Percentage and types of morphological abnormalities found in canine spermatozoa were evaluated by three investigators using three stains (Giemsa-Wright stain [Diff-Quik], eosin Y/nigrosin [Hancock], and eosin B/nigrosin [Society for Theriogenology morphology stain] with conventional light microscopy, compared to phase contrast microscopy on unstained samples. The percentage of spermatozoa with abnormal heads, midpieces, and tails varied by technique and by investigator. Average percentages of morphologically normal spermatozoa were significantly higher in samples stained with Diff-Quik and samples examined by phase contrast microscopy than in samples stained with Hancock or Society for Theriogenology morphology stains. No effect of investigator on the percentage of morphologically normal spermatozoa was assessed. Results suggest that staining or preparation technique may alter the morphology of canine spermatozoa artifactually.

The effects of stains and investigators on assessment of morphology of canine spermatozoa.

El objetivo fue examinar la morfologia de los espermatozoides con diferentes metodos de tincion y encontrar los mejores metodos para su estudio. Fueron seleccionados para el estudio, de manera aleatoria 67 pacientes, quienes ingresaron a la Unidad de tecnicas de reproduccion asistida. En la primera parte del estudio, se realizaron y tineron frotis con hematoxilina eosina (HE), Azul de Toluidina (AT), Giemsa, tincion de Wright, Hematoxilina Ferrica de Weigert, Anaranjado G, tincion eosina-anilina, metodo de Shorr, Papanicolau, metodo Berg, tincion verde brillante, anaranjado de acridina (AO) y tincion verde Janus. En la segunda parte del estudio, se realizaron frotis de 10 pacientes con normozoospermicos y se tineron con HE, AT, Metodo Shorr y Papanicolau. Se realizaron cuatro mediciones: ancho de la cabeza, longitud de la cabeza, parte media y cola, sobre 200 espermatozoides con morfologia normal. Se compararon las tinciones que mostraban mejor la morfologia. La evaluacion de la compactacion no fue posible en los frotis tenidos con los metodos de Shorr, Berg y AO. Una mejor evaluacion de la copactacion podria hacerse en otras tinciones. En la segunda parte los valores menores correspondieron a la tincion de AT en relacion a la medicion de la cabeza de los espermatozoides. Hubo una diferencia significativa en la longitud de la cabeza con tincion de AT. Las mediciones en los frotis con tecnicas de Shorr y Papanicolau fueron similares, con valores mas altos bajo tincion de Papanicolau. Se concluyo que los valores de la medicion morfologica en espermatozoides humanos podrian ser alterados segun el metodo de tincion utilizado.

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Assessment of Spermatozoa Morphology under Light Microscopy with Different Histologic Stains and Comparison of Morphometric Measurements

Fertility prognosis for infertile couples

SUMMARY: The aim was to examine the morphology of spermatozoa with different staining methods and aimed to find the better staining methods for morphology of spermatozoa in our study. Randomized 67 patients taken for the study who were admitted to Assisted Reproductive Techniques Unit. In the first part of the study, smears were stained with Hematoxylin Eosin (HE), Toluidin Blue (TB), Giemsa, Wright, ferrous Weigert haematoxylin stain, Orange G, eosin-aniline blue dye, Shorr Method, Papanicolau, Berg Method, Light Green stain, Acridine Orange (AO) and Janus Green dyes. In the second part of the study, smear preparations of 10 patients with normozoospermic were stained with HE, Toluidin Blue (TB), Shorr Method and Papanicolau. Four measurements were made including the middle piece, head length- head width and tail length for 200 spermatozoa with normal morphology. Comparisons were made between the stains that which showed a better morphology. Condensation assessment was not possible in smears stained with Shorr, Berg, Method, AO. Better assessment of condensation could be made in other stains. In the second part the smallestvalues belonged to of TB stain according to measurements of head of the spermatozoa. There was a significant difference at the head length with TB stain. Although measurements of Shorr and Papanicolau areclose to each otherand the largestvalues belonged to Papanicolau dye. It was concluded that measurement values in human sperm morphology could alter with the used staining method. ˚

Assessment˚˚ of˚˚ Spermatozoa˚˚ Morphology˚˚ under˚˚ Light˚˚ Microscopy˚˚ with˚˚ Different˚˚ Histologic˚˚ Stains˚˚ and Comparison˚˚ of˚˚ Morphometric˚˚ Measurements ˚ Evaluación˚ ˚de˚˚ la˚˚ Morfología˚˚ de˚˚ los˚˚ Espermatozoides˚ ˚bajo˚˚ Microscopía˚˚ Óptica˚˚ con Diferentes˚˚ Tinciones˚˚ Histológicas˚˚ y˚˚ Comparación˚˚ de˚˚ las˚˚ Mediciones˚˚ Morfométricas

The impact of woman's age and sperm parameters on fertilization rates in IVF cycles

A method combining Janus green B and Thymol blue stains the anterior part of the head, the nuclear membrane, middle piece, and tail of spermatozoa light green and the nucleus deep purple. The method provides excellent stained preparations for the evaluation of sperm morphology by phase contrast microscopy. It produces significantly less abnormal spermatozoa compared with the Papanicolau stain.

P V Bagger

Papers

Complete Staining of Human Spermatozoa and Immature Germ Cells Combined With Phase Contrast Microscopy