The value of canine semen evaluation for practitioners

doi:10.1016/j.theriogenology.2007.04.017

Theriogenology

Volume 68, Issue 3, August 2007, Pages 329-337

https://doi.org/10.1016/j.theriogenology.2007.04.017 Get rights and content

Abstract

Compared to human medicine, little is known in canine medicine regarding specific findings on semen evaluation and their correlation with fertility. Suggestions to optimize quality of semen evaluation in veterinary practice include creating standardized protocols for evaluation of all semen parameters and updating those protocols as needed; creating some form of quality control for the clinic laboratory; educating owners about our inability to predict with 100% accuracy whether dogs with poor semen quality never could impregnate a bitch or whether dogs with excellent semen quality always could impregnate a bitch; and generating protocols for further diagnostic work-up for those dogs with abnormal semen quality.

Section snippets

What do the numbers mean?

Veterinary practitioners may perform semen analysis as part of a complete breeding soundness examination, to evaluate suitability of semen for AI, preservation by chilling or freezing, or to investigate subfertility or infertility. Unfortunately, there is a paucity of data associating the parameters measured during semen evaluation with the issues veterinarians really need to evaluate, i.e. testicular function, fertilizing capability of spermatozoa, and likelihood that pups will develop

Collection and evaluation of semen

Collection of canine semen by manual ejaculation will not be described. The reader is asked to review pertinent literature regarding appropriate equipment, technique, and environment to enhance collection of semen from dogs. Although it is generally accepted that a dog should not be condemned on the results of one semen evaluation, there are no published guidelines for timing of semen collection relative to sexual activity, as there are in humans. The World Health Organization, to promote

Volume

Dog semen is ejaculated in three fractions. The first (pre-sperm) fraction is small in volume and contains few to no spermatozoa. The second (sperm-rich) fraction comes from the epididymes and testes. The third (prostatic) fraction consists solely of prostatic fluid and also contains few to no spermatozoa. The volumes of the first and third fractions, especially the latter, are variable, and the volume of the third fraction is controlled by the person collecting the sample, as they choose to

Color

Color evaluation is subjective but may guide the clinician toward other tests to be performed. A clear sample contains no spermatozoa. Cloudy or milky samples probably contain spermatozoa but always should be checked microscopically; occasionally, a dog with azoospermia will shed excessive numbers of fat droplets into the sample, giving the appearance of normal semen. Yellow semen is indicative of urine contamination and is also seen in humans with icterus or after ingestion of certain

pH

Controversy exists regarding the value of measurement of pH in canine semen. Reported values for normal pH in non-fractionated canine semen vary from 6.4 to 6.8 [11], [12], [13]. One author recommended that evaluation of pH be performed immediately after collection using accurate equipment (presumably a pH meter) and strongly discouraged use of a “dipstick” method [14]. In humans, it is recommended that pH be evaluated within an hour of semen collection and that pH paper be used [9]. The author

Motility

For canine semen, motility is better maintained if samples are kept at room temperature than at body temperature [11], [14]. Quick temperature fluctuations should be avoided. Room temperature may play a factor; one report of canine semen evaluation in a tropical region suggested that lack of motility in some samples was due to lack of air conditioning in the room where the sample was collected [12]. Percentage progressively motile spermatozoa from a given dog is not affected by frequency of

Morphology

Percentage morphologically normal spermatozoa (MNS) is not altered in dogs with frequent semen collection [16]. Age may have a role; in humans, percentage MNS declines after 45 years of age [17].

Preparation of the morphology slide and staining technique artifactually alter fertility [27], [28], [29], [30], [31]. One study compared preparation of slides by placing a drop of semen on a glass slide, laying another slide directly on top of it, and pulling the two slides apart, as in a squash prep;

Concentration and total number

Concentration is the parameter measured when performing semen evaluation in dogs but has little value as an indicator of semen quality. Concentration is inversely related to volume collected [40]. Concentration multiplied by volume is the total number of spermatozoa in the ejaculate; total number of spermatozoa is dependent on testicular size [41]. In dogs, normal total number of spermatozoa is greater than 300 million.

Total number of spermatozoa decreases with frequent semen collection,

Miscellaneous tests

None of the techniques described below are useful for semen evaluation by the average practitioner, with the possible exception of assessment for other cell types and the hypo-osmotic swelling test. Some, such as use of centrifugation gradients, can be employed to enhance quality of semen before chilling or freezing.

Assessment for other cell types

Other cell types that may be present in semen include prostatic or urethral epithelial cells, immature germ cells, red blood cells, and inflammatory cells. Quantification of epithelial cells may be accomplished by determining number of the cells of interest (N) per 100 spermatozoa. Concentration of the cell of interest in millions/mL equals the number of the cells of interest times concentration of spermatozoa in that sample divided by 100 [9].

As many as 2000 white blood cells/μL may be present

Live-dead staining

Live-dead staining relies on variable appearance of spermatozoa that take up stain. It is assumed that spermatozoa that take up stain have damaged plasma membranes and are therefore designated as non-functional, or dead. Eosin-nigrosin stains are those most commonly described. Problems with this test include inability to consistently classify spermatozoa with partial staining and interference with staining if glycerol or fat globules are present in the seminal fluid [49]. The author is unaware

Hypo-osmotic swelling (HOS) test

The hypo-osmotic swelling test involves submersion of spermatozoa into a hypo-osmotic medium. Those spermatozoa that have intact plasma membranes will swell as fluid moves into the sperm cell; this will cause swelling and coiling of the tail [3]. Hypo-osmolar solutions described include sodium citrate (7.35 g) and fructose (13.51 g) in 1000 mL of distilled water, and 100 mM sucrose solution [50], [51]. Incubation time before examination varies. In one study, maximum percentage swollen spermatozoa

Measurement of components of seminal fluid

The components of seminal fluid reported as measured as a component of semen evaluation in dogs include glandular products, proteins, and electrolytes [1], [55], [56], [57], [58], [59]. Components may break down at variable rates after semen collection due to metabolism by spermatozoa and enzyme degradation, and may vary with interval from previous ejaculation, degree of sexual excitement, and health status of accessory sex glands [1].

Heparin-binding proteins have been identified in semen of

Filters, centrifugation gradients, fluorescent staining, and DNA analysis

Various filters have been described for use in evaluation and improvement of canine semen quality [60], [61], [62]. Filters may be used to assess for normal function of spermatozoa by evaluating motility via determination of extent to which spermatozoa can penetrate the filter, or by binding abnormal spermatozoa. While percentage MNS may be much better in semen after filtration, total number of spermatozoa present in the filtrate may be much lower than that in the original sample. In one study

Quality control

Standardization and quality control within laboratories enhances accuracy of analysis. Standardization among laboratories better allows researchers and practitioners to compare laboratory practices and to determine if evidence in the veterinary literature from different laboratories is comparable. Commercial veterinary laboratories work hard to ensure continuing quality control. Individual practices rarely address this issue.

Types of error that may occur include random error, which are chance

Interpretation of findings

Values determined in semen analysis by any means, computer-aided or more subjective, are meaningless unless taken in context. Factors that must be considered include the signalment of the animal; inbred status of the animal; time since semen was last collected; and perhaps season of the year. With this background knowledge, conclusions may be drawn regarding the likelihood of a dog being able to impregnate females.

Age was associated with fertility in humans; semen quality declined after 45

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The lack of the influence of various species of Mycoplasma spp. on canine semen quality
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Mycoplasmas colonize fish, reptiles, birds and mammals, being commensals or causing diseases, sometimes severe in ruminants, swine, poultry, or wildlife animals. So far, 15 species of canine Mycoplasma spp. have been described. Conflicting results have been presented regarding the pathogenicity of Mycoplasma spp. Although many virulence factors of these bacteria have been described, they still require attention. The main aim of our study was to evaluate the presence of known canine Mycoplasmas in the male reproductive tract of clinically healthy dogs. The second aim was to check if Mycoplasma spp. cause any abnormalities in semen quality that could have further consequences and to propose the schemes for managing the carriers.
83.3% of examined dogs were Mycoplasma spp. -positive dogs, and most of them were the carriers of more than one species. Six dogs had azoospermic ejaculates. The total spermatozoa numbers were similar in Mycoplasma -positive and negative groups. Motility was slightly higher in Mycoplasma spp.-negative group, but the difference was not statistically significant. There was no significant difference in semen characteristics between the carriers and Mycoplasma spp.-negative dogs. Neither the individual species nor the number of species strains had a significant effect on sperm morphological parameters as well as viability.
Semen quality parameters are not correlated with the species found on the prepuce. Over 70% Mycoplasma spp.- positive dogs have more than one species of this bacteria. Despite finding mycoplasmas in azoospermic dogs, we suggest that they were not the cause of infertility. Mycoplasma spp. could be a part of normal microbiota in canine prepuce in individuals without any clinical signs.
Diagnostic tests in canine andrology - What do they really tell us about fertility?
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Citation Excerpt :
Proper examination techniques and interpretation of the findings are in turn essential skills for small animal practitioners who work with breeders [10]. This is especially true in the context of semen transfer, preservation by chilling or freezing, or investigation in cases of suspected subfertility or infertility [11,12]. The result of a breeding soundness evaluation should give reliable insights into the breeding potential of a dog.
Dog breeders often require breeding soundness evaluations which include andrological examinations of the genital organs, hormone measurements, and semen analyses. During the past decades, a considerable number of research results have been published, allowing diagnoses of specific andrological conditions and fertility assessment.
For specific examinations, however, no standard procedures have been defined and for some parameters different reference ranges have been published. Therefore, examination results from different facilities are difficult to compare and profound conclusions regarding health and fertility of a male dog are not always possible.
Conventional semen examination, however, is still useful in identifying deviations or no deviations from normality, especially if confounding factors are taken into account and if the exam is repeated in case of inconclusive findings. A standardization of examination procedures and reference ranges would help to harmonize the exchange of examination results and interpretation of the findings.
Insights into the influence of canine breed on proteomics of the spermatozoa and seminal plasma
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This study aimed to characterize the proteome of spermatozoa and seminal plasma of 4 purebred dogs (Golden Retriever, Great Dane, Bernese Mountain Dog, and Maremmano-Abruzzese Sheepdog). The ejaculate of 13 dogs was collected, and sperm characteristics were subjectively evaluated. Seminal plasma and sperm cells were separated and prepared individually for mass spectrometry. Data were evaluated by univariate and multivariate statistical analysis. A total of 162 proteins were identified, 47 in spermatozoa, 109 in seminal plasma, and 6 in both samples. Serum albumin in spermatozoa and tubulin alpha-3E chain, acrosin binding protein, and tubulin alpha-3 chain in plasma seminal were statistically relevant. Serum albumin and acrosin binding protein improve the sperm capacitation, acrosome reaction, and seminal quality. The tubulin family proteins are related to structural cell organization and flagella movement, and their presence in seminal plasma may be related to sample handling. According to cluster formation, a high association was observed among Bernese Mountain Dog and Great Dane, Golden Retriever, and Maremmano-Abruzzese Sheepdog for sperm proteins. For seminal plasma proteins, Bernese Mountain Dog, Great Dane, and Maremmano-Abruzzese Sheepdog were related. Further studies on breed-specific proteins in the semen of purebred dogs need to be performed to clarify its fertility roles.
For the first time spermatozoa proteins of dogs are described. The comparison of spermatozoa and seminal plasma proteins of four purebred dogs were performed. These results supporting that differences in semen protein profile of different canine breeds exist, which can improve the biotechnologies of reproduction in this species.
Assessment of Dog Testis Perfusion by Colour and Pulsed-Doppler Ultrasonography and Correlation With Sperm Oxidative DNA Damage
2020, Topics in Companion Animal Medicine
The assessment of testicular artery blood flow by colour and pulsed-Doppler ultrasonography is an important diagnostic technique to assess vascular perfusion. Recently, it has been suggested as a good predictor of sperm quality. On the other hand, through the alkaline Comet Assay, it is possible to quantify sperm oxidative DNA damage. The aim of this study was to evaluate the relationship between routine sperm parameters, testicular artery blood flow and oxidative DNA damage in canine sperm. Testicular ultrasonography and sperm collection were performed on 12 male dogs, with the animals being allocated into 2 groups, according to the classification of the ejaculates’ quality, as normozoospermic (N; n = 7) or non-normozoospermic (OAT; n = 5). Seven dogs aged between 1.5 and 8.0 years old were included in group N and 5 dogs, aged between 2.0 and 11.0 years old, were included in group OAT. The sperm-rich fraction of the ejaculates was evaluated for sperm routine parameters and DNA damage by comet assay. Colour and pulsed-Doppler ultrasonography were used to evaluate the blood flow of the supratesticular and marginal arteries of right and left testis. Group OAT presented higher levels of sperm oxidative DNA damage (A.U.) in comparison to group N (N:11.7 ± 9.9; OAT:34.2 ± 6.1; P< .001). The peak of systolic velocity was positively correlated with sperm concentration (r = 0.685; P= .005). The resistive and pulsatility indexes (RI and PI) of the supratesticular artery were negatively correlated with sperm membrane integrity (HOST⁺) (r = −0.594; P = .042; r = −0.612; P = .035, respectively). The end diastolic velocity (EDV) of the supratesticular artery was positively correlated with sperm concentration (r = 0.748; P = .005) and negatively correlated with sperm oxidative DNA damage (r = −0.766; P = .004). Our results suggest that the assessment of the testicular artery blood flow by colour and pulsed-Doppler ultrasonography could be a good predictor of sperm quality in dogs in terms of sperm concentration, membrane integrity and sperm oxidative DNA damage.
Proteomic data of seminal plasma and spermatozoa of four purebred dogs
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Semen contains several proteins that are important to fertilization and to identify reproductive failures. There are proteins that are specie-specific expressed, although differs among several breeds. This article provides experimental data describing the protein profile of seminal plasma and spermatozoa of four healthy purebred dogs: Golden Retriever (n=3), Bernese Mountain Dog (n=4), Great Dane (n=3), and Maremmano-Abruzzese Sheepdog (n=3), housed at São Paulo state, Brazil. Semen samples were collected by manual stimulation of the penis in a presence of a teaser bitch, when possible. The seminal plasma and sperm cells were separated by centrifugation and prepared for mass spectrometry. The gene ontology annotation of the proteins found is described. This is the first time that proteomic profile of the semen of purebred dogs is described. These data are a valuable resource to improve the biotechnologies of reproduction applied to canid species.
The Effect of N-N-Dimethylformamide on the Membrane Characteristics of Canine Spermatozoa After Cryopreservation, and its Relationship With Post-Thaw Motility
2020, Topics in Companion Animal Medicine
Citation Excerpt :
The dogs were subjected to physical and breeding soundness exams at the Section of Theriogenology and Herd Health, School of Veterinary Medicine, Universidad Nacional de Colombia. Only ejaculates from dogs with total motility > 70%, normal sperm morphology > 50%, and total sperm number > 300 × 106 sperm were used for freezing procedures.20 The dogs selected for the trial were healthy at the time of examination and semen collection and showed good libido.
Some studies have demonstrated that glycerol is superior to amides in preserving sperm motion characteristics of canine sperm; however, little is known about the effect of these cryoprotectants on the membrane characteristics of canine spermatozoa after freezing/thawing. In this study, the effects of using either N-N-dimethylformamide (DMF) or glycerol (GLY) on the integrity and function of the canine sperm, after cryopreservation were determined. We hypothesized that the use of a multiparametric approach for assessing the effect of DMF on the membranes of canine sperm would explain the lower values reported for post-thaw motility. Ejaculates from 12 dogs were collected, split into 2 groups, and frozen using a tris-fructose-citrate-egg yolk-based extender containing either 7% (v/v) GLY or 7% (v/v) DMF. Frozen straws (n = 120) were thawed and analyzed for subjectively-assessed sperm progressive motility, normal morphology, plasma membrane integrity, plasma membrane function (HOST+), acrosome membrane integrity, high mitochondrial membrane potential, and simultaneous assessment of sperm membrane integrity and function by a triple-staining fluorescent procedure. Overall, sperm motility and membrane intactness/function were higher when GLY was used as a cryoprotectant, as compared to DMF (P < .05). A model to explain the variation in progressive motility using the values obtained from the sperm integrity and function parameters was designed. The percent HOST+ sperm and high mitochondrial membrane potential sperm were mostly associated with the changes observed in the progressive motility (r² = 0.84; P = .043) when either GLY or DMF were used as cryoprotectants. These results may explain the overall reduced sperm quality observed after cryopreservation, as a reflection of sublethal damage sustained by the sperm membranes.

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The value of canine semen evaluation for practitioners

Abstract

Section snippets

What do the numbers mean?

Collection and evaluation of semen

Volume

Color

pH

Motility

Morphology

Concentration and total number

Miscellaneous tests

Assessment for other cell types

Live-dead staining

Hypo-osmotic swelling (HOS) test

Measurement of components of seminal fluid

Filters, centrifugation gradients, fluorescent staining, and DNA analysis

Quality control

Interpretation of findings

Anim Reprod Sci

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Fertil Steril

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Theriogenology

A critical review of methods for evaluation of spermatogenesis from seminal characteristics

J Androl

Semen characteristics and fertility in dogs

Vet Rec

Oestrus cycle stage dependent effects of male and female blood plasma and vaginal fluid on dog semen motility parameters

Theriogenology

Tests of sperm function and male infertility

Res Reprod

Time- and temperature-related alterations in seminal plasma constituents after ejaculation

Int J Androl

Evaluation of the “Minitube SpermVision Computer-Based Automated System” for dog semen analysis

Theriogenology

WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction

Clinical significance of hematospermia

Mayo Clin Proc

Studies on dog semen. I. Morphological characteristics

J Reprod Fertil

Semen characteristics of normal and vasectomized dogs

Indian J Exp Biol

Studies on evaluation of dog semen

Livest Advisor

The evaluation of semen quality in the dog and the effects of frequency of ejaculation upon semen quality, libido, and depletion of sperm reserves

Cornell Vet

Sperm concentration and normal sperm morphology decrease and follicle-stimulating hormone level increases with age

BJU Int

New semen quality scores developed by principal component analysis of semen characteristics

J Androl

Computer-assisted analysis of motility, velocity and linearity of dog spermatozoa

J Reprod Fertil

A three-step statistical procedure to identify sperm kinematic subpopulations in canine ejaculates: changes after cryopreservation

Reprod Domest Anim

Use of the sperm quality analyzer (SQA) for the assessment of dog sperm quality

Reprod Domest Anim