It mainly dependends on the size of the dog, the size of the prostate gland, the animal age, the frequency of semen collection, the level of erotisation, and the volume of 3 rd fraction collected. A decrease of semen volume is observed in cases of benign prostatic hyperplasia, prostatic cysts, inflammatory lesions of prostate and testicles, inflammation of epididymis, vas deferens or urethra and at weak libido.
The colour of whole ejaculate depends on the volume of third fraction of ejaculate collected, on the concentration of spermatozoa per mL and the potential presence of non-germ cells in the ejaculate. When analysing the colour, one should be aware of the method of collection, as colour varies with the fraction to be analysed and the fact that analysis may been performed on the whole semen or on fractioned semen.
The normal colour of whole ejaculate is greyish-white. Pathological colours include: green-greyish typical for the presence of the pus in semen; red or pink-specific for erythrocytes contamination haemorrhages from urethra or corpora cavernosa, prostatitis ; yellow specific for urine contamination; and brown, if in the presence of blood.
Any kind of semen contamination, such as hair or mud, exclude the specimen from further procedures including artificial insemination or semen preservation. It is therefore important to check the region of praeputial opening before semen collection and to clean it. The presence of sediment consisting of sperm cells at the bottom of the tube is a normal feature if the semen is left for several minutes. One of the most important step of conventional semen assessment is the subjective evaluation of progressively motile spermatozoa Spz under contrast-phase microscope.
The evaluation is performed under the objective of x20 to x If the highly concentrated sperm-rich fraction is collected separately, the semen should be extended with saline or Tris-buffer to a concentration allowing the observation of particular, single sperm cells. The assessment is based on the evaluation of the average percentage of progressively motile spermatozoa in a few different fields of the specimen. A decrease in the percentage of motile spermatozoa may results from temperature shock, contamination with water, urine, blood or lubricants but also from long sexual abstinence and systemic or infectious diseases, such as brucellosis.
Sperm agglutination is always pathological and is frequently found in cases of infectious diseases. Concentration and total sperm count. In order to find the sperm count per mL, the number of spermatozoa in the one or four large squares depending of the chamber is multiplied by For the assessment of sperm concentration more sophisticated equipment could also be used, such as the spectrophotometer, flow cytometer or computer assisted semen analyser Rijsselaere et al.
A large variety in the total number of spermatozoa per ejaculate is observed in different breeds. It varies between 50 x10 6 up to x 10 6 Spz Linde-Forsberg, ; Oettle Small breeds do not produce as many spermatozoa as large breeds, as sperm cell production is related to the weight of the testicular tissue.
The number of spermatozoa per ejaculate also varies according to age, testicular weight, sexual activity and the size of the dog Amann, The total number of spermatozoa in the ejaculate may be decreased in young and older dogs and in inbred males. Apprehension, absence of the teaser bitch, painful prostate, spine rear limbs may also negatively influence the number of spermatozoa ejaculated.
Sperm morphology. The morphology may be assessed under contrast-phase microscope, but usually the evaluation is performed under light microscope on stained slides. Smears of undiluted or diluted ejaculate are examined microscopically for the presence of structural abnormalities of spermatozoa. The semen is smeared on a glass slide in a similar manner to that of blood, air dried and stained.
The semen may be also stained with a nigrosin-eosin stain. A drop of this stain is gently mixed with a drop of semen on a pre-warmed slide before being smeared, and allowed to air dry. Evaluation of sperm morphology should be completed microscopically using oil immersion, using an objective of x or x A minimum number of spermatozoa should be counted and evaluated for the presence of abnormalities.
The percentage of cells with particular morphological defects and of normal cells are calculated. Traditionally sperm cells abnormalities are divided into primary defects - originating from abnormalities of spermatogenesis and secondary defects - originating from abnormalities of semen maturation, transit through the ductal system and specimen preparation. According to another classification sperm abnormalities may be divided into major defects, negatively correlated with fertility, and minor defects, unassociated with fertility Table 6 Oettle, When a spermatozoon presents more than one abnormality, it should be classified according to the most important abnormality or with the most prevalent one, if they have equal significance Oettle, The assessment of the percentage of live and dead spermatozoa is based on the assumption that dead spermatozoa possess disintegrated plasma membrane allowing eosin penetration.
Thus the percentage of eosin positive cells stained with nigrosin-eosin stain is considered as percentage of dead cells. The evaluation of the percentage of live and dead spermatozoa and the percentage of morphological defects may be performed on the same nigrosin-eosin stained slides. In the past 2 to 3 decades, several strategies were developed to escape the subjectivity in the semen evaluation, related to the experience and skills of the observer, the method of specimen preparation, staining technique and number of cells evaluated, and wich is particularly important when the fertility potential of preserved sperm cells has to be ascertain.
Moreover, implementation of such methodologies, not routinely usable in the small to median veterinary clinics due to their costs, allows accurate comparisons between laboratories worlwide and minimizes occurence of large errors.
Furthermore, advanced semen assessment is essential whenever the semen has to be preserved, in particular for freezing. Advanced semen assessment techniques are sumarized on table 7. In general, the results obtained with these methods are better correlated with the AI outcome than the results of traditional semen evaluation. The use of adequate number of viable sperm cells per dose. On next sections the major issues on timing the AI and available techniques of semen deposition on the bitch genital tract will be discussed.
Obtaining successful pregnancies and adequate number of offspring per litter depends upon the correct timing for mating, as well as for insemination, particularly because bitches are mono-estrous, presenting usually one to two reproductive cycles per year. Although relationship among behavioral, hormonal and physiological events for the average bitch exists, considerable individual variation are also currently found on what concerns the duration of the estrogenic and early luteal stages proestrus and estrus and of the anestrus Concannon et al.
The bitch usually presents a relatively long follicular phase and considerable variability exists in the onset of estrous behavior and acceptance of the male, making it difficult to determine occurrence of the LH surge and onset of ovulation in this species unless specific methods for timing the ovulation and estimating the fertile period are used Linde-Forsberg, Furthermore, in this species, ovulation of immature oocytes primary oocytes, before extrusion of the first polar body determines the need for a maturation period in distal oviducts that may last for hours Concannon, , ; Tsuitsui, ; Tsutsui et al.
Those particularities in the reproductive physiology may explain why the major cause for infertility in the bitch is the inappropriate breeding management Goodman, ; Linde-Forsberg, Consequently, careful planning of mating time by timing ovulation is a key step in canine artificial insemination.
These evaluations should be performed in sequence and with days intervals for the majority of females if the bitch has been reported to present short heat period, of about 6 to 9 days, is possible that daily evaluations may be needed.
On the vaginal cytology, epithelial cells of the vagina change their form in response to estrogen impregnation, and passes from small round cells with a clearly visible cytoplasm in non-estrogenic stages, to larger, cornified, angular shaped-cells with small pyknotic nucleus, almost to the point of disappearing, under the influence of estrogens Figure 1.
Progesterone semi-quantitative immunoenzymatic assays are available for clinical routines, but although rapids, these test lack accuracy. A recent study showed that, in dogs, semi-quantitative methods for progesterone determination are less accurate than the quantitative methods, in particular at intermediate plasma progesterone concentrations Moxon et al.
According to this study, the tested semi-quantitative assay estimated higher progesterone concentration than. RIA radioimmunoassay , which could suggest that the fertilization period had commenced earlier than it was actually the case. In addition to those assays, quantitative radio or chemiluminescent assays can also be used, even if not always available in the house lab, since cross-reactivity exist to the molecule between different species, for example with human progesterone.
Schematic representation of the major morphological changes of the predominant epithelial cell in the vaginal cytology during the dog estrous cycle. Although ovarian ultrasound examination is a reliable and accurate method to determine ovulation in most domestic females, in the bitch fat accumulation in ovarian bursa that encloses the ovaries may difficult the value of the technique.
Consequently, follicular dynamics evaluation through ultrasonography US in dogs is still experimental and must follow a very precise protocol, which accuracy increase with the use of frequent examinations. In a recent study, Fontbonne reported that US was accurate enough to detect the occurrence of ovulation and obtain comparable numbers of ovarian structures between US examinations and macroscopic visual count on the surface of the ovaries after surgical removal, even if only one daily examination was performed.
However, that author accepts that features of ovulation may be difficult to visualize in large breeds and in overweight animals. Pre-ovulatory follicles may present different aspects at US. Usually they appear as round to slightly triangular anechoic structures, sometimes slightly flattened, giving a honeycomb aspect to the ovary Figure 2.
Persistence of non-ovulatory follicular structures was perceived in US images after ovulation. Also, in the immediate post-ovulation period, until 24 hours after US changes of the ovaries at ovulation, hypoechoic structures were observed in most cases Figure 2.
Ultrasonographic scans of canine ovaries before and after LH surge and ovulation. US are compared with images of longitudinal sections of canine ovaries of similar stages of follicle developement.
It is possible to use vaginal endoscopy to determine the fertile period although it does not allow accurate timing of ovulation. However, this method requires expensive equipment. Nevertheless, it may give a huge contribution to the vaginal evaluation and detection of anatomical abnormalities that may impair proper reproductive performance. The fluctuation of estrogen and progesterone concentrations in the blood at consecutive stages of estrous cycle in the bitch results in specific morphologic changes of the vaginal mucosa.
The observation of the cranial part of the vagina is performed for this purpose. The deep introduction of the tip of endoscope into the narrow part of the vagina close to the cervix dorsal median postcervical fold or paracervix, is of less diagnostic value Pineda et al.
Vaginoscopic examination is performed using a rigid endoscope mm in diameter, with diagnostic sheath and a length of cm or longer. The examination should be done on the standing animal. Usually there is no need of administration of sedatives. When the tip of the optics reaches the vagina it should be repositioned at horizontal axis.
During proestrus the increase of the estrogen concentration results in the oedema of the vaginal mucosa. Vaginoscopy reveals rounded folds in the vagina.
The mucosa of the folds is turgid, pink in colour and with a smooth surface. Normally the bloody discharge is also visible in the vagina.
Sometimes, periodic blood outflow from the cervix, through the paracervix may be observed. The lumen of the vagina is narrow, which can be appreciated when the endoscope is advanced cranially.
At the last days of proestrus and at beginning of estrus, the decrease of estrogen concentration and increase of progesterone P 4 level is noted. It results in the collapse of vaginal folds.
Formerly turgid and smooth, the mucosa, becomes wrinkled and shrunked. Vaginal folds become smaller. Maximal intensity of shrinkage of vaginal mucosa is observed between 3 and days of estrous cycle. This time the loss of fluid from the tissue of vaginal mucosa and submucosa is great and the shape of vaginal folds become angulated with sharp angles at the top of folds.
As the result, the lumen of the vagina is wider in comparison to proestrus. During diestrus vaginal folds become flat and round. The mucosa is red and small petechia may be visible at places touched by the tip of the endoscope. This is due to the fact that epithelium of the vagina is thin and consists of only cell layers in diestrus and anestrus.
An opaque, thick mucus is sometimes visible on the surface of epithelium Figure 3. Vaginal endoscopy of the bitch. In the bitch, when timing the day of ovulation as accurately as possible is essential to guarantee adequate fertility in natural mating systems, it becomes even more important to determine precisely when to inseminate bitches according to the sort of semen to be used fresh, chilled or frozen semen , as usually semen longevity and sperm cells survival decreases with time.
When fresh or chilled semen is used, insemination should be performed on the day of ovulation, and a second insemination must be schedule for 2 days later. However, scheduling for the artificial inseminations may be slightly adjusted according to the experience of the operator, the place for semen deposition and the limitation on the number of inseminations.
Consequently, regimes for canine AI may vary with authors Root Kustritz, Table 8 condenses the available information on the AI schedules for fresh, chilled and frozen semen. Graphic representation of the fertile period and the ideal moment for canine AI according to the type of semen.
If in the AI with fresh semen the success of the procedure is strongly related to the quality of semen used and the moment for AI Table 9 , when using chilled semen both the quality of semen and the site of semen deposition are important factors for success, whilst in the AI with frozen semen, the intra-uterine semen deposition is critical Table In dogs, during natural mating, occurs the projection of a considerable portion of the ejaculate into the uterus, through the cervical canal, during the coital tie England et al.
However, deep vaginal insemination shows acceptable results when using fresh semen, and also to some extent for chilled semen. As in other species, in dogs sperm cell number in the uterine lumen may be influenced by many factors, such as the moment of estrus, the type of breeding natural mating or insemination the method of insemination intravaginal or intrauterine , the type of semen fresh, chilled or frozen and sperm quality total and progressive motility and sperm speed , besides some individual variations Rijsselaere et al.
However, despite the influence of the intrauterine vs. Consequently, no potential differences or advantages exist between the vaginal endoscopic approach and laparoscopy when the intra-uterine insemination is intended, as no differences were found in the deposition of the semen in the uterine body or the cranial tip of the uterine horns Fukushima et al.
Nevertheless, abdominal laparoscopy or surgery is strongly discouraged on the basis of animal welfare issues, as non-healthy related invading procedure that should be avoided. No diferences were found in the litter size between these groups, which were very similar in age and parity of the bitches. Results for the AI procedures with fresh and chilled semen and 2 AI per animal, 48h apart.
Deep vaginal insemination is probably the widestly used method for insemination with fresh semen when the technique is performed by the breeder or in small budget clinics. For vaginal AI a simple plastic catheter of proper length may be used, to which a plastic disposable syringe containing the semen is attached. Or a commercial catheter in flexible latex tube presenting an inflatable balloon at the tip, like the Osiris gun, may be used; when inflated, this kind of device has the advantage of increasing the probability for intrauterine transport of the semen and of preventing semen backflow Farstadt, ; Linde Forsberg, a.
Before AI procedures start, cleaning of the perineal area, in particular the peri-vulvar area, is needed. As transabdominal palpation is usually used to guide or ascertain the vaginal catheter position, the owner of the female should be instructed to bring the animal with an empty stomach, which facilitates the procedure Linde Forsberg, a.
The bitch is placed in a standing position on an examination table or on the floor according to the size of the female. To avoid catheterization of the urethra the urethral opening in the bitch is located at the pelvic brim , particular attention should be paid not to unintentionally introduce into the urinary bladder.
The insemination catheter is carefully introduced in the vagina of the bitch, first steeply upwards until the pelvic brim has been passed, and then in a horizontal angle, when it is carefully pushed further ahead Farstad, In alternative, the vulva may be elevated to just below the anus as the bitch does when stimulated by the male dog Linde Forsberg, a.
At this point, the position of the AI catheter must be learn by palpation, and orientated. If the catheter is in the urinary bladder, the cranial part of the vagina and the cervix may be palpable above the catheter and also the tip of the catheter stands out more clearly, due to the thinner walls of the urinary bladder in comparison to those of the vagina Linde Forsberg, a. After certification that the catheter is correctly placed, it is moved onward through the cranial portion of the vagina delimited by the dorsal medial folds.
In smaller or primiparous bitches this point can be difficult to overcome, and may not be possible to pass the catheter into the cervical fornix. Except for those females, the AI catheter should be further introduced until it reaches the paracervical area, which can be palpated as a 1- to 2-cm-long, firm structure that ends at the cervix a firm, rounded to ovoid structure, freely movable.
The semen is deposited once the catheter has been located in the paracervical area, close to the external cervical os. This position facilitates transabdominal palpation of the cervix and ensures that the semen will not be expelled through backflow. According to earlier reports, the bitch should be maintained in the same position up to period of time varying from 5 to 20 min after AI. However, reducing the interval of elevated hindquarters to 1 min seems not affect fertility Pinto et al.
Also, feathering or stroking of the vulvar or perineal region is reported by several authors as form of stimulating the semen transport into the uterus, in an attempt to mimic the vaginal stimulation by the thrusting movements of the dog during natural mount. However, the contribution of such procedures to the exit of the technique has not been proven yet. Intrauterine insemination may be performed by using non-surgical transcervical catheterisation Linde-Forsberg, ; Linde-Forsberg and Forsberg, , ; Linde-Forsberg et al.
M Silva et al. The majority of European centers working on small animal reproduction prefer transcervical intrauterine insemination TCI due to reasons associated with animal welfare. However, catheterisation of uterine cervix in the bitch is a difficult procedure and demand skill and experience.
The semen of lower quality, such as frozen-thawed or that collected from subfertile dogs have to be deposited intrauterine to assure satisfactory results of artificial insemination Linde-Forsberg et al. The conception rates after intravaginal insemination with frozen-thawed semen are significantly lower when compared with the results of intrauterine insemination. The method of non-surgical transcervical intrauterine insemination was first time described in Andersen, The technique has been adapted from the artificial insemination performed in foxes.
As evidence of their belief, nearly everyone in the village can name a person whose unexplained death was clearly the result of a toxic puppy pregnancy. After one year-old college graduate had an encounter with a stray dog that scratched him on the leg six months earlier, he became extremely wary of dogs because he was deathly afraid that one might knock him up.
Importantly, he also underwent a month of behavioral reconditioning with a dog while being treated as an inpatient. The views expressed are those of the author s and are not necessarily those of Scientific American.
Already a subscriber? Sign in. Thanks for reading Scientific American. Create your free account or Sign in to continue. See Subscription Options. It turns out there is more to your genome than genes. In fact, genes are regulated so that they are on or off at different times, and in different amounts. And this is really important for making a person a person, and a dog a dog. Imagine you want to make a grilled cheese sandwich. Things have to happen in a specific order.
Put the butter in the pan first, then make the sandwich, then put it in the pan, then flip it, et cetera. Similarly, to make a person or a dog, everything needs to happen in a certain order.
You need to have a head before you have hair, right? And fingers before you have fingernails! To make sure this order happens the right way, genes get turned on and off in different amounts and in a very specific order by things called transcription factors. Transcription factors attach to genes to turn them on and off, a little or a lot. Even though human and dog genes are similar, they are not regulated the same. This is the set of genes that makes your arms and legs, or in the case of a dog four legs.
The genes themselves are pretty similar in dogs and people. What gives a dog four legs and a human two arms and two legs has more to do with which genes are turned on when and to what level. You might think that having both of these orders at the same time would be fine—that maybe one would win, ending up with either two arms and two legs, or with four legs. The dog instructions for making limbs would prevent the human way from working right. And vice versa: the human instructions for making limbs would stop the dog way from working right.
So what would probably happen is that no limbs would be made at all, or the wrong number or location of limbs would be made because the human and dog instructions are competing with each other. And without limbs developing the right way, all development would stop and the developing embryo would die. Limb development is just one really important example of how different regulation of genes in humans and dogs would prevent making a Mog. There are many, many other gene sets in humans and dogs that would act like this.
By Amanda Jacobson, Stanford University. He did not have a dog dad and human mom.
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