disinfection. The PSs were synthesized and photophysical parameters, such as UV-Vis absorption spectra and singlet oxygen quantum yield (ΦΔ) were presented. Photoinactivation of BoHV-1 was first shown in cell culture and then confirmed in artificially infected bovine semen and then the phototoxicity of PSs against spermatozoa was evaluated. All PSs were effective in BoHV-1 inactivation; however, the photosensitizer containing two chlorine atoms, showed to be more efficient due to the shorter time required for complete viral inactivation. The slight alterations in sperm kinetics were observed, but remained within those acceptable by regulatory agencies for animal reproduction. Although the methodology used in this work only included bovine semen, we emphasize that the proposed photodisinfection methodology can be adapted and applied to a wide range of biological materials and microorganisms of animal or human interest.

Ovarian evaluation of Girolando (Holstein × Gir) heifers submitted to a GnRH-PGF2α-GnRH protocol in the dry or rainy seasons in the tropical savannah.

The Girolando breed is used in pasture-based dairy production systems in Brazil to associate the high production of Bos taurus to the rusticity and thermal adaptation of Bos indicus. This study was designed to evaluate the physiological response to a gonadotropin-releasing hormone (GnRH)-prostaglandin F2α (PGF2α)-GnRH protocol to synchronize the ovulation in 40 Girolando heifers of a pasture-based dairy production system and its relationships with the temperature and humidity index (THI) during the dry (DS) and rainy season (RS) in the tropical savannah-Brazil's cerrado biome. Responses were characterized by follicular and corpus luteum number and diameter, ovulation (D9), and pregnancy rates after first AI. Total follicle number (8.1 ± 0.3 × 8.8 ± 0.3), D9 ovulatory follicle diameter (11.9 ± 0.4 × 10.1 ± 0.4 mm), corpus luteum diameter (8.6 ± 1.3 × 3.9 ± 1.5 mm), corpus luteum score (3.7 ± 0.8 × 1.8 ± 1.0), corpus luteum diameter after AI (9.6 ± 1.6 × 3.9 ± 1.5 mm), and corpus luteum score after AI (3.2 ± 0.4 × 0.9 ± 0.6) in DS and RS differed (P < 0.01). D9 ovulation rate was 40 % (DS) and 20 % (RS), without differences (P > 0.05). Pregnancy rate was 45 % (DS) and 11 % (RS), with differences (P < 0.01). THI differed between DS and RS (P < 0.01). THI may interfere in the follicular and luteal dynamics and in the response of Girolando heifers to the GnRH-PGF2α protocol in the tropical savannah, thus reducing the chances of pregnancy at the first artificial insemination.

Blastocoel fluid removal and melatonin supplementation in the culture medium improve the viability of vitrified bovine embryos.

In this study, we investigated the effects of melatonin supplementation in the culture medium and blastocoel fluid removal (BFR) before vitrification on the quality and viability of in vitro-derived bovine embryos. After fertilization, presumptive zygotes were assigned to one of the following treatments: control, in vitro standard culture (IVC) medium; IVC + M10-9, IVC medium supplemented 10-9 M melatonin; or IVC + M10-9 BFR, IVC medium supplemented with 10-9 M melatonin plus BFR on day 7 (D7) of culture. D7 blastocysts were vitrified by the Cryotop method and, after 5 mo of storage, were warmed and incubated for an additional 72 h. The re-expansion rate was evaluated after 2 and 24 h, and the hatching rate was evaluated after 24, 48, and 72 h. At 72 h, the total number of cells (TNC); number of apoptotic cells (NAC); and expression of genes related to oxidative stress (HSPA5), cell metabolism (SLC2A3), cell repair (MSH6), placentation (KRT8 and PLAC8), and implantation (FOSL1) were assessed in the blastocysts. Less than 30% of the control blastocysts re-expanded until 2 h, whereas more than 85% of the IVC + M10-9 and IVC + M10-9 BFR blastocysts re-expanded (P < 0.05). The hatching rate of IVC + M10-9 BFR blastocysts increased at all time points (P < 0.05), reaching 66.8% at 72 h of incubation. The TNC was similar among treatments (P > 0.05), regardless of vitrification/warming and re-cultivation. The NAC:TNC was smaller for melatonin-treated blastocysts (P < 0.05). BFR increased HSPA5 (P = 0.0118) expression and did not affect SLC2A3, MSH6, KRT8, and FOSL1 expression (P > 0.05). In conclusion, melatonin (10-9 M) supplementation in the culture medium and BFR on D7 of culture increased the hatching rate 24, 48, and 72 h after warming of the vitrified embryos, indicating an improvement in cryotolerance.

Spatial distribution and risk factors for sheep toxoplasmosis in Goiás, Brazilian Cerrado Region.

The present study aimed to evaluate the spatial distribution and risk factors for infection by Toxoplasma gondii in sheep in the state of Goiás, located in the central-western region of Brazil. Through the immunofluorescence antibody test (IFAT), the seroprevalence of anti-T. gondii antibodies was analyzed in 1000 blood serum samples obtained from sheep in all macro and micro regions of the state of Goiás. Data related to sex, age of the animals, size of the farm, type of farm, water source, veterinary assistance, replacement of the herd, presence of domestic cats, presence of wild cats and presence of other wild animals were obtained at the sampling time. The differences between the seroprevalences obtained in relation to the variables analyzed were estimated using Pearson's chi-square test (χ2). The odds ratio (OR) values for each risk factor evaluated were statistically analyzed with a confidence interval of 95%. Positivity for IgG anti-T. gondii was observed (titer ≥64) in 34.3% (343/1000) of the samples, which ranged from 26.9% (31/115) to 44.2% (53/120) and from 21.8 (12/55) to 55.2% (16 / 29), respectively in the analyzed mesoregions and microregions. In all investigated regions of the State of Goiás, serum-reactive animals were detected with the age of the animals, the source of water, the form of replacement of the herd and the presence of domestic cats and wild animals risk factors statistically associated with the occurrence of T. gondii in animals.

Immunoconjugates to increase photoinactivation of bovine alphaherpesvirus 1 in semen.

Artificial insemination and in vitro embryo production are increasingly used to improve the reproductive efficiency of herds, however success of these techniques depends on the sanitary quality of the semen. Insemination centers commonly use antibiotics in their routine procedure, but they are not able against viruses. In this paper, we demonstrate a new approach for disinfecting virus in bovine semen using photoimmunoinactivation, an adaptation of the photodynamic inactivation (PDI) methodology. The photosensitizers (PSs), hematoporphyrin (HP) and zinc tetracarboxy-phthalocyanine (ZnPc) were conjugated to Immunoglobulin Y (IgY) anti-bovine alphaherpesvirus 1 (BoHV-1) and used for PDI against the BoHV-1 viruses in cell culture and compared to the unconjugated PSs. Both treatments proved to be efficient, but a significant decrease in the irradiation time required to completely eliminate the virus was observed in the samples treated with the immunoconjugates. Photophysical measurements help us to understand the coupling between PSs and IgY and the evaluated production of singlet oxygen. Following the cell culture test, the same approach was applied in semen artificially infected with BoHV-1. The immunoconjugates were also efficient for complete virus inactivation up to 5 min of irradiation and proved to be safe using several parameters of sperm viability, demonstrating the feasibility of our strategy for disinfection viruses in semen.

Histopatology of the reproductive tract of Nellore pubertal heifers with genital ureaplasmosis

ABSTRACT In order to study and characterize the lesions in the reproductive tract of Nellore heifers naturally infected with Ureaplasma diversum and presenting granular vulvovaginitis syndrome (GVS), fragments of uterine tube, uterus, cervix, vagina and vulva of 20 animals were evaluated. The macroscopic lesions of the vulvovaginal mucosa were classified in scores of "1" mild, until "4", severe inflammation and pustular or necrotic lesions. The histopathological evaluation was performed using scores of "1" to "4", according to the inflammatory alterations. The fragments with severe microscopic lesions (3 and 4) were from the uterine tubes and uterus, which showed leukocytes infiltration and destruction and/or necrosis of epithelium. Alterations in the lower reproductive tract fragments were mild, but characteristics of acute inflammatory processes. The histopathological findings of the reproductive tract of females naturally infected with Ureaplasma diversum are consistent with injuries that compromise the environment from the local where spermatozoa acquires ability to fertilize an oocyte until those where the oocyte is fertilized. Therefore, animals with GVS should be identified early in the herd, because, besides the reduction in the fertility rates caused by tissue damages, they can contribute to disseminate the microorganism. Key words: bovine, tissue evaluation, reproduction, Ureaplasma diversum.

Assessment of cow and farm level risk factors associated with Ureaplasma diversum in pasture-based dairy systems - A field study

ABSTRACT Potential risk factors for Ureaplasma diversum in the vaginal mucus of 1,238 dairy cows were included in a multivariate logistic regression model, based on the cow level (i.e., granular vulvovaginitis [+GVV], yearly milk production [4500 kg or more], pregnancy, predominance of Bos taurus [+Bos Taurus], score of corporal condition [at least 2.5], concomitant positivity for Escherichia coli [+E.coli]), and farm level i.e., milking room hygiene (-Milking room), dunghill location, and replacement female). Ureaplasma diversum was present in 41.1% of the samples. Independent risk factors for U. diversum were +GVV (odds ratio [OR], 1.31); +Mycoplasma spp (OR, 5.67); yearly milk production (4500 kg or more) (OR, 1.99); +Bos taurus (OR, 1.68); +E. coli (OR, 4.96); -milking room (OR, 2.31); and replacement females (OR, 1.89). Ureaplasma diversum vaginal colonization was strongly associated with Mycoplasma spp., E. coli, and number of pregnant cows.

[Primers design and optimization of PCR and nested-PCR assays for the specific detection of Tritrichomonas foetus]. / Construção de iniciadores e otimização de ensaios de PCR e de nested-PCR para a detecção específica de Tritrichomonas foetus.

Tritrichomonas foetus is a pathogenic protozoan that causes a venereal disease in cattle known as bovine genital tricomonosis. In spite of the efficacy to recognize the target genomic DNA, the protocols so far developed for the diagnosis of this organism by PCR promote some inespecific amplifications or they are unable to discriminate T. foetus against other species within the genus. The objective of this study was to assess and optimize PCR and nested-PCR assays for the specific diagnosis of T. foetus, using novel primers selected from the alignment of sequences of the genes 18S rRNA, 5.8S rRNA, 28S rRNA and of the internal transcribed spacers of the rDNA (ITS1 and ITS2). A pair of primers was constructed for the genus-specific amplification of a 648 bp fragment and two others to amplify T. foetus species-specific fragments of 343 and 429 bp. No cross amplification was observed against Bos taurus genomic DNA neither against the DNA of usual bovine genital pathogens. Both, single and nested-PCR assays, presented analytical sensitivity to detect at least two T. foetus organisms.

Embryonic stem-like cells derived from in vitro produced bovine blastocysts

The aim of this work was to study the derivation of bovine embryonic stem-like (ES-like) cells from the inner cell mass (ICM) of in vitro produced blastocysts. The ICMs were mechanically isolated and six out of seventeen (35 percent) ICMs could attach to a monolayer of murine embryonic fibroblasts (MEF). Ten days after, primary outgrowths were mechanically dissected into several small clumps and transferred to a new MEF layer. Cells were further propagated and passaged by physical dissociation over a 60 days period. The pluripotency of the bovine ES-like cells was confirmed by RT-PCR of Oct-4 and STAT-3 gene markers. The colonies were weakly stained for alkaline phosphatase and the mesoderm and endoderm differentiation gene markers such as GATA-4 and Flk-1, respectively, were not expressed. Embryoid bodies were spontaneously formed at the seventh passage. Results showed that bovine ES-like cells could be obtained and passaged by mechanical procedures from the fresh in vitro produced blastocysts.

Construção de iniciadores e otimização de ensaios de PCR e de nested-PCR para a detecção específica de Tritrichomonas foetus / Primers design and optimization of PCR and nested-PCR assays for the specific detection of Tritrichomonas foetus

Tritrichomonas foetus é um protozoário patogênico responsável por doença venérea em bovinos conhecida por tricomonose genital bovina. A tricomonose bovina é uma doença venérea causada pelo protozoário cujo habitat natural é o trato genital. Os protocolos já desenvolvidos para o diagnóstico deste parasito por PCR, apesar de serem eficazes na identificação do DNA genômico alvo, promovem algumas amplificações inespecíficas ou são incapazes de distinguir T. foetus das outras espécies do gênero. O presente trabalho foi desenvolvido com o objetivo de estabelecer e otimizar protocolos de ensaio de PCR e nested-PCR para o diagnóstico específico de T. foetus, empregando-se novos iniciadores, selecionados do alinhamento das seqüências dos genes 18S rRNA, 5,8S rRNA, 28S rRNA e dos espaços transcritos do rDNA (ITS1 e ITS2). Um par de iniciadores foi construído para amplificação gênero-específica de um fragmento de 648 pares de base e outros dois para a obtenção de produtos espécie- específicos de 343 e 429 pb. Nenhuma reação cruzada foi observada frente ao DNA genômico de Bos taurus ou de microrganismos responsáveis por infecções genitais. A sensibilidade dos ensaios de PCR e de nested-PCR apresentados neste estudo permitiu um limiar de detecção de até dois parasitos.

Tritrichomonas foetus is a pathogenic protozoan that causes a venereal disease in cattle known as bovine genital tricomonosis. In spite of the efficacy to recognize the target genomic DNA, the protocols so far developed for the diagnosis of this organism by PCR promote some inespecific amplifications or they are unable to discriminate T. foetus against other species within the genus. The objective of this study was to assess and optimize PCR and nested-PCR assays for the specific diagnosis of T. foetus, using novel primers selected from the alignment of sequences of the genes 18S rRNA, 5.8S rRNA, 28S rRNA and of the internal transcribed spacers of the rDNA (ITS1 and ITS2). A pair of primers was constructed for the genus-specific amplification of a 648 bp fragment and two others to amplify T. foetus species-specific fragments of 343 and 429 bp. No cross amplification was observed against Bos taurus genomic DNA neither against the DNA of usual bovine genital pathogens. Both, single and nested-PCR assays, presented analytical sensitivity to detect at least two T. foetus organisms.

Fertilidade real e intervalo de partos de vacas nelore PO sob manejo extensivo e sem estação de monta na região centro oeste do Brasil / True fertility and calving interval of nelore po cows under extensive management and without breeding season in middle west of Brazil

Este estudo foi desenvolvido com o objetivo de avaliar as fontes de variação que interferem no índice fertilidade real (FR) e o efeito do ambiente no intervalo de partos (IDP) de vacas Nelore PO criadas em sistema extensivo de produção na região Centro-Oeste do Brasil. Os dados analisados foram ordem de parto (OP), idade ao parto(IP), peso ao nascimento do bezerro (PN), peso à desmama (PD), ano e mês do parto (AP, MP) e sexo do bezerro (SB) de546 vacas, calculando-se o IDP e fixando-se o efeito aleatório da mãe (EAM). IDP e PD foram utilizados para calcular o índice de fertilidade real. O IDP médio de 452,68 ± 117,10 dias foi influenciado (P<0,05) pela ordem de parto, idade aoparto, peso ao nascer e efeito aleatório da mãe. A FR média de 148,6 ± 34,5kg sofreu influência (P<0,05) de ano e mês do parto, ordem de parto, sexo do bezerro e idade da mãe.

This study was designed to evaluate the variation sources that interfere in the true fertility indexand the effect of herd environment in the calving interval of Nelore P.O. cows raised in extensive production systems inMiddle-West of Brazil, without breeding season. The analyzed data were parity order (OP), calving age (IP), calf birthweight (PN), weaning weight (PD), calving year and month (AP, MP) and calf sex (SB) of 546 cows, calculating the calving interval (IDP) and fixing the aleatory effect of mother (EAM). IDP and PD were used to calculate the index oftrue fertility (FR). IDP (452,68±117,10 days) was influenced (P <0,05) by OP, IP, PN and EAM. FR average(148,6±34,5kg) was influenced (P <0,05) by AP, MP, OP, S and IM.

Estudo experimental do enxerto autólogo de ovário em cadelas submetidas a ovariectomia e ovariosalpingohisterectomia / Experimental ovarian autograft in bitches submitted to ovariectomy and ovariohysterectomy

A pesquisa mostra os resultados obtidos em 10 cadelas mestiças das quais, 6 foram castradas e enxertadas com fragmento autólogo de ovário e 4 formaram os grupos de controle, somente castradas. Foram avaliadas no período pós-operatório de 12 meses através exames clínico e citológico, dosagem sérica de progesterona, observaçäo macroscópica do local de enxertia e exame histológico de fragmento do enxerto ovariano. Durante a fase pós-operatória de observaçäo, as fêmeas somente castradas e as enxertadas nao mostraram alteraçoes das mamas, com relaçäo ao peso comporal, utilizando o teste de Wilcoxon as somente castradas demonstraram diferença estatísticamente significante (p=0,0156). No tocante a citologia vaginal as fêmeas enxertadas apresentaram as quatro fases do ciclo estral. Através o teste de Wilcoxon concluiu-se que ocorre diferença significativa nas dosagens séricas de progesterona entre as fêmeas somente castradas (p=0.0082) e as enxertadas (p=0.0156). Após período de 12 e 36 meses foi realizada avaliaçäo macroscópica e exame histológico dos enxertos ovarianos, que mostraram diferentes etapas de foliculogênese.