Detection of HIV and HCV RNA in semen from Brazilian coinfected men using multiplex PCR before and after semen washing

INTRODUCTION: Prolonged survival of patients under HAART has resulted in new demands for assisted reproductive technologies. HIV serodiscordant couples wish to make use of assisted reproduction techniques in order to avoid viral transmission to the partner or to the newborn. It is therefore essential to test the effectiveness of techniques aimed at reducing HIV and HCV loads in infected semen using molecular biology tests. METHODS: After seminal analysis, semen samples from 20 coinfected patients were submitted to cell fractioning and isolation of motile spermatozoa by density gradient centrifugation and swim-up. HIV and HCV RNA detection tests were performed with RNA obtained from sperm, seminal plasma and total semen. RESULTS: In pre-washing semen, HIV RNA was detected in 100% of total semen samples, whereas HCV RNA was concomitantly amplified in only one specimen. Neither HIV nor HCV were detected either in the swim-up or in the post-washing semen fractions. CONCLUSIONS: Reduction of HIV and/or HCV shedding in semen by density gradient centrifugation followed by swim-up is an efficient method. These findings lead us to believe that, although semen is rarely found to contain HCV, semen processing is highly beneficial for HIV/HCV coinfected individuals.

Sperm tail flexibility test: a simple test for selecting viable spermatozoa for intracytoplasmic sperm injection from semen samples without motile spermatozoa

PURPOSE: The objective was to describe the results of the injection of immotile spermatozoa with flexible tails when only immotile spermatozoa are present in the semen sample. METHODS: A retrospective study was conducted to analyze the procedure results for 10 couples who participated in our intracytoplasmic sperm injection program. The sperm tail was considered flexible when it moved up and down independently of the head movement, and it was considered inflexible when the movement occurred together (tail plus head). The fertilization and pregnancy rate were analyzed. RESULTS: The normal fertilization rate (presence of 2 pronuclei) was 30.3% (40/132), and the abnormal fertilization rate (presence of less than or more than 2 pronuclei) was 6.81% (9/132). A total of 52 embryos were obtained with 9 transfer procedures performed (pregnancy rate: 11.12%). CONCLUSIONS: The sperm tail flexibility test (STFT) is an easy and cost-effective way for selecting viable immotile spermatozoa and can be used as an alternative method for determining the viability of spermatozoa. This test seems to be a simple and risk-free method when compared to the swelling test.

Enxerto homólogo congelado de diafragma na correção dos defeitos da parede abdominal de ratos

OBJETIVOS: Análise morfológica e biomecânica do enxerto homólogo congelado de diafragma para correção de defeito da parede abdominal em ratos. MÉTODOS: Os animais foram distribuidos em controle (20 ratos Wistar) e experimento (30 ratos Wistar). Os do grupo controle foram submetidos à laparotomia mediana e sutura da parede abdominal; já os do grupo experimento, à ressecção da parede abdominal e reconstrução com enxerto homólogo congelado de diafragma. Os animais foram submetidos à eutanásia no 3° e 6° mês de pós-operatório e avaliados quanto à presença de complicações pós-operatórias, integridade do enxerto, presença de aderências, avaliação tensiométrica, avaliação histopatológica com H/E e com sirius-red (colágeno tipo I e III). RESULTADOS: Houve integração do enxerto em todos os animais sem complicações. Aderências foram semehantes entre os grupos controle e experimento após três e seis meses. Observaram-se maior força máxima, força de ruptura e tensão nos animais do grupo experimento aos três meses de pós-operatório (p=0,001; p=0,012 e p=0,001, respectivamente). Na correlação entre as diferentes variáveis estudadas houve correlação estatisticamente significante entre força máxima e tensão nos grupos controle e experimento (p=0,001 e p= 0,001), e nos subgrupos três e seis meses (p=0,002 e p=0,001). Correlacionaram-se força máxima e colágeno tipo I (p=0,04) e Índice de Maturação do Colágeno (IMaC) e força máxima (p=0,03) ambos somente no grupo controle, mas nos subgrupos 3 e 6 meses (p=0,045 e p=0,038). O número de monomorfonucleares e força máxima também apresentou significância estatística tanto para o grupo controle quanto para o experimento (p=0,005 e p=0,004, respectivamente). CONCLUSÃO: O enxerto homólogo congelado de diafragma mostrou ser boa alternativa no reparo de grandes defeitos da parede abdominal em ratos.

Toxoplasma gondii in semen of experimentally infected swine

Eight reproductive boars were divided into three groups and inoculated with Toxoplasma gondii [GI (n=3) 1.5x10(4) oocysts strain P; GII (n=3) 1.0x10(6) tachyzoites strain RH; and GIII (n=2) non-inoculated control]. Clinical, hematological, parasitemia and serological tests and studies of the parasite in the semen through bioassay and PCR, and in reproductive organs (Bioassay and immunohistochemical analyses) were conducted to evaluate the toxoplasmic infection. Blood and semen were collected on day -2, -1, 1, 3, 5, 7, 9, 11, 14 and weekly up to 84 days post-inoculation (DPI). No clinical or hematimetric alteration was observed in the boars. Parasitemia was detected in one boar inoculated with oocysts at the 7th DPI and in another boar infected with tachyzoites (GII) at the 3rd and 49th DPI. Serological tests revealed antibodies against T. gondii in animals inoculated with oocysts or tachyzoites at the 7th DPI with dilutions of 1:256 and 1:64, which reached peaks of 1:4096 at day 11 and 9, respectively. The bioassays revealed the presence of the parasite in semen samples of a boar inoculated with oocysts (GI) at 3, 49 and 56 DPI and from two boars infected with tachyzoites (GII), one animal at 5 and two animals at 49 days DPI. Mice inoculated with semen from the control group (GIII) remained serologically negative. PCR analysis showed T. gondii DNA in the semen of Boar 1 and Boar 3 inoculated with tachyzoites and oocysts, respectively. The immuno-histochemical tests showed T. gondii in the reproductive organs of Boar 1 and Boar 2, inoculated with tachyzoites and oocysts, respectively. These findings suggest the possible occurrence of venereal transmission of T. gondii in swine.

Transmission of porcine circovirus 2 (PCV2) by semen and viral distribution in different piglet tissues

Porcine circovirus infections are caused by the porcine circovirus 2 (PCV2). Among six different clinical manifestations involving respiratory, enteric, nervous and reproductive signs, the postweaning multisystemic wasting syndrome (PMWS) is the most important and studied disease. However, reproductive failures associated with PCV2 have been increasingly reported. Some studies have shown the possible contamination of sows by semen of PCV2 positive boars. In order to investigate the transmission of PCV2 by contaminated semen and its ability to infect the sow and piglets, 20 PCV2 negative sows were inseminated, 10 with negative boar semen and 10 with previously nested-PCR tested positive boar semen. The sows were weekly monitored and blood samples were collected. Based on the results, 4 out 20 sows were selected (1 sow was PCR negative and inseminated with a negative semen, 2 sows were PCR negative and inseminated with a positive semen and 1 sow was PCR negative and inseminated with a positive semen, but became PCR positive around the 30 days of pregnancy). After weaning, 12 male piglets, 3 of each sow, were selected and maintained under isolation. In order to investigate which organs harbored the virus, the young pigs were necropsied around 9 months of age. Samples of serum collected monthly were tested by immunocitochemistry (ICC), and all 12 pigs serum converted. Samples of lymphoid, systemic and reproductive organs were analyzed by nested-PCR and immunohistochemistry (IHC). Evaluation of the samples by nested-PCR, revealed that several tissues were positive in 10 of 12 pigs, mainly the lymph nodes, bone marrow and spleen. Various samples were positive by IHC in 8 of 12 piglets, being the lymph nodes, tonsils and bulbourethral glands the most frequently positive. Thus, the results of testing different samples, in the 3 tests (ICC, nested-PCR and IHC) were complementary. These results show that PCV2 transmission through semen to the sows and piglets may occur and may also represent a potential risk for the herd.

Toxoplasma gondii in experimentally infected Bos taurus and Bos indicus semen and tissues

Eighteen young steers were inoculated with Toxoplasma gondii and randomly distributed into three groups of six animals each: GI, 2.5x10(5) "P" strain oocysts, GII, 5.0x10(6) "RH" strain tachyzoites, and GIII (Control). Clinical, serological and parasitemia exams were realized. Parasite investigation by bioassay and PCR was realized on semen and fragments of skeletal musculature, lymph nodes, brain, retina, spleen, liver, lung, testicle, epididymis and seminal vesicle. Blood and semen samples were collected on days -2, -1, 1, 3, 5, 7, 14 and weekly thereafter, up to postinfection day (PID) 84. The inoculated steers (GI and GII) presented hyperthermia from PID 3 to 16. Antibodies against T. gondii were detected through the indirect fluorescence antibody test (IFAT) on PID 5 (1:16) in both inoculated groups (oocysts and tachyzoites), reaching peaks of 1:4096 on PID 7. Parasitemia outbursts occurred in all infected bovines, principally from PID 7 to 28, independent of the strain and inoculate used. Bioassays revealed the presence of parasites in semen samples of animals infected with oocysts (GI) and tachyzoites (GII) on several experimental days between PID 7 and 84. Tissue parasitism by T. gondii was diagnosed by bioassay and the PCR technique in several organ and tissue fragments. These findings suggest the possibility of sexual transmission of T. gondii in the bovine species.

Detection and dynamics of porcine circovirus 2 shedding in semen using conventional and real-time PCR

The dynamics of porcine circovirus type 2 (PCV2) shedding in semen of naturally infected boars was studied. Semen was collected serially each 15 or 20 days during 62 days from 5 boars from a herd and from 11 boars from an artificial insemination center. All boars were positive for PCV2 DNA by nested polymerase chain reaction of raw semen in at least two sampling dates, and most of them had detectable shedding in all sampling dates. Real-time quantitative PCR was performed in 23 samples. All samples showed low amounts of PCV2 DNA, ranging from 98 to 652 PCV2 copies/mL. No differences between the frequencies of PCV2 DNA shed in semen were found considering herds and age of boars. PCV2 shedding in the semen can occur continuously or intermittently up to 60 days in naturally infected boars at 12 to 42 months old in absence of PCV2 clinical signs. These results demonstrate sporadic and long-term shedding patterns of low amounts of PCV2 DNA in semen from naturally infected boars.

Cysteine addition on short-term cooled boar semen preservation and its relationship with swine field fertility

Artificial insemination is routinely used in the swine industry to reduce the costs of production through to increase the efficiency of the refrigerated boar semen process. The objective of this study was to evaluate the effect of different levels of cysteine (CYS) added to the Beltsville Thawing Solution (BTS) extender semen during cooling for up to 72 hours. Ejaculated from three boars were collected with the gloved-hand technique and semen aliquots were diluted in BTS as follow: BTS only (BTS), BTS + 0.1mM cysteine (CYS0.1), BTS + 0.5mM cysteine (CYS0.5), BTS + 1.0mM cysteine (CYS1.0), BTS + 2.5mM cysteine (CYS2.5), BTS + 5.0mM cysteine (CYS5.0), BTS + 10.0mM cysteine (CYS10.0), and BTS + 20.0mM cysteine (CYS20.0). Evaluation of sperm integrity were analyzed using 0.5mg/ml propidium iodide (plasma membrane), 100µg/ml isothiocynate-conjugated Pisum sativun agglutinin (acrosomal membrane) and 153µM 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (mitochondria potential) after semen dilution at specific times (0, 24, 48 and 72 hours). Additionally, we also evaluated the effects of 5.0 mM CYS addition in the BTS extender on the maintenance of sperm quality and their influence on fertility in the swine production. After artificial insemination, animals were evaluated based on the estrous return and the number of piglet's born. Cysteine at concentrations of 10.0 and 20.0mM resulted in more pronounced reductions even at the time zero. Semen viability decreased to levels below 10% at these high levels of CYS in the first 24 hour of storage at 17ºC. At the end of the storage time, less than 65% of sperm cells had intact plasma membrane in all groups. The sperm viability decreased significantly when the semen was added at high concentrations of CYS (time "0"; CYS10.0 and CYS20.0; p<0.05), when compared to the other CYS concentrations. The BTS (10.20±0.39) treated group showed a lower rate of estrus return when compared to other (BTSCYS; 86.05±039), and it showed also the highest total number of piglets borne per treatment (12.71±3.38 vs. 9.00±3.38, respectively). In conclusion, the addition of CYS in the BTS semen extender did not maintain spermatic viability of boar cooled spermatozoa and it results in a higher percentage of return to estrus and lower number of piglets borne.

Mycoplasma agalactiae in semen and milk of goat from Pernambuco state, Brazil

In goat and sheep flocks, mycoplasmosis is a disease that may cause severe economical losses associated with polyarthritis, mastitis, agalactia, conjunctivitis, pneumonia and reproductive failure. The latter may involve repeat breeding, granular vulvovaginitis, infertility and abortions. The aim of the present study was to assess the occurrence of Mycoplasma agalactiae (Ma) in semen and milk samples from naturally infected goat in the semiarid region from Pernambuco State, Northeast from Brazil. Thirty-nine semen samples and 81 milk samples were submitted to DNA extraction using a commercially available kit and following the manufacturer's instructions. The polymerase chain reaction (PCR) was then performed in accordance with protocols described in the literature. The results of the present study revealed the presence of Ma in the DNA of 17.9% (7/39) of the semen samples and 3.7% (3/81) of the milk samples. The results obtained in the present study confirm the elimination of the DNA of Ma in the semen and milk samples. The presence of this agent in goat flocks is considered very risky in terms of reproductive disorders and contagious agalactia outbreaks in the Northeast region of Brazil.

Effect of psychological stress on the L-arginine-nitric oxide pathway and semen quality

It has been reported that mental stress causes abnormality of spermiogram parameters. We investigated the effect of psychological stress on the L-arginine-nitric oxide (NO) pathway. Semen samples were collected from 29 healthy fourth semester medical students just before (stress) and 3 months after (non-stress) the final examinations. Psychological stress was measured by the State Anxiety Inventory questionnaire. After standard semen analysis, arginase activity and NO concentration were measured spectrophotometrically in the seminal plasma. Measurements were made in duplicate. During the stress period, sperm concentration (41.28 ± 3.70 vs 77.62 ± 7.13 x 10(6)/mL), rapid progressive motility of spermatozoa (8.79 ± 1.66 vs 20.86 ± 1.63%) and seminal plasma arginase activity (0.12 ± 0.01 vs 0.22 ± 0.01 U/mL) were significantly lower than in the non-stress situation, whereas seminal plasma NO (17.28 ± 0.56 vs 10.02 ± 0.49 µmol/L) was higher compared to the non-stress period (P < 0.001 for all). During stress there was a negative correlation between NO concentration and sperm concentration, the percentage of rapid progressive motility and arginase activity (r = -0.622, P < 0.01; r = -0.425, P < 0.05 and r = -0.445, P < 0.05, respectively). These results indicate that psychological stress causes an increase of NO level and a decrease of arginase activity in the L-arginine-NO pathway. Furthermore, poor sperm quality may be due to excessive production of NO under psychological stress. In the light of these results, we suggest that the arginine-NO pathway, together with arginase and NO synthase, are involved in semen quality under stress conditions.

Características químicas, microbiológicas e sensoriais de leite de cabra congelado

O objetivo deste trabalho foi avaliar as modificações químicas, microbiológicas e sensoriais do leite caprino pasteurizado e congelado durante armazenamento por 90 dias. Foram realizadas análises para caracterização química da matéria prima utilizada nos experimentos (gordura, acidez Dornic, densidade, extrato seco total, pH e ácidos graxos livres-AGL) e caracterização microbiológica (contagem total, psicrotróficos, coliformes totais e fecais). Utilizou-se pasteurização lenta a 63°±1°C por 30 minutos para as amostras de leite seguido de armazenamento em freezer à temperatura de -18°C±1°C. Nos tempos 0, 30, 60 e 90 dias de congelamento foram efetuadas análises químicas (pH, acidez e AGL), microbiológicas (contagem total, psicrotróficos e coliformes) e sensoriais (sabor e aroma característico, sabor e odor estranho e aparência geral). Também, realizou-se análise sensorial do leite nos tempos zero e com 90 dias de armazenamento, após descongelamento e homogeneização em liquidificador por dois minutos. Foi observado que o congelamento prolongado do leite pasteurizado não alterou significativamente suas características químicas e microbiológicas. Apenas a acidez apresentou decréscimo significativo. No entanto, a qualidade do leite do ponto de vista sensorial apresentou modificações significativas, com perdas de sabor e aroma característicos e declínio acentuado da aparência geral durante o armazenamento. A homogeneização do leite em liquidificador, após o descongelamento melhorou a aparência geral e a aceitação do produto pela equipe de provadores.

Efeito de diferentes tratamentos pré-congelamento sobre a qualidade do morango var. Chandler congelado

A adequabilidade da variedade de morango Chandler ao processo de congelamento e a influência da utilização de tratamentos pré-congelamento foram avaliadas. Tanto na matéria-prima como no produto final foram realizadas medidas objetivas de textura, pH, acidez total titulável, teor de sólidos solúveis, teor de ácido ascórbico e perda de líquido por exsudação. Subjetivamente foram avaliados o sabor, a textura e a cor do produto final. O preparo dos frutos para congelamento compreendeu os procedimentos normais de limpeza, corte em metades e os tratamentos de pré-congelamento, que utilizaram: sacarose, cloreto de cálcio, pectina e aplicação de calor. Os produtos foram acondicionados em latas com capacidade para 450g (74,6 x 95,2mm), revestidas internamente com verniz epóxi fenólico, que foram recravadas e em seguida congeladas a -25°C. O produto final foi armazenado por 45 dias a -18°C e avaliado após 12h de descongelamento em temperatura de 5°C. Os resultados mostraram que a variedade Chandler apresenta características adequadas ao congelamento e que a adição de cálcio e pectina é útil a este tipo de processamento, colaborando para a diminuição da perda de líquido por exsudação, assim como redução de perdas de ácido ascórbico e não interferindo no sabor e na cor do produto.

TEORES DE HISTAMINA E QUALIDADE FÍSICO-QUÍMICA E SENSORIAL DE FILÉ DE PEIXE CONGELADO

A qualidade de 120 amostras de diferentes tipos de filé de peixe congelado foi avaliada com relação às características sensoriais, pH, teores de bases voláteis totais e histamina e presença de gás sulfídrico. Os resultados da análise sensorial indicaram boa qualidade dos peixes com valores médios de 5,52 (CV £ 13%) tanto para odor quanto para aspecto geral. Com relação a bases voláteis totais (BVT), teores médios de 62,71 mg N/100 g foram encontrados, com valores significativamente maiores (> 80 mg N/100 g) para namorado (Pseudopercis numida) e cação (famílias Carcharhinidae e Squalidae) e menores que 68 mg N/100 g para os demais tipos de peixe. Os valores médios de pH variaram de 6,11 em abrótea (Urophycis brasiliensis) a 7,49 em castanha (Umbrina sp.). Baseado nestes resultados, 79, 39 e 62% das amostras não atenderiam aos limites estipulados pela legislação vigente para BVT, pH e gás sulfídrico, respectivamente. Histamina foi encontrada em 37% das amostras com teores médios de 0,00 para linguado (Paralichthys sp. e Pleuronectes sp.) a 0,50 mg/100 g para namorado. A presença de histamina não foi detectada em abrótea. Foram observadas diferenças significativa entre os valores de BVT, pH e histamina das amostras de peixe agrupadas por famílias. Para uma melhor avaliação do potencial de utilização e estabelecimento de limites críticos para estes parâmetros como critério de qualidade, sugere-se que amostras sejam monitoradas desde o momento da captura. Os teores de histamina detectados nas amostras não seriam, por si só, capazes de causar intoxicação.

Estabilidade físico-química e sensorial do suco de limão Tahiti natural e adoçado, congelado

Suco de limão Tahiti adoçado com 60% de sacarose (p:p) e puro foram congelados e armazenados por 60 dias. Análise de variância, comparação de médias e regressão foram usadas para avaliar o efeito da adição de sacarose e tempo de armazenamento nas propriedades físico-químicas e sensoriais dos dois tipos de sucos. Sessenta por cento de sacarose foi escolhido pois simples diluição produziu limonada com boa aceitabilidade. O suco natural foi adoçado e diluído na mesma proporção que o suco adoçado antes das análises. A concentração de sólidos solúveis, acidez titulável e ácido ascórbico, e pH do suco não foram afetados pelo tempo de armazenamento. Houve escurecimento do suco durante o armazenamento, não detectado na análise sensorial, mas determinado pelo método espectrofotométrico. Atributos sensoriais de sabor e odor de limão passado e percepção de doçura foram afetados por tempo de armazenamento.