Evaluation of fertilizing potential of frozen-thawed dog spermatozoa diluted in ACP-106 using an in vitro sperm--oocyte interaction assay.

The aim of present study was to evaluate frozen canine semen with ACP-106 (Powder Coconut Water) using an in vitro sperm--oocyte interaction assay (SOIA). Ten ejaculates from five stud dogs were diluted in ACP-106 containing 20% egg yolk, submitted to cooling in a thermal box for 40 min and in a refrigerator for 30 min. After this period, a second dilution was performed using ACP-106 containing 20% egg yolk and 12% glycerol. Samples were thawed at 38 degrees C for 1 min. Post-thaw motility was evaluated by light microscopy and by using a computer aided semen analysis (CASA). Plasma membrane integrity and sperm morphology/acrosomal status were evaluated by fluorescent probes (C-FDA/PI) and Bengal Rose respectively. Moreover, frozen-thawed semen was analysed by a SOIA. Subjective post-thaw motility was 52.0 +/- 14.8% and it was significant higher than the total motility estimated by CASA (23.0 +/- 14.8%) because this system considered the egg yolk debris as immotile spermatozoa. Although normal sperm rate and acrosomal integrity evaluated by Bengal Rose stain was 89.6 +/- 3.1% and 94.3 +/- 3.1%, respectively, post-thaw percentage of intact plasma membrane was only 35.1 +/- 14.3%. Regarding SOIA, the percentage of interacted oocytes (bound, penetrated and bound and/or penetrated) was 75.3%. Using regression analysis, it was found significant relations between some CASA patterns and data for SOIA. In conclusion, the freezing-thawing procedure using ACP-106 was efficient for maintain the in vitro fertility potential of dog spermatozoa.

Influence of temperature during glycerol addition and post-thaw dilution on the quality of canine frozen semen.

Contents The aim of the present study was to compare the influence of room temperature (27 degrees C) and 4 degrees C during glycerol addition on canine semen cryopreservation and verify the effect of different post-thawing dilutions on canine semen. Ten ejaculates from five stud dogs were collected by digital manipulation. Semen samples were evaluated and further divided into two aliquots. The first aliquot was extended in Tris-egg yolk-glycerol at 27 degrees C and the second one received glycerol at 4 degrees C. Samples were frozen and stored in liquid nitrogen. After 1 week, samples were thawed and submitted to evaluations of progressive sperm motility, morphology, acrosomal integrity, hypo-osmotic swelling (HOST) and thermoresistance tests. For thermoresistance test, aliquots were divided in two portions: one portion was kept undiluted (1 : 0) and the other one was diluted in a 1 : 4 ratio (one part semen to four parts extender). No differences were observed between temperatures for glycerol addition regarding seminal parameters evaluated. Furthermore, post-thawing dilutions demonstrated similar effect on canine semen longevity. Correlations among post-thaw sperm motility and HOST and results from thermoresistance test were observed for both temperatures for glycerol addition. In conclusion, glycerol could be added to canine semen at room temperature (27 degrees C) or at 4 degrees C. Moreover, there is no need to extend canine semen after thawing for the thermoresistance test, but if we need to increase the inseminating volume for artificial inseminations, the addition of extender will not damage the semen.

Comparison between different dilution rates on canine semen freezing using Tris-buffer with the addition of egg-yolk and glycerol

Compararam-se a concentração espermática padronizada e a expansão volume: volume na diluição do sêmen canino para congelação. O sêmen de seis cães, submetidos a duas coletas por estimulação manual, foi avaliado e diluído em tris acrescido de gema de ovo e glicerol, de acordo com duas diferentes diluições. A primeira baseou-se na concentração espermática padronizada de 200x106 espermatozóides/ml, e a segunda mediante diluição volume: volume, na proporção de uma parte de sêmen para uma de diluidor. O sêmen foi congelado, armazenado em nitrogênio líquido e descongelado após uma semana. A motilidade e o vigor espermáticos foram avaliados a cada etapa do processo e aos 15 e 30min após descongelação. A morfologia espermática foi avaliada após coleta e descongelação. Nenhuma diferença foi observada entre os tratamentos após a descongelação quanto à motilidade, vigor, porcentagem de espermatozóides morfologicamente normais e longevidade. Ambas as taxas de diluição podem ser eficientemente utilizadas na congelação do sêmen canino.

Quality of canine semen submitted to single or fractionated glycerol addition during the freezing process.

Glycerol is the cryoprotector most frequently used to freeze semen from different species. The objective of the present study was to compare the effect of single and fractionated glycerol addition on canine semen quality after thawing. Sperm fractions from 12 stud dogs were collected, evaluated, extended in Tris plus egg-yolk and separated into two aliquots to which glycerol was added in one step (single) or in three steps at 5-min intervals (fractionated). Semen was frozen and stored in liquid nitrogen and thawed after 1 week. A thermoresistance test was performed over a period of 120 min at 37-39 degrees C to evaluate the percentage of mobile spermatozoa (motility) and the status of motility (vigor) after thawing. There were no significant differences between the two methods of glycerol addition-immediately after thawing, and during the thermoresistance test-in sperm motility, vigor or morphology. A significant reduction in motility and vigor was found at 15 min after thawing and these parameters continued to decline until 120 min. In conclusion, glycerol can be added to canine semen in single or fractionated manner, but the single addition method is the easiest and the most practical to use.

Relaçäo entre perímetro escrotal e concentraçäo espermática em cäes, clinicamente normais, da raça Pastor Alemäo / Relationship between scrotal circumference and spermatic concentration in normal clinical German Shepherd dogs

The aim of this study was to estimate the correlation between scrotal girth and spermatic concentration in German Sheepherd dogs. Thirteen German Sheepherd dogs were used and 44 semen samples were obtained by digital manipulation. The ejaculate was separated in its three fractions. The sperm rich fraction was kept for evaluation and determination of spermatic concentration by spectrofotometry. The parameters were expressed as mean and standard deviation. Spearman correlation test was used to estimate the correlation between spermatic concentration and scrotal girth. The mean spermatic concentration was 568.45±314.91x10(6) sptz/ml and the mean scrotal girth was17.6±1.6 cm and the correlation was r = 0.10. It can be conclude that scrotal girth can not be an appropriate measurement as indicative of spermatic concentration in German Sheepherd dogs