Dog Semen Research Articles

Effect of Oral Administration of Selenium and Vitamin E on the Quality of Fresh, Refrigerated and Frozen Semen in French Bulldog Breed Dogs

Background: New methodologies have been developed seeking to maximize pregnancy rate in female dogs created in commercial kennels, and also in order to maintain the quality of canine semen after dilution, refrigeration or freezing. One of the main factors that generate damage to sperm is oxidative stress, to minimize sperm damage, selenium and antioxidants like vitamin E are administered, by oral administration, seeking to improve the quality of semen. The objective was to study the effect of vitamin E and selenium, by oral administration, in the quality of fresh, refrigerated and frozen semen in adult dogs French Bulldog breed.Materials, Methods & Results: Semen samples were collected from 5 adult dogs, French Bulldog breed, being 2 semen drawing before the daily oral supplementation with vitamin E and selenium (ESE®) and semen drawing at 20, 40 and 60 days after the beginning of oral supplement. The ejaculated samples were diluted in TRIS - fructose citric acid (3.28 g TRIS-hydroxy-methyl-amino-methane, 1.78 g of citric acid monohydrate and 1.25 g of D - fructose, dissolved in 100 mL of distilled water and added of 20% egg yolk and 6% of glycerol. The characteristics evaluated in fresh semen were: volume (mL), color, appearance, concentration (x106 / mL), sperm motility (%), sperm strength (1 to 5) and morphology (%). For refrigerated and frozen semen were analyzed: sperm motility (%), sperm strength (1-5) and morphology (%). Diluted semen samples were centrifuged at: 1500 g/10 min and “pellets” formed by sperm of each ejaculated, detached from the tube wall were diluted homogeneously in the diluent TRIS type up to the final volume of 1.5 mL. After that, packaged in 0.5 mL French straws, kept under refrigeration at 5ºC/4 h, placed in nitrogen vapor at -120ºC/15 min, and dipped in liquid nitrogen at -196ºC and then stored on identified rachis and stored in liquid nitrogen container until the time of thawing in water bath at 37°C/30 s for semen microscopic analysis. Data from fresh, refrigerated and frozen semen were statistically analyzed by analysis of variance and the average compared by 5% of Tukey test. Fresh semen sperm concentration differed (P < 0.05) between the samples, rising after 40 days after the beginning of oral supplementation with selenium and vitamin E. For the spermatic strength, better score (P < 0.05) was observed at collection 4, in 40 days after the beginning of oral supplementation to dogs. For fresh and refrigerated semen, the total defects, defects of head, acrosome and tail did not differ (P > 0.05) between the samples. Total sperm defects and minor head and tail defects did not differ (P > 0.05) between the samples in post-thawing. Regarding the acrosome defects after thawing, there was a significant reduction (P < 0.05) in samples performed 40 and 60 days after the beginning of oral supplementation with selenium and vitamin E.Discussion: Attention should be paid for what purpose the extenders within the refrigeration or freezing biotech will be used. The managed supplement, by oral administration, containing selenium and vitamin E, influenced beneficially raising the sperm concentration in fresh semen and decreasing the acrosome defects in frozen semen. Oral administration of supplementation with selenium and vitamin E is recommended for improving the quality of fresh and frozen semen in dogs.

Effects of Soy Lecithin Extender on Dog Sperm Cryopreservation

ABSTRACTSemen cryopreservation is an essential biotechnology in canine reproduction and during the cryopreservation process commonly egg yolk are used. The discrepancy in the egg yolk composition and the potential risk of disease dissemination are obstacles for semen exportation and use. Therefore, studies aiming to substitute egg yolk are extremely important. In this context, soy lecithin contains a low-density lipoprotein fraction, is an interesting alternative. Thus, the objective of this study was to compare extenders based on soy lecithin (several concentrations and forms) with egg yolk during the cryopreservation process of dog sperm. For this purpose, we used twelve dogs. Semen was evaluated at different time points (after refrigeration, glycerolization, and thawing), by motility analysis (CASA) and functional tests (e.g., membrane integrity—eosin/nigrosin, acrosome integrity—fast green/Bengal rose, mitochondrial activity—3’3 diaminobenzidine, Chromatin susceptibility to acid-induced denaturation—SCSA, and susceptibility to oxidative stress—thiobarbituric acid reactive substances). The results indicated that egg yolk and lower concentrations of lecithin had similar effects on mitochondrial activity and motility. Thus, soy lecithin is a potentially viable alternative to egg yolk for the cryopreservation of dog semen.

Quality of Labrador- Retriever dog semen on short-term preservation in different extenders

The objective of the present study was to find the comparative efficacy of three extenders to preserve semen of Labrador-Retriever (LR) dogs at 5o C for a short term. The semen samples of LR dogs were collected by digital manipulation method and extended at the rate of 1:4 in Tris-Egg Yolk- Citric Acid-Glucose (TEYCAG), Tris-Egg Yolk- Citric Acid-Fructose (TEYCAF) and Egg Yolk-Citrate-Glycine-Glucose (EYCGG) extenders by split sample technique. Semen was evaluated at 0, 24, 48, 72, 96 and 120 hours of preservation. Mean motile, live and HOST-reacted sperm and acrosomal, head, mid piece and tail abnormalities of spermatozoa varied significantly (PP less than 0.01) between extenders and between preservation periods. The interactions between extender and preservation period were also significant (P less than 0.01) except for HOST-reacted and head abnormalities of sperm. The highest mean motile, live and HOST-reacted sperm were recorded in TEYCAG extender which did not differ significantly from that of TEYCAF extender. Mean per cent sperm acrosomal and tail abnormalities were significantly (PP less than 0.05) lower, and the incidences of mean sperm head and mid piece abnormalities were also lower in TEYCAG, but not significantly from that in TEYCAF irrespective of hour of preservation. Per cent motile, live and HOST-reacted sperm were significantly (PP less than 0.05) lower and sperm acrosomal, head, mid piece and tail abnormalities were significantly (PP less than 0.05) higher in EYCGG as compared to that in TEYCAG and TEYCAF irrespective of hour of preservation. It was concluded that the semen of LR dog sustained good quality during preservation up to 5 days at 5oC suitable for successful artificial insemination and would be preserved better in TEYCAG and TEYCAF extenders than in EYCGG extender, since more than 50 per cent sperm motility and live sperm were maintained up to 120 hours of preservation in the former two extenders.

Freezing dog semen using −80 °C ultra-freezer: Sperm function and in vivo fertility

Long term storage of canine frozen semen is conventionally performed in liquid nitrogen (LN2). However, previous works in freezing canine semen using a −80 °C ultra-freezer (−80°C-UF) showed no differences on sperm quality after thawing. The main objective of this study was to compare the effects of the freezing techniques using LN2 or -80°C-UF on sperm function and in vivo fertility of frozen–thawed dog semen. The sperm-rich fraction of the ejaculate was collected separately from five Chihuahua breed, and each one divided into two aliquots, and frozen and stored in LN2 or -80°C-UF. Sperm function was analyzed for motility and viability, acrosome integrity, mitochondrial function and phosphatidylserine translocation by flow cytometry before and after cryopreservation. A total of 10 bitches were intravaginal inseminated (IVAI; LN2 frozen-thawed semen = 5 and −80°C-UF frozen–thawed semen = 5). Pregnancy status was confirmed 30 d after IVAI by transabdominal ultrasonography and live born puppies at term were recorded. Sperm function parameters were affected for both freezing protocols. Differences (P < 0.05) were found between freezing and storage methods in most of the parameters of sperm function analyzed, except in the phosphatidylserine translocation. The percentages of pregnancies were not different between the two freezing and storage protocols used. Semen freezing and storage using −80 °C UF is an effective technique for long-term preservation of canine spermatozoa.

In Iraq, First Documentation of Canine Brucellosis by Application of Three Techniques (Rapid test, Indirect ELISA and 16S rDNA Inter-spacer PCR)

Brucella canis is a Gram-negative organism infecting, mainly, the genital organs of both sexes and resulted in several reproductive problems. This bacterium is excreted in urine, milk, fetuses or semen of infected dogs, and transmitted to sensitive dogs through sexual, oral, nasal and conjunctival routes. In general, the routine detection of infection is done by serological tests and the confirmation through isolation of causative agent by the culture. Previously, many global studies documented the excellent efficacy of polymerase chain reaction in detecting the bacterial DNA, perfectly and high accuracy. The present study is the first Iraqi document that dealt with the diagnosis of B. Canis, serologically by Rapid test and Indirect ELISA test, and demonstration the infection, molecularly, in seropositive dogs by “16S rDNA inter-spacer PCR” technique. Serologically, the study revealed that 14 (5.76%) and 31 (12.76%) dogs were positive with Rapid test and Indirect ELISA, respectively, while only 5 (16.13%) dogs were positives molecularly. As well as, the received serologic data exhibited that all positive samples with rapid tests were, also, positives by indirect ELISA. At level of P  0.05, the statistical differences were reported within the applied techniques, positive dogs with most common risk factors (sex and age). In relation to sex, the results appeared that the females had an infection rate more than males; while in association to age, the infection rates were similar in both first groups (&lt;4 and 1-4 years) and increased apparently in the last aged group ( 4 years).

A retrospective clinical study of endoscopic-assisted transcervical insemination in the bitch with frozen-thawed dog semen.

Since the conclusion of data collation from previously published work, a further 352 inseminations using frozen-thawed dog semen by endoscopic-assisted transcervical insemination (EIU) have been performed by the author. Insemination was performed on the second day in which crenulation of the anterior vagina was detected in conjunction with a progesterone concentration of >10ng/ml. All semen samples were analysed for total number of sperm, total motility and progressive motility using computer-assisted semen analysis (CASA). The insemination dose was based on the progressively motile normal spermatozoa (PMNS). Insemination was performed on all bitches as previously described using a ureterorenoscope. Additional extender was inseminated subsequent to the semen to expand and fill the uterus. The semen and additional extender were inseminated slowly over a period of 15-20min. Pregnancy was determined by B-mode ultrasound equipped with a 7.5-MHz probe whilst standing and/or via the whelping rate. The number of sperm inseminated ranged from 9×106 PMNS to 519×106 PMNS, with progressive motility values ranging between 20% and 80%. The overall pregnancy rate was 68% (238/352). When stratified by PMNS, pregnancy rates were as follows: >150×106 PMNS - 76% (110/145), 100-150×106 - 68% (87/128) and <100×106 PMNS - 52% (41/79). Pregnancy rate was significantly higher when >150×106 PMNS (p=.003) or 100-150×106 PMNS (p=.027) were inseminated compared to <100×106 PMNS. These data are concordant with previous reports recommending the insemination of >150×106 PMNS to maximize pregnancy rate. These results indicate that one optimally timed EIU insemination results in similar pregnancy rates to previous publications of one optimally timed, or two or more non-optimally timed inseminations using the Norwegian catheter.

Quality of fresh and chilled-stored raccoon dog semen and its impact on artificial insemination efficiency.

BackgroundThe aim of the study was to evaluate the quality of fresh raccoon dog semen and raccoon dog semen stored at 4 °C. The qualitative evaluation was based on apoptosis in the sperm cells, which was tested by the Annexin V/Pi assay, the TUNEL method and JC-1. In addition, the suitability of the semen for insemination and its effect on reproduction in females were determined in relation to the time of storage.ResultsDuring cold storage of the semen, in the samples from all groups a gradual decrease was noted in the percentage of live cells, and an increase in the percentage of cells with abnormal morphology, exhibiting changes typical of late-stage apoptosis (V+/PI+), and of necrotic cells (V−/PI+). There was a significant increase in the percentage of ApoBrDu + sperm cells, while the mitochondrial membrane potential of the sperm decreased significantly after 12 h of storage at 4 °C in the case of lower-quality semen and after 48 h in the case of semen of good quality. As the percentage of sperm with DNA and cell membrane damage increased and the mitochondrial membrane potential decreased, there was an increase in AspAT and acrosin activity. The increase in the percentage of apoptotic sperm in the raccoon dog semen stored at 4 °C resulted in a decrease in the number of females with cubs.ConclusionsIdentification of apoptotic changes in sperm by flow cytometry using the annexin assay, the TUNEL assay and evaluation of mitochondrial membrane potential can be recommended for determination of the suitability of raccoon dog semen for artificial insemination. The study shows that fresh raccoon dog semen should not be used for insemination more than 48 h after collection in the case of semen of very high quality, or after more than 24 h in the case of semen of poorer quality. Cytometric methods of semen analysis should also be used to evaluate various extenders of raccoon dog semen and methods of cryopreservation in terms of ensuring sperm viability, fertilization capacity, and suitability for insemination.

Dog sperm head morphometry: its diversity and evolution.

Dogs have been under strong artificial selection as a consequence of their relationship with man. Differences between breeds are evident that could be reflected in seminal characteristics. The present study was to evaluate differences in sperm head morphometry between seven well-defined breeds of dog: the British Bulldog, Chihuahua, German Shepherd, Labrador Retriever, Spanish Mastiff, Staffordshire Terrier, and Valencian Rat Hunting dog. Semen samples were obtained by masturbation and smears stained with Diff-Quik. Morphometric analysis (CASA-Morph) produced four size and four shape parameters. Length, Ellipticity, and Elongation showed higher differences between breeds. MANOVA revealed differences among all breeds. Considering the whole dataset, principal component analysis (PCA) showed that PC1 was related to head shape and PC2 to size. Procluster analysis showed the British Bulldog to be the most isolated breed, followed by the German Shepherd. The PCA breed by breed showed the Chihuahua, Labrador Retriever, Spanish Mastiff, and Staffordshire Terrier to have PC1 related to shape and PC2 to size, whereas the British Bulldog, Valencia Rat Hunting dog, and German Shepherd had PC1 related to size and PC2 to shape. The dendrogram for cluster groupings and the distance between them showed the British Bulldog to be separated from the rest of the breeds. Future work on dog semen must take into account the large differences in the breeds’ sperm characteristics. The results provide a base for future work on phylogenetic and evolutionary studies of dogs, based on their seminal characteristics.

Corrigendum: Environmental chemicals impact dog semen quality in vitro and may be associated with a temporal decline in sperm motility and increased cryptorchidism.

Scientific Reports 6: Article number: 31281; published online: 09 August 2016; updated: 16 September 2016.

Environmental chemicals impact dog semen quality in vitro and may be associated with a temporal decline in sperm motility and increased cryptorchidism.

Adverse temporal trends in human semen quality and cryptorchidism in infants have been associated with exposure to environmental chemicals (ECs) during development. Here we report that a population of breeding dogs exhibit a 26 year (1988–2014) decline in sperm quality and a concurrent increased incidence of cryptorchidism in male offspring (1995–2014). A decline in the number of males born relative to the number of females was also observed. ECs, including diethylhexyl phthalate (DEHP) and polychlorinated bisphenol 153 (PCB153), were detected in adult dog testes and commercial dog foods at concentrations reported to perturb reproductive function in other species. Testicular concentrations of DEHP and PCB153 perturbed sperm viability, motility and DNA integrity in vitro but did not affect LH stimulated testosterone secretion from adult testis explants. The direct effects of chemicals on sperm may therefore contribute to the decline in canine semen quality that parallels that reported in the human.

Avaliação in vitro do sêmen criopreservado de cães naturalmente infectados por Leishmania sp.

RESUMO O objetivo deste trabalho foi avaliar a qualidade in vitro do sêmen criopreservado de cães naturalmente infectados por Leishmania sp. Foram coletadas amostras de sêmen de 12 cães, sendo seis positivos (GI) e seis negativos (GII) para leishmaniose visceral (LV), semanalmente, totalizando quatro coletas por animal. O sêmen criopreservado foi avaliado pelo teste de termorresistência rápida (TTR), nos tempos zero, 30 e 60 minutos, pela análise computadorizada (CASA) e por meio de sondas fluorescentes; esta última técnica com o intuito de avaliar a integridade das membranas espermáticas. Houve diferença estatística pela técnica de TTR no parâmetro motilidade progressiva, no tempo 0min (68,33% GI e 72,50% GII), e no vigor espermático (2,67 GI e 3,0 GII), no tempo 30min. Quanto ao CASA, houve diferença estatística apenas na motilidade total (27,50% GI e 57,08% GII), embora os demais parâmetros seminais tenham apresentado valores relativos diminuídos nos cães do GI. Nas análises com sondas fluorescentes, foram observadas diferenças estatísticas entre os grupos quanto à integridade das membranas plasmática e acrossomal e ao potencial mitocondrial das células espermáticas. Concluiu-se que a LV pode comprometer a qualidade do sêmen criopreservado de cães parasitados.

Cryopreservation of Dog Semen in a Tris Extender with 1% or 2% Soya Bean Lecithin as a Replacement of Egg Yolk.

Egg yolk is usually included in extenders used for preservation of dog semen. Lecithin is an interesting animal-protein free alternative to egg yolk for semen preservation. The aim of our study was to evaluate soya bean lecithin for cryopreservation of dog semen. Five ejaculate replicates were divided in three equal parts, centrifuged and each pellet diluted with one of the three Tris-based extenders containing 20% egg yolk, 1% soya bean lecithin or 2% soya bean lecithin. Extended semen was loaded in 0.5-ml straws, cooled and diluted a second time and frozen in liquid nitrogen vapours. Sperm motility parameters (CASA), acrosome integrity (FITC-PNA/PI) and sperm membrane integrity (C-FDA) were evaluated 5 min post-thaw and after 2 and 4 h of incubation. Total motility was significantly better in the egg yolk extender than in any of the lecithin-based extender and was better in the 1% lecithin extender than in the 2% lecithin extender. Sperm membrane integrity was significantly better in the egg yolk extender than in any of the lecithin-based extenders but did not differ significantly between the 1% and 2% lecithin extenders. Acrosome integrity was significantly better in the egg yolk extender than in the 2% lecithin extender but did not differ between the egg yolk extender and the 1% lecithin extender or between the two lecithin extenders. In conclusion, egg yolk was superior to lecithin in our study. The extender with 1% lecithin preserved sperm motility better than the extender with 2% lecithin.

The Quality of Frozen-thawed Canine Semen With Respect to Semen Extender Composition and Sequence of Ejaculate Collection in Dogs

The aim of this study was to evaluate the effect of clarified egg yolk addition to semen extender, and the semen collection sequence on the quality of frozen-thawed semen in dogs. Semen was collected from 6 dogs in a time interval of 24 hours. As parameter of the quality of frozen-thawed (F-T) semen, the motility by computer assisted sperm analysis (CASA) and plasma membrane integrity by hypo-osmotic swelling test (HOS) were evaluated. All kinematic parameters of sperm motility were higher in F-T samples containing the whole in comparison to the clarified egg yolk. The sequence of semen collection affected sperm movement characteristics of native as well as F-T semen, but it was not possible to determine whether the fresh semen from the 1st or 2nd collection is of higher quality. All motility parameters of sperms frozen with extender containing the whole egg yolk were significantly higher in the case of the 2nd collection. The situation was not so clear in the case of clarified egg yolk addition, but the velocity values were higher in F-T samples from the 2nd collection. In contrast to proven differences in motility, the effect of the addition of clarified egg yolk and the sequence of semen collection were not projected at all on the quality of plasma membrane of canine sperms evaluated by HOS test.

Artificial Insemination Intravaginal in bitch breed Dogo Argentino using chilled semen: Case report

This paper reports the reproductive biotech Artificial Insemination (IA) held a bitch Dogo Argentino breed, a year and a half old, nulliparous, resident in the city of Teresina, PI, using chilled semen from a dog of the same breed, residing in Recife , PE. The dog semen was collected by digital manipulation, diluted in Tris-yolk, chilled to 4°C, identified, stored in isothermal box and sent by air transport for the city of Teresina. The IA was performed intravaginally, using chilled semen for 24 hours of collection as a single dose, according to the results of vaginal cytology and hormonal dosage bitch. Forty days after IA abdominal ultrasonography was carried out confirming pregnancy. It concludes that the use of IA with chilled semen is a viable alternative that allows the fertilization of dogs living in geographically distant areas.

Some factors influencing canine sperm motility

This study aimed to evaluate whether sperm motility (MOT) in dog semen is influenced by dog age, breed, or number of sperm collections. The study group consisted of four Shih Tzus and five Border Collies, aged 1–8 years. Sperm was collected from each dog once every seven days, for a total of six weeks. Sperm motility did not differ (P &gt; 0.05) based on age and breed. Mean MOT at the first collection (67.4% ± 6.9%) was lower (P &lt; 0.05) than that at the third, fifth, and sixth collections (86.2%; 88.9%; 89.1% ± 6.9%, respectively). Increased MOT was observed after the third collection, demonstrating a positive correlation with the number of sperm collections and sperm motility (P&lt; 0.05). Accordingly, our results indicate that sperm processing efficiency is increased after the collection of three ejaculates.

Prognostic value of a pre-freeze hypo-osmotic swelling test on the post-thaw quality of dog semen

Throughout cryopreservation, sperm are exposed to major osmotic challenges. Only intact membranes of sperm cells are able to regulate these volumetric changes, which can be determined by the hypo-osmotic swelling test (HOS test). Correlations between the HOS test and conventional semen variables are inconsistent. Therefore, the objectives of this study were (1) to examine relationships between HOS test results and standard semen variables before freezing and after thawing and (2) to evaluate the prognostic value of the HOS assessments on post-thaw quality of dog semen. Semen of 35 dogs was collected and analyzed before freezing and after thawing following a 7-day freeze–thaw interval. Conventional semen variables such as sperm cell motility, membrane integrity morphology were evaluated and the HOS test was conducted with results from this test being recorded. In fresh semen the HOS test was positively correlated with progressive motility of sperm cells: r=0.52, sperm cell membrane integrity: r=0.50 and normal sperm cell morphology: r=0.46 (P<0.05). In frozen–thawed semen, the data obtained with the HOS test were positively correlated with progressive sperm cell motility: r=0.67 and membrane integrity: r=0.86 (P<0.05). The data obtained with the HOS test in fresh semen were positively correlated with sperm cell membrane integrity: r=0.50 normal sperm cell morphology: r=0.55 and data from the HOS test (r=0.43; P<0.05) with frozen–thawed semen. For the prediction of individual cryopreservation capacity, results from assessment of the fresh semen variables of good and poor semen quality were statistically compared. Based on these results, it is not possible to predict the quality of frozen–thawed dog semen using the HOS test.

Afterword to Sperm morphometrics today and tomorrow special issue in Asian Journal of Andrology.

Early in his investigations, Leeuwenhoek (1670s) deduced that spermatozoa were alive and an integral part of semen, rather than artifacts or parasites. He eventually observed spermatozoa in the semen of men, dogs, horses, birds, fishes, amphibians, molluscs, and many insects, and concluded that they must be a universal feature of male reproduction. The huge differences in sperm form among species have been discussed in relation to evolutionary changes dictated by the egg and its investments. Spallanzani (1800s) was the first scientist to develop successful methods for artificial insemination, first with amphibians and later with dogs. With these experiments, he showed that physical contact between intact spermatozoa and ova was necessary to achieve the fertilization. Some years later (1820s), Prevost and Dumas performed the defining experiment to identify correctly the function of spermatozoa in reproduction.

Sperm quality and selected biochemical parameters of seminal fluid in dogs with benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) in dogs is most commonly associated with age and increasing concentrations of dihydrotesterone, a hormone that stimulates growth and secretion of the prostatic epithelial cells. During this process, the biochemical composition of prostatic secretion changes, which can affect the quality of semen and limit the ability of the sperm to contribute to fertilization. Therefore, the present study was conducted to examine possible correlation between BPH and biological quality of semen. The study was performed in 11 sexually mature dogs of various breeds. Animals were divided into two groups: healthy dogs (Group I; n=5; mean age 4.32; SEM=1.28) and dogs with BPH (Group II n=6; mean age 6.16; SEM=0.65). Semen and prostate secretions were collected and evaluated in this study. Standard semen examinations were conducted in the ejaculates collected; moreover, the extent of apoptosis and DNA defragmentation was determined. The selected biochemical parameters were determined in the prostate secretion. According to the examination results, there were no significant differences in standard semen parameters between the two groups of dogs. Nevertheless, morphological tests of semen in dogs with BPH demonstrated elevated percentages of primary defects in spermatozoa. A significant increase (P=0.01) in DNA defragmentation of sperm was found in dogs with BPH. Moreover, changes in the biochemical composition of prostate secretion were demonstrated. In dogs with BPH, pH of prostate secretions was greater (P=0.03), concentrations of cholesterol increased while concentrations of Zn and Cu decreased. The study findings reveal that BPH does not change semen quality in dogs.

Freeze-dried dog sperm: Dynamics of DNA integrity

Freeze-drying (FD) has been proposed as an alternative method to preserve spermatozoa. During the FD procedure, sperm DNA might become damaged by both freezing and drying stresses caused by the endonucleases, the oxidative stress and the storage conditions. We examined the DNA integrity of dog sperm freeze-dried with two kinds of chelating agents in FD buffers and storage at two different temperatures. Ejaculated sperm from four dogs were suspended in basic medium (10mM Tris–HCl buffer+50mM NaCl) supplemented with 50mM EGTA or with 50mM EDTA and then freeze-dried. Sperm samples were stored at 4°C as room temperature, and the analysis of DNA damage was performed after a month and 5months of storage using a Sperm Chromatin Dispersion test. We found four different sperm populations according to the size of the halos around the sperm head: (1) absent halo, (2) <6μm, (3) 6–10μm, (4) >10μm. All of them coexisted in each freeze-dried dog semen samples and differed significantly among different treatments. The highest percentage of spermatozoa with halo >10μm was obtained when the semen samples were freeze-dried in EDTA medium and stored at room temperature for five months. Results suggested that both, the kind of chelating agent as well as storage temperature and period, influenced DNA integrity of freeze-dried dog sperm.

Effect of dialysis of dog semen on sperm characteristics and some biochemical components of seminal plasma.

The aim of this study was to investigate the effect of dog semen dialysis on sperm characteristics and some biochemical components of seminal plasma. Whole ejaculates were dialyzed against Tris-citrate-fructose extender for a 5 h period at room temperature (using semi-permeable cellulose tubing of 12-14 kDa molecular weight cut-off). It has been demonstrated that the long-term dialysis of dog semen causes a significant decrease in sperm quality parameters and disrupts the biochemical properties of seminal plasma. This procedure requires further improvement.