Canine Reproductive Pathophysiology

• Canine Reproductive Pathophysiology, presented by Meera Gatlin DVM, MPH, DACVPM.

Introduction

• Contact Meera Gatlin via email; her office is in the Wildlife Building, room #115.
• There is no syllabus; exam reply is on PPT or lectures.
• TIDA lessons are due on Canvas by April 4, 2025.
• Completing TIDA modules online earns a certificate like the IAT for the USDA program.

Learning Objectives - Canine

• The canine reproductive tract in the bitch and dog relating to estrus, breeding, and copulation will be reviewed.
• Hormonal changes occurring in the canine estrous cycle and their link to behavioral and clinical changes will be understood.
• Cytological changes will be visually identified through each stage of the canine estrus cycle.
• The importance of canine ovulation timing for optimal breeding management will be discussed.
• The importance of hormonal physiology in health concerns like pyometra, ovarian remnant syndrome, and benign prostatic hypertrophy, and in spay/neuter guidelines will be conveyed.
• Parameters used to evaluate canine semen will be listed.
• The type of placentation found in the canine will be described.
• The physiology of pregnancy and parturition will be understood.
• The learning Objectives apply to topics discussed today and next week

Reproductive Anatomy : The Bitch

• Canine ovaries are caudal to the corresponding kidneys and located within the ovarian bursa, sized at 1-3cm.
• Ovarian artery and vein, primary follicles, maturing follicles, corpus albicans, retrogressive corpus luteum, corpus luteum (progesterone), Graafian follicle (oestrogen), and a ruptured follicle are involved in all stages of ovarian activity.
• Uterine tubes (AKA Fallopian Tubes, Oviducts): the transport of oocytes from the ovary to the uterus.
• Ovarian end: Infundibulum with fimbriae.
• Middle: Ampulla, where fertilization occurs.
• Uterine end: Isthmus with crypts for sperm docking.
• Uterotubular Junction prevents backflow from the uterus.
• The uterus is bicornuate, suspended by the broad ligament, has long uterine horns containing the developing embryos, and a short uterine body where the two horns meet.
• The cervix is a short, thick-walled muscular sphincter connecting the uterus with the vagina, located within the abdomen, with an average size of 1.5 - 2 cm.
• The vagina is oriented along the longitudinal axis, the epithelial lining undergoes hormonal changes during estrus.
• Cervix is not visible or reachable, it is located within the abdomen
• During proestrus, the vaginal mucosa is edematous and pink.
• During estrus, the vaginal mucosa becomes a pale pink and reduction in edema causes a crenulated appearance.
• During diestrus, the vaginal mucosa appears patchy and hyperemic, and tissue is irritable.
• The vestibule is between the lips of the vulva and the vagina.
• A canine will typically have 10 (range 8-12) mammary glands, two thoracic, two abdominal, and one inguinal on each chain, arranged in bilaterally symmetric rows.
• Mammary glands are often symmetrical, but some variation can occur.

Canine Puberty

• Puberty in canines occurs between 6 and 24 months of age and influenced by breed, size, environment, and body condition.
• Familial reproductive history will help with predictions.
• First heats are less predictable and can be split.
• Term "heat" refers to the entire estrous cycle.

Canine Hormones

• FSH = ovarian follicular development.
• Estrogen prepares the reproductive tract for coitus.
• Estrogen (-) feedback on FSH stimulates LH.
• LH = start of estrus, transition to luteal phase
• follicle ruptures, forms CL, which release P4
• P4 causes behaviors and prepares repro tract for fertilized ova
• If pregnant, P4 from CL inhibits GnRH to prevent more follicles and maintain pregnancy.
• If not pregnant, CL regresses, lifting the negative feedback and restarting the cycle.
• P4 = progesterone

The Canine Estrous Cycle

• Canine estrous cycle is monoestrous and typically happens twice yearly but can range from 1-4 times a year.
• It is non-seasonal.
• 4 phases: anestrus, proestrus, estrus, metestrus or diestrus.
• Term "Diestrus" is preferred over "metestrus".
• Proestrus (Follicular phase) lasts for 9 days, the bitch is attractive to males but unwilling to stand to be mated.
• There is vulvar swelling with serosanguinous discharge.
• The bloody, tinged discharge in proestrus is not a period
• Hormone levels: Estradiol peaks and Progesterone begins to rise in late proestrus.
• Follicles are developing, Mixed epithelial cells, Extracellular bacteria, Red Blood Cells, White Blood Cells in early to mid-proestrus.
• Range: 0-27 days
• Estrus lasts for 9 days, the bitch is receptive to mating.
• The vulva softens but is still swollen and the vaginal discharge gradually lightens in color.
• Hormone levels: Estradiol drops to basal concentrations, Progesterone increases rapidly and LH (luteinizing hormone) surge occurs.
• Follicles ovulate primary oocytes which must undergo a final meiotic division to secondary oocytes and CL (corpora lutea) develops.

• 90% cornified epithelial cells, +/- Extracellular bacteria, Reducing RBC, with rare to no WBC, Range: 4-24d

• Diestrus (Luteal phase) lasts for 2 months, and the bitch is not receptive to mating.
• The vulva decreases in size and there may be some vaginal discharge.
• There may be +/- mammary development with lactation in late diestrus, hormones low, and CL present.

• 50% parabasal and intermediate epithelial cells with WBC may be present (even abundant) in early diestrus and reduced RBC.

• Corpus luteum is present regardless of pregnancy status
• Diestrus period is roughly the same length, regardless of pregnancy status
• The anestrus phase lasts for four months.
• The bitch is not receptive to mating and the vulva continues to reduce in size.
• Hormone levels: Estradiol low and Progesterone low.
• FSH (follicle stimulating hormone) and LH pulses increase in late anestrus.
• Antral follicles in various stages of development/atresia occur
• CLs regressing, >90 parabasal and intermediate epithelial cells, Few extracellular bacteria, Few WBC, no RBC.

Correlation of Hormones and Stages

• Note how the 4 stages correlate with the hormones, with rising Estrogen levels, then first rise in Progesterone that continues in the Diestrus stage.
• There is a fertile period.
• Ova mature, oocyte maturation, LH surge and a reminder that the dotted line is rising progesterone and dashed line is falling estrogen levels.
• Unique in canine ovulation.
• Pro-estrus is dictated by Estrogen and Estrus is dictated by Progesterone

Oocyte Maturation

• Ovulation occurs 2 days after LH surge.
• Eggs ovulate as primary oocytes in prophase I (immature).
• Require approx. 48-60 hrs to complete metaphase II.
• Eggs are only mature 4 days after LH surge (start of fertile window).
• Eggs remain fertile for 48-72 hrs following maturation.

Veterinarian Tools for Estrous Understanding

• 3 tools for vets to help understand hormones: vaginal cytology, progesterone testing, and LH testing

Vaginal Cytology

• Useful tool any vet can use to evaluate progression of estrus cycle.
• Bioassay for estrogen levels (not quantified), inexpensive and easily performed when bitch is in estrus.
• Sampling should not be attempted when the bitch is in anestrus
• Sample vagina (not vestibule) using moistened cotton swab and part lips, advance dorsally and cranially SLOWLY.
• It is important to advance the swab up and over the pelvic brim
• Spin the swab to collect cellular material, roll sample onto slide in unidirectional fashion, and Diff-quick stain.
• Phases
• 4 phases of cells: 2 Non-cornified - parabasal and intermediate; 2 Cornified - superficial and anucleate.
• The four phases of cells can be remembered with the acronym PISA
• Parabasal cells "fried egg" seen in diestrus and anestrus and not uncommon in early proestrus, conspicuously absent in estrus.
• Intermediate - Small – round or oval with a moderately sized nuclei that are large – slight polygonal shape with large nucleus / cytoplasm ratio seen in proestrus.
• Superficial - Early cornification seen in late proestrus into estrus with a polygonal or “cornflake” shape evident with an undefined or very small nuclei.
• Anucleate - Full cornification seen in estrus with Polygonal or “cornflake” shape evident with no Nucleus is absent.
• Neutrophils are often seen in diestrus following copulation, can be seen in proestrus, especially with the RBCs present, as the vagina is NOT a sterile environment.
• Mixed population of extracellular bacteria is normal in the vagina, and should evaluate for intracellular bacteria in neutrophils.
• Vaginal cytology a bioassay for estrogen levels and should show Parabasal cell to Neutrophil Superficail cell through the cycle.

Serum Progesterone Testing

• Serum Progesterone and Implication to Estimated Day until Ovulation and Best fertile days
• <1 ng/mL - Proestrus
• 1.0-1.9 ng/mL - At or near LH surge is +2 days (+4 days best)
• 2.0-3.9 ng/mL - LH surge has likely already occurred +1 day (+ 3 days best)
• 4.0-10 ng/mL - (avg is 5.0), At or near ovulation, (o days, +2 days best)

• 10 ng/mL - Ovulation has occurred, Immediately.

The Boys

• Male dogs have a penis with musculocavernous tissue, where the Os penis aids in intromission.
• At the base of the penis has a Bulbus Glandis engages that engages the copulatory lock, unique to canine species.
• Elasticity at base of penis allows 180 degree turn for copulatory lock

Prostate

• Only accessory sex gland in the dog, located retroperitoneal, bilobed, surrounds the urethra at the neck of the bladder and provides most of the ejaculatory volume.
• Testes have oblique positioning within the scrotum with the long axis dorso-caudal
• Head of epididymis oriented cranially and tail (cauda) of the epididymis is oriented caudally.
• Testicular decent into the scrotum typically happens within the first week of life and they reach their final position at approximately 35 days.
• Males are not classified as cryptorchid until 6 months when inguinal rings are fully closed.
• Check for two testicles in male puppies during visits for shots

Sexual Behavior

• Age of first ejaculation Varies by breed but approximately 1 year of age and can occur after a year in some larger breed dogs.
• Secondary Behaviors in male dogs are Humping, Marking and Leg lifting that are not always sexual in nature and can be related to anxiety and/or dominance.
• Castration does not always resolve secondary male behaviors because there can be a learned component to the behavior
• Natural Breeding will occur where The penis enters the vagina prior to becoming erect.
• Os penis provides rigidity and Vulva deflects prepuce, penis becomes fully erect that causes swelling of the bulbus glandis during erection, with the tie lasts for approximately 10-15 minutes.
• Do not try to separate two dogs if they are copulatory locked or tied because it can cause injuries to both dogs.
• Mating occurs at first by mounting, then butt to butt and they remain tied for 10 to 15 minutes

Semen Collection/Evaluation

• Needed: Teaser bitch and/or scent swab, Artificial vagina (AV) that connection to glass or plastic collection tube or sleeve.
• Need stimulus of the male dog to collect Sperm
• Restraint/collection via a Calm/quiet environment, Good footing, and a Strong gloved hand, and NO lube
• Retraction of the sheet during stimulus is important, because if the sheet is constraining the engorging bulbous glandis, it can be painful and cause a bad experience, making it difficult to collect future sperm.
• Note
  • Color
  • Opacity
  • Volume: where volume is only relevant to final sperm count and not a reflection of performance Volume is the only relevance to a performanc, all test should be done in 3 fractions.
• Sperm Concentration measured with Hemocytometer cassettes
  • Densimeter
  • Computer assisted sperm analysis (CASA)
  • Nucleocounter
• Total Sperm Count by Concentration (per mL) X volume = total sperm/ejaculate.
  • Ideally 10 million sperm / lb body weight.
• Motility
  • Evaluate diluted on a warmed stage at high power
  • Total motility vs progressive (purpose and direction). Ideally >70% progressively motile.
• Morphology
  • Evaluate individual sperm shape Requires fixation and staining (eosin-nigrosin) Ideally >70% normal morphology. If there is a high percentage of defects, it is a sign of what is going on with testicular health

Breeding Management

• Important components are The Bitch's ovulatory cycle, Stud dog / sperm availability with Processing lifespan, and Type of breeding.
• Oocyte Maturation as indicated from cycle diagtam.
• There is a very narrow fertile window and timing is essential for breeding whether or not it is natural or artificial

Semen Prep and Lifespan

• Semen collection then evaluation for use within
  • Fresh semen, used directly for insemination with for only 20 minutes, and only viable for 5-7 days in bitch
  • Chilled preservation transport lasts for are good about 48-72 hour inside a female dog. It is most commonly used when shipping semen for breeding
  • Cryopreservation best last
• Sperm in general require physiologic temperature
• During a semen collection, if one only wants to get prostatic fluid, it can be done by only collecting the post-fraction sample

Types of canine breeding

- natural selection

• artificial, such as side by side fresh frozen vaginally, via the trenscervix, surgical or interuterine

Natural Service

• where the when and what of dogs mating can be done via the male and female dogs breeding in short bursts for up to 14 days
• there can be issues with transporting
• Transporting dogs or genetic material can be a challenge. Natural service may not be possible in situations with large sexual dimorphism, prior injuries, unwillingness to stand, or anatomical challenges

Procedures of Insemination

• Vaginal insemination (VAI) is where Semen is deposited in vaginal tract using pipette or catheter.
  • Trans-cervical insemination (TCI) calls for a rigid fiberoptic endoscope deposit directly into the uterine body by catheterizing the cervix with fresh, chilled, or frozen specimen. It is minimally invasive and normally requires little to no sedation.
    • Requires training
• Requires hand-eye coordination, but is rewarding to do if you like to play video games
• Intrauterine / surgical insemination (IUI) Semen injected into the horns of the uterus via an abdominal surgical incision via. a fresh, chilled, or frozen specimen
• IUI is invasive and requires full anesthesia, avoid this choice if possible

Physiology of Pregnancy in Canines

• Fertilization occurs in the uterine tubes.
• Transuterine migration allows to embryos to spread evenly through the uterus prior to implantation.
• Canine embryos implant ~16-18 days following LH surge.
• Type of placentation in Canines is zonary and endotheliochorial.
• Progesterone is from CL NOT placenta
• Diestrus is the SAME length in gravid and non-gravid bitches occurs before Pregnancy diagnosis or Parturition.
• Gestational Length is about 65 days from LH Surge with calculation from breeding dates that are inaccurate.
• 63 days from ovulation. If you ovariectomize a pregnant does, they will lose the pregnancy because there will be no progesterone supporting it

pregnancy diagnositics

• Palpation is best before about 31 days then gets challenging
• Ultrasound best about 28 days and is useful for checking for Viability by check.ing the fetus and heart rate
• Relaxian that can be released with gravit utters that can be good to show after about 28 days in sample
• radiology can start after 45 days.
• Pregnancy can be diagnosed with palpation, ultrasound, relaxin, and radiographs. Confirm pregnancy with ultrasound during days 25-28 It is reasonable to get fetal counts with ultrasound
• Gestational aging can come from fetal measurements
• Radiographs can be used to get a more accurate fetal count ~ days 55-60

Canine Parturition

• Canines can drop as high as degrees in temperate so the revaluation needs to be good with
• Relaxation of gravid uterus
• Mammary development or early lactation
• Most predicting methods are fairly inaccurate, and this is with state the labor is starting which is the stages that occur,
• Comperative drop and rebound for temperature
• Myometrial uterine contractions are not externally visible from out point.
• Position puppies for the delivery
• Early cervical dilatation and are not palpable to
• Recommendations are puppies are delivered about every hour, to avoid complications in maternal health, as well as neonatal mortality
• Stage three is the passing of the placenta. This alternates with stage two

Harmones and Health

• Harmones influence pyometra which is uterine infection.
• Hormonaly and infectioned by E.coli and closed with fluid, and open
• In pyometra hormones can play a significant role, like easter gain and sensitivity to progesterone

Pyomerta

• Higher progesterone level to decrease uterine contraction Patients are more like the typically order then more like the eighter with a lot of fever Treatment is spay or medial management, using alizin to help decrease levels of the infection

Ovarian Remnant Syndrome

• Functioning ovarian tissue causes the return of extra after spay Assigns of estrus evident Be able to test for high progesterone and get the animals health from the How do you avoid any level is gone

Benign Prostatic Hyperplasia

• BPH is Prostate cancer that can affect intact males/dogs that causes a secondary testing level It's in older dogs older than five years of age Non-painful condition but can we promote it to get the best

Spay and Neuter

Understanding the reproduction can be important and have the body have a good change Has a very high quality to reduce heat and Easel have a procedure that is fast pace Recent from the past decade shows that the hormonal balance can help change the urinary tract, and to help balance joints

Canine Health Information Center (CHIC)

• Created by the Orthopedic Foundation for Animals
• Information is specific to purebred dogs
• It is health testing information for breeding
• It engages testing healthy dogs for breeding

Pre-ovulatory Follicular Luteinization

• Is unique to dogs. It is an increase in progesterone prior to ovulation
• The serum progesterone levels are anywhere from 0.2 to 1.5 ng during pre-ovulatory follicular luteinization
• There is an LH surge around 2 ng
• Two days after the LH surge there is ovulation
• The progesterone has a doubling effect. Two days after the LH surge for about 5 ng and 10ng when ovulation happens