محاضرات د . نزيه . ماجستير توليد

         محاضرات د . نزيه .  ماجستير توليد

                     
                        Anatomy of reproductive system

 The female reproductive organs include paired gonads (ovaries), which produce both female gametes (ova) and hormones; paired uterine tubes, which capture the ova on their release from the ovaries and convey them to the uterus; the uterus, in which the fertilized ova are retained and nourished until prenatal development is complete; the vagina, which serves both as copulatory organ and as birth canal and the vestibule, which continues the vagina to open externally at the vulva.
The ovaries

The ovaries possess both gametogenic and endocrine functions. Each ovary is a solid, basically ellipsoidal body, although commonly made irregular by the projection from the surface of large follicles and corpora, the irregularity is naturally greatest in polytocous species, in which follicles ripen in bitches. Ovaries are usually found in the dorsal part of the abdomen, close to the tips of the horns of the uterus, as they do not shift far from their place of development. Each ovary is suspended within the cranial part (mesovarium) of the broad ligament, the common suspension of the female reproductive tract. The rapid enlargement undergone by those follicles selected to come to maturity in the current cycle is mainly due to the accumulation of the fluid by which the ova are swept out on ovulation, the cavity within the ruptured follicle, though it may initially fill with blood, is soon occupied by hypertrophy of the granulosa and theca cells that originally lined the space. This produces a solid body known as the corpus luteum (yellow body) on account of its color. Corpora lutea are transient structures that wax and wane between one estrous period and the next. Degeneration of the corpora lutea is characterized by vacuolization of the cytoplasm of the luteal cells due to lipid accumulation and nuclear shrinkage. Although transient, they are important as the source of progesterone, just as the ripening follicles are the source of estrogen. Corpora lutea finally regress and are replaced by connective tissue scars, corpora albicantes.
The uterine tubes


 The uterine tubes are narrow and generally very flexuous. They capture the ova released from the ovaries and convey them toward the uterus because they also convey the sperm in their ascent, fertilization normally occurs within the tubes. The free cranial extremity takes the form of a thin walled funnel infundibulum placed close to the cranial pole of the ovary. The free edge of the funnel is ragged, and the tags (fimbriae) come into contact with, and sometimes adhere to the surface of the ovary. A small (abdominal) orifice in the depth of the funnel leads to the longer tubular part that is divided into two more or less equal segments. The proximal one, known as the ampulla, is followed by the more convoluted and narrower isthmus, the isthmus joins the apex of the horn of the uterus at the uterotubal junction, a region of very variable appearance.
The uterus
The uterus is the enlarged part of the tract in which contain embryos, where they establish a means of physiological exchange with the mother‟s blood stream, and where they are protected and nourished until ready to be delivered to the outside world. In all domestic mammals the median part of the uterus has two segments. The caudal, very thick-walled segment. The horns (cornua) vary greatly in length, and it is longest in polytocous species. Their disposition also varies; they are characteristically wound in ruminants, straight and divergent in mares and bitches, and cast into intestine-like loops in sows. The body and horns of the uterus typically lie within the abdomen above the mass of intestines. The uterus possesses serosal, muscular and mucosal coats that are known as the perimetrium, myometrium and endometrium respectively. The serosal covering reaches the uterus by extension from the supporting broad ligament.
The cervix

The cervix generally lies within the pelvic cavity, interposed between the rectum and the bladder. The cervix projects, caudally into the vagina, this heavy, smooth, except during estrus and parturition, this mucus seen at estrus secretion of cervical goblets cells. Its provides a sphincter controlling access to and from the vagina. Mucus secreted by cervical glands plugs the canal at most


times, the passage is open only at estrus and immediately before, during and for a short time, after parturition.
The vagina

 The vagina, consists of two parts The cranial part, which represent reproductive passage that runs from the cervix to the entrance of the urethra, the caudal part, the vestibule extends from the urethral orifice to the external vulva and combines reproductive and urinary functions. The two parts together constitute the female copulatory organ and birth canal. The vagina is a relatively long, thin- walled tube that is distensible in length and width. It occupies a median position within the pelvic cavity, related to the rectum dorsally and the bladder and urethra ventrally. The surface is smooth but circular and longitudinal folds may form when the walls of the inactive organ collapse inward. The intrusion of the cervix into the cranial part of the vagina reduces the lumen of this part to a ring-like space known as the fornix.
The vestibule and vulva

The vestibule much shorter than the vagina, lies mainly if not entirely caudal to the ischial arch, which is a circumstance that permits it to slope ventrally to its opening at the vulva. In bitch, the urethral opening is raised above the general level of the vestibular floor in others, such as the cow, it is associated with a sub urethral diverticulum more caudally, the vestibular walls are marked by the entrances of the ducts of vestibular glands. In bitch the glands are small but numerous and the duct orifices form linear series.

Reproductive hormones
The trends in endocrine changes are shown the main feature which distinguishes them from other species previously described is the prolonged luteal phase, illustrated by the persistence of high progesterone levels in the peripheral blood. It is noticeable that progesterone levels start to rise before ovulation has occurred, which confirms the morphological evidence of preovulatory

 luteinization of the mature follicles 60–70 hours before ovulation. This preovulatory rise in progesterone ma provide the stimulus for the bitch to accept the male. In addition, it can also be used as a method to determine the timing of artificial insemination in that it should not be delayed long after concentrations > 2–3 ng/ml are observed in peripheral plasma. Estrogens rise rapidly just before the onset of standing estrus, and are rapidly followed by the LH peak, which lasts much longer than that of other species; ovulation occurs 24–96 hours after this. FSH concentrations at estrus reach a peak, coincident with that of LH. Prolactin appears to have a negative correlation with progesterone; thus, as progesterone levels fall towards the end of metestrus or pregnancy, prolactin increases; it is the major luteotrophic hormone in this species.

Puberty
Puberty in the female dog is defined as the occurrence of the first estrus (onset of receptive behavior), most puppies attain puberty between 8 and 19 months of age, with a range of 3.5 to 24 months for both males and females, onset of puberty is also influenced by body condition and breed. Pubertal estrus occurs variably at 6–14 months in most breeds, with means positively correlated with breed size.

Seasonality
Canids have only oestrus cycle per year and are seasonal, where as dog has one or two, sometimes even three oestrus cycle per year and no obvious seasonality. Recent researchers indicates that fertility may be lower during the warm season. Domestic bitches are  nonseasonally monoestrous. As a result of this unique reproductive physiology, bitches spontaneously ovulate only once or twice per year and ovulation can occur at any time of the year. However, there are a few exceptions, such as the Tibetan Mastiff and the Basenji breeds.


Estrus cycle
 Reproductive activity in the bitch differs from the polycyclic pattern of other species in that there are no frequent, recurring periods of heat. All bitches have a prolonged period of anoestrus or sexual quiescence between successive heats irrespective of whether they have been pregnant or not; this pattern has been described as monocyclic. The average interval between successive estrous periods is 7 months, but it is variable, and there is some evidence that the breed of the bitch can have an effect. Mating does not appear to influence the interval, although pregnancy caused a slight increase. There does not appear to be any seasonal effect on reproductive function since there is a fairly even distribution of the occurrence of estrus throughout the year. The estrous cycle is traditionally divided into four phases. Proestrus. The bitch has a true proestrus characterised by the presence of vulval oedema, swelling and a sanguinous discharge. Some fastidious bitches show no evidence of discharge as they is attractive to males but will not accept the male. Estrus. The bitch will accept the male and adopts the breeding stance. The vulva becomes less oedematous and the vulval discharge becomes clearer, less sanguinous and less copious. The duration of proestrus and estrus combined is about 18 days, i.e. 9 days each. However, this can be very variable, some bitches showing very little sign of proestrus before they will accept and stand for the dog and others producing a copious sanguinous discharge during true estrus. Some bitches also show evidence of sire preference, which can affect the timing. Ovulation usually occurs 1 or 2 days after the onset of estrus, although, using laparoscopy, it has been observed that some follicles continue to ovulate up to 14 days later. Metestrus. This stage starts when the bitch ceases to accept the dog; however, there is dispute about its duration. Some consider that it ends when the corpora lutea have regressed at 70–80 days whilst others measure it in relation to the time taken for repair of the endometrium, 130–140 days. Anoestrus. At the end of metoestrus the bitch passes into a period of anoestrus without any external signs. The same is also true after parturition following a normal pregnancy. This phase lasts about 3 months before the bitch returns to proestrus.

 The inter estrous interval is the time from the onset of proestrus to the subsequent onset of proestrus and includes proestrus, estrus, diestrus and anestrus. These phases reflect, respectively, follicular phase rise in estrogen, the initial luteal phase rise in progesterone and decline in estrogen, the remainder of the luteal phase, and the interval between loss of luteal function and onset of next cycle.
The first indication that proestrus is approaching is the onset of slight swelling of the vulval lips. This generally precedes the commencement of bleeding by several days. Labial swelling is progressive during the proestrus period. Bleeding attains its maximum early in proestrus and continues at this level into the early part of true estrus. During the greater part of proestrus the bitch, although attractive to the dog, takes no interest in his attentions. She will not stand for him and generally attacks him if he attempts to mount her. A day or so before the end of proestrus, her attitude changes, and she shows signs of courtship towards the male. These comprise sudden darting movements which end in a crouching attitude with her limbs tense and her face alert. She barks invitingly, but as the dog approaches she moves suddenly again. She will not yet allow him to mount. With the onset of estrus the invitation to coitus is obvious. She stands in the mating position with her tail slightly raised or held to one side. She remains still while the male mounts and copulates. In the later stages of the copulatory tie, which occupies from 15 to 25 minutes, she becomes restless and irritable and her attempt to free herself may cause the male considerable physical embarrassment. After the first 2 days of estrus, sexual desire gradually recedes, but with the continued persuasion of the male she will accept coitus until the end of the period. Bleeding, although reduced in amount, generally continues well into estrus. More often, however, the discharge becomes yellow as estrus proceeds. Vulval swelling and tumefaction are greatest at the onset of the stage of acceptance. During the course of estrus the enlarged labia become softer in consistency. Some labial swelling continues into the first part of the metestrous phase.

Proestrus

Proestrus is diagnosed clinically by the onset of vulvar edema and/or serosanguinous discharge. The average duration of proestrus in mature bitches is 9 days with a normal range of 0 to
 27 days. Proestrus is defined as that stage of the estrous cycle where noticeable signs of serosanguinous (thin, bloody) vaginal discharge begin. Varying degrees of bloody vaginal discharge are typically seen. The nature of the discharge is typically bright red and voluminous at the onset of proestrus and then becomes less voluminous towards the end. During proestrus, females are attractive to male dogs, but generally refuse mating. Pheromones, which are present in the female‟s vaginal secretions and urine, are responsible for this attractiveness. Sexual reflexes such as flagging of the tail (elevation of the tail away from the vulva and swaying of the hips from side to side) in response to touching the perineal region begin in proestrus. Hormonally, the proestrual bitch is under the influence of estrogen and this is the dominant hormone during this stage of the cycle. Estrogen is responsible for most of the clinical signs seen in bitches during proestrus, because this hormone stimulates growth and activity of the glandular epithelium of the uterus and promotes swelling and increased vascularity of the lining of the uterus (mucosa). The serosanguinous vaginal discharge is a direct result of these effects because the junctions between the cells lining the capillaries leak and permit passage of red blood cells into the uterine lumen which is eventually discharged vaginally after passage through the cervix. Serum progesterone concentrations during proestrus are at basal levels (<0.5 ng/ml) and then start a gradual rise at the end of proestrus. Preovulatory follicular luteinization (transformation of the estrogen secreting follicle to a progesterone secreting structure), unique to the canine is responsible for this increase in progesterone concentration. The effects of estrogen dominance during proestrus are also reflected when vaginal cytology is evaluated. Sloughed vaginal epithelial cell types vary during proestrus and are usually accompanied by numerous red blood cells.

Estrus

 Estrus in the canine is characterized by the bitches’ willingness to allow mounting and intromission. The estrus phase begins with this acceptance of the male and ends when the bitch no longer permits a mating. The average duration of estrus in the bitch has been reported to be 9 days with a range of 4 to 24 days. The character of the vaginal discharge is also different in estrus in most bitches and is classically straw colored due to the diminishing presence of red blood cells. Some bitches, however, may continue to have a serosanguinous vaginal discharge through the estrus period. Hormonally, the bitch appears to be receptive to the male in estrus due to declining estrogen levels and increasing progesterone levels. Estradiol continues its decline from peak values of late proestrus to intermediate values of 10–20 ng/ml. Estrus in the bitch occurs in response to the decline in estradiol that normally begins shortly before the LH surge and continues throughout estrus. Serum progesterone concentrations gradually and steadily rise during estrus. At the start of estrus, progesterone concentrations are typically near 1.0 ng/ml and reach levels near 2.0 ng/ml at the preovulatory LH (Luteinizing Hormone) surge. At the time of ovulation two days later, the serum progesterone concentration is typically in the range of 4.0 to 10 ng/ml. Progesterone concentrations continue to rise after ovulation and reach peak levels in diestrus. Estrus onset is facilitated synergistically by the rapid rise in progesterone resulting from the LH surge. Cytologically, estrus can be defined by the presence of greater than 90 percent of the vaginal epithelial cells being superficial cells.
Metestrus

Metestrus, the post-estrous portion of the luteal phase, was initially defined behaviorally as starting when estrous behavior ceases, metestrus begins a day 6–11 from estrus. The end of metestrus and anestrus onset are variably defined as when uterine endometrium has undergone histological “repair”, when serum progesterone declines to levels persistently below 1 or 2 ng/ml. Serum progesterone increases to peaks of 15–80 ng/ml.


Diestrus


 Diestrus is the phase of the cycle that follows estrus and is characterized by progesterone dominance, the duration averages 56 to 58 days in pregnancy and 60 to 100 days in the non pregnant bitch. Diestrus is generally considered to occur clinically when the estrus bitch first refuses a mating and less likely to be attractive to males at this time. Progesterone secretion is maximal approximately 2 to 3 weeks after the beginning of diestrus and reach peaks of 15 to 90 ng/ml at this time. After this peak, progesterone gradually declines over the remainder of diestrus. It has been suggested that the onset of diestrus be defined by vaginal cytology rather than mating behavior because events such as whelping can be more accurately timed.
Anestrus

The duration of anestrus differs between and within dog breeds, indicating a genetic basis for anestrus length. This variation depends on breed, health, age, time of year, environment, and multiple other factors. Canine anestrus involves the absence of overt evidence of ovarian activity, is considered to be lasting a minimum of 7 weeks after progesterone declines below 1–2 ng/ml, and averages 18–20 weeks. It may initially be important for the normal endometrial repair that is completed around day 120–130, the mid-luteal phase, low in early anestrus and absent by day 120.
Changes in the vagina and uterus

Vaginal smear

The cyclical changes which occur in vaginal cytology. Vaginal smears stained with either a simple stain such as Leishman‟s or a trichrome stain such as Shorr‟s can, with practice, be used to determine the stage of the estrous cycle. The advent of the „Diff-Quik‟ staining method has greatly simplified the procedure. At the onset of proestrus, large numbers of erythrocytes are present; however, when true estrus occurs the number of erythrocytes is reduced, and the smear consists of superficial cell types from the stratified squamous epithelium, such as anuclear cells, cells with pyknotic nuclei and large intermediate cells. Towards the end of estrus, numbers of


polymorphonuclear neutrophil leucocytes appear in the smear, and these become the dominant cell type during metestrus. In anoestrus nucleated basal and intermediate cells of the stratified squamous epithelium, together with a few neutrophils, form the characteristic smear.
 Vaginal epithelium

The vaginal epithelium during anoestrus is of the low, columnar, cuboidal type and comprises two or three layers only. During proestrus the epithelial cells change to the high, squamous, stratified type and persist in this form until the later stages of estrus. The stratum corneum and the layers immediately beneath it are lost by desquamation during the proestrous and estrous periods, leaving a low, squamous structure which becomes converted to columnar epithelium in 1–3 weeks after the end of heat. During metestrus (and pregnancy) the epithelium is of a higher columnar type than during anestrus. During proestrus, estrus and early metestrus the epithelium and lamina propria are infiltrated with large numbers of neutrophils which eventually escape into the vaginal lumen.
Endometrium

The endometrium shows considerable change during the estrous cycle. The endometrial glands in proestrus and estrus are loosely coiled with very obvious lumina and deep epithelial lining. During metestrus the glands become larger, the lumina smaller and the coiled parts in the basal layer of the endometrium more tortuous. As the bitch passes into anoestrus, there is a reduction in the amount and degree of coiling of the glands. At about 98 days after the onset of estrus, i.e. in metestrus, there is evidence of desquamation of the endometrial epithelium; however, by about 120–130 days the epithelium has been restored by proliferation of cells from the crypts of the endometrial glands.
Changes in the ovaries

During anoestrus the ovaries are oval and slightly flattened. In a bitch of medium size they measure approximately 1.4 cm from pole to pole and 0.8 cm from the attached to the free border. No appreciable follicles can be seen, although on section the minute remnants of the CLs of the previous cycle may be seen as yellow or brown spots. In the young bitch, the surface of the ovaries is smooth


 and regular, but in the aged animal it is irregular and scarred. At the commencement of proestrus, developing follicles have already attained a diameter of 0.5 cm. They progressively enlarge, until at the time of ovulation their size varies from 0.6 to 1 cm. By this time the ovary is considerably enlarged, its size depending on the number of ripe follicles present, and its shape is irregular due to the projection of the follicles from its surface. Owing to the thickness of the follicle wall it may be difficult to distinguish between follicles and CLs. Prior to ovulation, the surface of the follicle shows a slightly raised papule, pin-head-sized, and the epithelium covering it is brown, which  contrasts with the flesh color of the remainder of the follicle. A remarkable feature of the ripening follicle of the bitch is the thickness of its wall, due to hypertrophy and folding of the granulosa cells, which can be seen on section with the naked eye as evidence of preovulatory luteinization. Ovulation is spontaneous and normally occurs 1 or 2 days after the onset of the period of acceptance. Most of the follicles rupture over a period of 48 hours. The oocyte is capable of being fertilized for up to 108 hours after ovulation. The CL at first contains a central cavity, but becomes filled by compact luteinized cells by the 10th day after ovulation, by which time the body has attained its full size (0.6– 1 cm). CLs now comprise by far the greater mass of the ovary. As a rule an approximately equal number of CLs are found in each ovary, although occasionally there are wide differences. (In this connection it is interesting to note that the numbers of fetuses in the respective cornua in pregnancy frequently differ from those of the CLs in the ovaries on the respective sides.) Embryonic migration into the cornua on the opposite side would appear to be common. On section, the CL is yellowish pink; it remains unchanged in the non-pregnant bitch until about the 30th day after ovulation, after which it slowly atrophies. Visible vestiges may be present throughout anoestrus. During pregnancy the CLs persist at their maximum size throughout, but regress fairly rapidly after parturition.
Ultrasound appearance of the ovaries

Using a transabdominal approach via the flank, with the bitch standing and a 7.5 MHz real- time linear array transducer, were able to identify ovarian structures at the onset of proestrus; these

 were circular and anechoic and were obviously developing antral follicles. When the bitch was in estrus, they had increased in size, reaching a maximum of 4–13 mm in diameter on day 13 (day 0 being onset of proestrus). The walls of the follicles became thickened from day 10 onwards, presumably due to preovulatory luteinisation; there was no evidence of follicular collapse associated with ovulation. At 25–30 days after the onset of proestrus the ovaries were difficult to identify.




Ovulation
Ovulation occurs in response to an abrupt end of proestrus gonadotropin surge resulting in a 1– 3 day elevation in LH and a 1–4 day elevation in FSH. Ovulation has been timed to occur about 48– 60 h after the LH surge. In bitches, and in contrast to most other mammals, oocyte maturation occurs in the distal uterine tubes ±2 days post ovulation, as in foxes.

Fertilization
Dogs are highly fertile animal, with fertility rates reaching 95% of mated bitches. The high fertility is likely because fertile mating can occur in as early as 5 days before ovulation or as late as 6 days after ovulation and because of prolonged life spans of intrauterine sperm (up to 7 days) and oocytes (up to 7-8 days, including up to 5 days after oocyte maturation). The optimal time of fertilization which occurs between 2 to 4 days after ovulation, when the oocytes are fully mature and have not underwent degeneration. Plasma progesterone is elevated from the day of the LH surge until 18 h before parturition, peaks around day 20-30 and declines slowly thereafter, is solely of luteal origin, and is dependent on both LH and prolactin secretion. The circulating levels of progesterone appear to far exceed those required to maintain pregnancy in dogs; in ovariectomized pregnant bitches maintained on exogenous progesterone. Pregnancy was maintained even though serum concentrations of progesterone were below 2 ng/ml for several days in some animals.