Prevalence, Risk Factors and Impact of Subclinical Endometritis on Reproductive Performance of Nili-Ravi Buffalo

Subclinical endometritis (SCE) is the inflammation of endometrium without systemic illness, hence remains mostly undiagnosed and untreated. The early diagnosis necessitates the evaluation of important risk factors. The objective of this study was to identify the risk factors of SCE and their effects on reproductive performance of Nili-Ravi buffalo. 100 buffaloes approaching parturition were selected. During calving, various risk factors viz., type of calving, peri-parturient disorders, sex and birth weight of calf, gestation period and season of calving were recorded. Buffaloes were subjected to endometrial cytology using cytobrush method on 45 day postpartum (DPP) and divided into 2 groups viz., buffaloes ‘with SCE’ (>5% PMN; n=38) and ‘without SCE’ (≤5% PMN; n=62). Buffaloes in estrus were artificially inseminated and fertility parameters were recorded. The occurrence of SCE was significantly affected by calving assistance (OR=11.74; P<0.001), peri-parturient disorders (OR=6.87; P<0.05) and gestation period (OR=1.16; P<0.05). Sex, birth weight of calf and season of calving were not associated with SCE. The service period of buffaloes did not vary between two groups. The median days open was significantly (P<0.05) higher in buffaloes with as compared to buffaloes without SCE (141 vs. 117 d). Buffaloes with SCE had significantly (P<0.05) lower first service conception (21.1 vs. 43.5%) and took more (P<0.05) mean number of services per conception (2.71 vs. 1.62) compared to buffaloes without the SCE. It may be concluded that risk factors around the time of calving control the onset of SCE which in turn has negative impact on reproductive performance of Nili-Ravi buffaloes

Magnetic activated cell sorting (MACS): utility in assisted reproduction.

Assisted reproductive techniques (ART) have now been extensively incorporated in the management of infertile couples. But even after rapid methodological and technological advances the success rates of these procedures have been below expectations. This has led to development of many sperm preparation protocols to obtain an ideal semen sample for artificial reproduction. Sperm apoptosis has been heavily linked to failures in reproductive techniques. One of the earliest changes shown by apoptotic spermatozoa is externalization of phosphatidyl serine. Magnetic activated cell sorting (MACS) is a novel sperm preparation technique that separates apoptotic and non-apoptotic spermatozoa based on the expression of phosphatidylserine. This has led to the incorporation of MACS as a sperm preparation technique. The review highlights the principle and mechanism of this novel technique and enumerates its advantages as a sperm preparation technique. Its utility in ART as an efficient tool for sperm recovery and its application in cryopreservation of semen samples is also explained.

Evaluation of acrosomal status and sperm viability in fresh and cryopreserved specimens by the use of fluorescent peanut agglutinin lectin in conjunction with hypo-osmotic swelling test

OBJECTIVE: In this study, we evaluated whether the hypo-osmotic swelling test (HOST) can be used as a vital marker in combination with peanut agglutinin (PNA) - labeling in fresh and cryopreserved spermatozoa. MATERIALS AND METHODS: Human sperm populations were exposed to a hypo-osmotic medium for 60 minutes, and then incubated in a 1 µg/mL solution of the fluorescent dye Hoescht 33258 (H33258) for 10 minutes. Excess stain was removed by washing in phosphate-buffered saline (PBS) solution, and the pellet was resuspended in 100 µL of culture medium. Twenty microliters of this solution were subsequently smeared on a microscope slide, and fixed in ice-cold methanol to permeabilize the sperm membranes. The fixed smears were finally incubated in a 40-µg/mL FITC-PNA solution for 20 minutes. Simultaneous assessment of acrosome and viability scores was done in a fluorescent microscope equipped with appropriate filters and phase contrast illumination. The same slide was examined for FITC-PNA labeling, tail swelling, and for Hoechst-33258 staining by interchanging the filters and phase contrast optics. RESULTS: In fresh specimens, HOST was found to provide viability assessments comparable to those obtained using the H33258 method (r = 0.95). However, the results of HOST and H33258 were not correlated in cryopreserved specimens (r = 0.22). There was no alteration of PNA-labeling due to the HOST or H33258. CONCLUSIONS: FITC-PNA labeling in conjunction with the visualization of the morphological change induced by exposure to hypo-osmotic solution provides a simple but effective method for establishing the state of acrosomal membrane and viability in fresh human spermatozoa, but this technique is not reliable for cryopreserved ones.

Identification of male factor infertility using a novel semen quality score and reactive oxygen species levels

OBJETIVO: Determinar se pacientes portadores do fator de infertilidade masculina podem ser precisamente identificados através do cálculo de um novo escore de qualidade de sêmen e pela medida de espécies reativas de oxigênio durante uma avaliação rotineira de infertilidade. MÉTODOS: Amostras de sêmen de 133 pacientes e de 91 doadores saudáveis foram avaliadas através de análise manual e computadorizada de sêmen. Um modelo de análise do componente principal foi empregado para calcular o escore de qualidade de sêmen, utilizando logaritmos base 10, multiplicados por ponderações variáveis de 9 parâmetros espermáticos. Os níveis de espécies reativas de oxigênio foram medidos através de testes de quimiluminescência. RESULTADOS: O escore de qualidade de sêmen apresentou sensibilidade de 80.45% e precisão de 77% para um "cutoff" de 93.1 na identificação do fator de infertilidade masculina. A área sob a curva "receiver operating characteristic" para o escore de qualidade de sêmen foi de 84.28% (95% intervalo de confiança: 65.22%-100%). Os níveis de espécies reativas de oxigênio [log10 (espécies reativas de oxigênio +1)] foram siginificativamente mais elevados nos pacientes portadores de fator de infertilidade masculina. A medica de espécies reativas de oxigênio apresentou sensibilidade de 83.47% e especificidade de 60.52% com uma precisão (definida como pacientes portadores do fator de infertilidade masculina com diagnóstico positivo e doadores corretamente excluídos) de 75% para um "cutoff" de 1.25 na identificação de pacientes portadores do fator de infertilidade masculina. A área sob a curva "receiver operating characteristic" para níveis de espécies reativas de oxigênio foi de 78.92% (95% intervalo de confiança: 72.60%-85.23%). Os escores de qualidade de sêmen correlacionaram negativamente com os níveis de espécies reativas de oxigênio tanto nos doadores e nos pacientes portadores do fator de infertilidade masculina. CONCLUSÕES: O escore de qualidade de sêmen e os níveis espécies reativas de oxigênio nas amostras de sêmen parecem associar-se fortemente com o fator de infertilidade masculina. Na medida em que os dois parâmetros mostraram-se mais sensíveis que parâmetros espermáticos individuais na identificação do fator de infertilidade masculina, deveriam ser incluídos na avaliação rotineira de infertilidade.