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Therio Practical Katelynn Gilbert  Table of Contents Palpation 101 Sheep Pregnancy Dystocia Small Animal Breeding Obstetrics Semen Analysis Fetotomy Stallion Analysis AI and Heat Dete...

Therio Practical Katelynn Gilbert  Table of Contents Palpation 101 Sheep Pregnancy Dystocia Small Animal Breeding Obstetrics Semen Analysis Fetotomy Stallion Analysis AI and Heat Detection Mare BSE Transcervical Insemination Misc notes Ram BSE  Palpation 101 Sheet: cows= cartilaginous cervix ○ C= cervical diameter ○ should be able to feel rings ○ UT= uterine tone ○ Internal cervical os= smaller than T1-T3, flaccid external cervical os ○ RH/LH= uterine horns ○ Internal cervical os= what we’re ○ RO/LO= ovaries measuring for cervical diameter F1-F3, CH, CL3-CL1, Cy ○ Broad ligament loosely attached (cystic), NSS (no significant to horns structures) Glove ○ pink= smaller, blue= larger ○ Turn glove inside out → use air to evert fingers  Palpation 101 Approaching the cow ○ Hooking method: pull on cervix ○ Don’t touch rump of cow → walk hand forward to ○ Grab just the tail and insert hand intercornual ligament → move into rectum hand up both horns and retract Should only have to insert hand up to uterus → start at base and work wrist to palpate cervix your way up → isolate ovaries Retraction and use thumb to identify ○ Find the pelvic brim → locate structures uterus ○ Sweeping method: insert hand → reach around to “sweep” up uterus into hand → retract → start at base and work your way up → isolate ovaries and use thumb to identify structures Know how to identify different follicles on ultrasound!!!  Dystocia Placing chains ○ Continue to twist ○ Don’t have to make half hitch ○ Calf should “flip over” ○ Place as high as possible, one on each limb ○ Place chains into Cornell detorsion rod ○ Knot or tie chain to rod ○ Twist detorsion rod until it becomes too hard to twist ○ Apply fulcrum (ex: broom handle)  Dystocia Uterine torsion 3P’s ○ Palpate broad ligament ○ Presentation ○ Feel which way it’s “going down” normal= cranial/caudal longitudinal → clockwise vs counterclockwise AKA anterior/posterior Retained limb abnormal= transverse ○ Front limb= both joints flex in ○ Position same direction normal = dorsosacral ○ Back limb= one joint flexes in one abnormal= dorsopubic, dorsoilial direction and the other joint ○ Posture flexes in the opposite direction normal= extended abnormal= flexed/retained  Dystocia Obstetrical management Forced extraction: the manual or ○ mutation= manipulating the mechanically assisted removal of the fetus into a deliverable position calf through traction. ○ repulsion= push the fetus back Fetotomy: the reduction and removal into the uterine cavity (more room to correct/maneuver) of the fetus by division and removal of ○ rotation= turning the fetus on its extremities and sections longitudinal axis Cesarean section: the surgical removal ○ version= turning the fetus on its of a fetus via laparohysterotomy. transverse axis Extension of extremities: the correction of flexural deformities of the extremities.  Dystocia Lubrication ○ J-lube= toxic if it leaks into the abdomen= use with caution if you may need to do a C-section  Obstetrics Head snare Chinese finger trap ○ Place with thumb and finger in ○ Goes over the hoof to help pull loop to place it behind the ears leg ○ Black part/Y-piece inserted into ○ Difficult to clean placental mouth to act as mouth gag fluid/tissues off of mesh ○ Point of tension when removing Rope calf to help keep head straight ○ Bowline knot → create loop to (but not trying to pull the calf go above the fetlock → make out by its head) half hitch → place below the fetlock → +/- create half hitch to use as handle ○ Difficult to clean (biosecurity) ○ Difficult to remove wet knots  Obstetrics Chain placement ○ Keep knots on top (if on bottom ○ Longer chains preferred can cause leg to bend and make ○ Place chain inside itself to create it more difficult to remove fetus) loop → place hand in loop but ○ Place handle about 6 inches away keep pinkie finger out (to from the vulva prevent chain from sliding down) → place loop above fetlock → make half hitch (outside of the body) → place another loop below fetlock  Obstetrics Calf jack ○ Butt plate placed over the top of the cow so it rests below the vulva ○ Take pre-placed chains → remove handle → attach chain to loop of calf jack ○ Pull “lever” so chain is tightened → push down on the end of the pole when cow starts to strain (use gravity to help pull calf out)  Obstetrics Stages of labor Examination ○ Stage 1: early labor, with a typical ○ Tie tail to the neck or have an duration of 4–12 hours. Its assistant hold it conclusion is marked by rupture ○ Fetal viability of the allantois. ○ Presentation ○ Stage 2: delivery of the calf. This ○ Degree of fetus-dam is also referred to as “true labor” disproportion and typically lasts 30 min to 4 hours, but the calf can often live 8–10 hours.4 ○ Stage 3: passage of the fetal membranes  Fetotomy Place chain on leg (one loop above ○ Pass wire introducer up and over and below fetlock) the shoulder of the fetus → try Fetotome to “catch” wire introducer → ○ Place wire introducer through remove wire from cow and feed fetotome → insert gigli wire into through fetotome wire one of the holes at the end of introducer → attach handle on the wire introducer and bring gigli wire though second hole → pull wire ○ Insert fetotome into cow with introducer through the fetotome fetotome head as close to fetus → place gigli wire handle on the as possible (make sure wires end of the wire → use second aren’t crossed) and 2 fingers wire introducer (the one that can width away from joint go inside the cow) and “hide” wire inside head of the tool →  Fetotomy Fetotome cont. Krey hook ○ Place chain around fetotome ○ Place doubled over chain on Krey handle hook → place free end in loop → ○ Place fingers on either side of pull loop to secure chain to Krey the head of the fetotome, with hook elbow of other arm raised high ○ Insert into cow closed → open ○ Wait until person holding the and attach to fetus → pull head of the fetotome says they’re ready ○ Move gigli wire with continuous movements (make sure not to stop until all the way through)  Fetotomy There must be sufficient space to Make sure you remove every piece of perform the cuts the fetus Anterior presentation cut sequence Examine the uterus for any tears, cuts, ○ Decapitation and neck or the presence of another calf amputation (allow shoulder to collapse and more room for next If the calf does not present any cut) abnormality such as schistosomus ○ Thoracic cut and evisceration refelxus or perosomus elumbis, and if (remove thorax and front legs) the calf is in a caudal presentation and ○ Abdominal cut is too large to be delivered vaginally, a ○ Pelvic bisection (split the legs fetotomy should be avoided because and pull them out) after cutting one or both rear limbs the thorax will be too large to be delivered and usually the calf cannot be rotated. A C-section would be indicated for these cases. Fetotomy equipment: (A) Utrecht fetatome; (B) threader or insertion coil and wire brush; (C) Lyss wire handles; (D) wire handles; (E) Shriever wire introducer or passer; (F) Hauptner wire introducer or passer; (G) Linde’s fetotomy palm knife; (H) Geunther’s fetotomy finger knife; (I) side cutters to sever wire; (J) T-bar obstetric handle; (K) Krey hook with obstetric chain attached; (L) wire; (M) Moore’s obstetric handle. (a) fetal head snare; (b) de-torsion bar or rod; (c) wooden dowel for applying torque to de-torsion bar; (d) double blunt eye hooks or Vienna scissor eye hooks (a) 152-cm obstetric chain; (b) 76-cm obstetric chain; (c) Krey hook with attached chain; (d) T-bar obstetric chain handle; (e) Moore’s obstetric handle; (f) components of disassembled fetal extractor  Fetotomy Fetotomy indications: Partial fetotomy= most common, ○ Dead fetus remove head, neck, or limbs ○ Uncorrectable fetal malposition After fetotomy ○ Large fetus (fetomaternal ○ Lavage uterus disproportion) ○ Antibiotics ○ Fetal monsters ○ Anti-inflammatories ○ Incomplete cervical dilation ○ Ecbolics (ex: oxytocin or lutalyse) Requires adequate space between uterine wall and fetus (i.e. if uterus is contracted around fetus then they’re not a good candidate)  AI and Heat Detection Cow estrus behaviors: increased Estrus detection: paint cows, tail-head locomotion and vocalization, elevation pressure monitors, estrus detector of tail “flagging,” attempting to mount animals (teasers, freemartins), females, standing to be mounted, clear surveillance cameras, pedometry mucus discharge from vulva, swelling Ovosynch + CIDR = timed AI= no need and reddening of vulva, rubbed hair on to observe signs of heat/estrus tail head and dirty flanks, sniffing ○ Day 0 give GnRH to create CL → genitalia, chin resting on other cows, put in CIDR (progesterone) head raised and lip curled, decreased ○ 7 days later remove CIDR + give feed intake shot of PGF2a to lyse CL Causes for anestrus: pregnancy, pre- ○ give GnRH 48 hours later to pubertal, lactation, postpartum, create new CL manipulation, stress, pathology, lutalyse= acts on/lyses the CL freemartin, senile/old, owner failure (not observed) CIDR device (progesterone)  AI and Heat Detection cycles= 21 days long Ovulation occurs about 10-24 hours after standing heat ends → morning/afternoon rule ○ See signs in morning → inseminate in afternoon ○ See signs in afternoon → inseminate next morning  AI and Heat Detection Liquid nitrogen handling ○ Max 10 sec above the frost line ○ Well-ventilated area → otherwise lower canister back ○ Use tongs to withdraw objects into the liquid nitrogen for 10-15 ○ Wear gloves when handling sec to cool completely Semen thawing ○ Remove straws from tank with ○ Clean equipment between use tweezers with 70% isopropyl alcohol ○ Minimize time spent removing semen from liquid nitrogen tank to reduce semen damage (i.e. time above the frost line)  AI and Heat Detection Semen thawing cont. ○ Larger volumes of water are ○ Recommended to thaw semen recommended if more than 5 at 94-98℉ (last semester 96℉ semen straws are thawed was the ideal choice) simultaneously ○ Don’t thaw semen straws in your ○ Dry straw with paper towel once pocket or in the cow because the removed from water bath thaw rates are too slow → ○ Cut semen straw at crimped end reduce the number of viable → place plastic sheath over the sperm in the sample semen and gun → place semen ○ If using several straws stir water gun close to your body until bath so straws don’t freeze ready to inseminate to avoid together cold-shocking semen ○ Don’t load too many guns simultaneously (could reduce conception rates)  AI and Heat Detection Beef cattle Cows must have adequate body ○ 24 day heat detection test condition to expect most of them Cow should be 50-days post to be cycling calving, free of ○ Keeping track of heat dates reproductive disorders, and average interval (in days) between not be pregnant detected heats is divided into the At the end of the 24-day “expected” interval or 21 days period, the number of cows ○ Surest sign of estrus is that of a cow or detected in heat is divided heifer that permits other animals to by the total number of mount her while she remains standing cows eligible to have (standing heat) estrous cycles observe the cattle carefully for about 30 minutes at least twice per day  AI and Heat Detection Semen placement The recto-vaginal insemination process is ○ semen should be placed in the used. body of the uterus just in front of ○ The inseminator places his hand in the the cervix. rectum and manipulates the ○ You can recognize the proper reproductive tract so that the gun site by the change in tissue passes through the vagina consistency—from firm and hard ○ Then it is manipulated through the in the cervix to soft and spongy cervical rings in the uterus. ○ Then held at the internal opening of ○ To deposit semen at this location the cervix for semen deposition requires the use of a special Other methods: Kamar Heatmount device called Cassou pipette, or Detectors, Estrotect Heat Detectors, “AI gun.” Bovine Beacon, tail head markers, chin-ball markers, and the HeatWatch II System  AI and Heat Detection Dairy cows BCS 1-5 Beef cows BCS 1-9 ○ ideal= BCS 5  Transcervical Insemination Dorsal median vaginal fold= landmark Simple restraint without sedation Frozen semen most common Can be applied to all bitches Cervix is intra-abdominal Performed with minimal staff Utilizes rigid endoscope requirements Guaranteed intra-uterine semen Can use fresh, chilled, and frozen deposition semen No surgery or GA 30 degree viewing angle (forward Repeat inseminations possible oblique)= limited view of tissues below Results as good as any other the horizontal plane insemination technique Need scope with working length of about 29cm  Transcervical Insemination Anatomy ○ Cervical tubercle in front of and ○ Avoid clitoris (ventral below dorsal median fold commissure of lips) when (seperated from dorsal median introducing instruments fold by the transverse fold) ○ Urethral tubercle ventral (just ○ Narrow paracervical area (due to caudal to the vestibulovaginal dorsal median fold) junction)= careful not to introduce instrument into urethral orifice instead of vagina ○ Longitudinal folds in vagina ○ Dorsal median fold  Transcervical Insemination Technique ○ Use monitor to confirm ○ Clean vulva placement in vagina ○ Position catheter so the tip is ○ Keep scope tip in vaginal lumen contained within the sheath to ○ proestrus/estrus= rounded avoid contamination vaginal folds and excess fluids ○ Lubricate sides of sheath can make advancing scope more ○ Introduce at dorsal commissure difficult as they fill the lumen of vulva (avoid clitoral ○ Dorsal median fold may obscure fossa/clitoris) vision ○ Avoid urethra when inserting ○ Small amount of air inserted into catheter to ensure all the semen is deposited  Transcervical Insemination External size/shape of bitch does not Causes of loss of visibility: accurately predict internal anatomy of ○ Fogging (especially when first a bitch (ex: vaginal length) introduced, can be cleared by Diameter of paracervix= limiting deliberately touching vaginal factor, use catheter to keep fold off of fold, or can remove then rinse scope and replace) Cervical tubercle= can vary in size and ○ Excess fluid (usually more shape profound in proestrus, if Cervical os= usually found in rosette of necessary can remove fluid with folds (don’t mistake area with dorsal catheter) median fold), normally ventral position ○ Thick vaginal discharges in center of tubercle but can be in different locations or change position  Transcervical Insemination Causes of backflow Inseminate slowly and use only small ○ Catheter position (not far volume of air to clear semen from enough into uterine body, tip catheter should pass through cervical Safety canal into body of uterus, mark ○ Risk of trauma from catheter or catheter to know how far in you endoscope are) ○ Risk of infection (ex: from env or ○ Catheter type (bullet shaped inadequately cleaned equipment) with terminal hole best, if two ○ May be more at risk in diestrus side holes then more tendency to (due to progesterone influence) flow back along the catheter) ○ Insemination volume (max 3mL)  Transcervical Insemination Frozen semen results ○ Intrauterine deposition of semen ○ Accurate timing of insemination ○ Semen quality ○ Bitch fertility Dorsal median fold  Ram BSE Eyes and legs important for breeding Scrotal circumference soundness ○ Tapes: yellow and white Scrotal palpation (Reliabull, has plunger that turns ○ Start at the top and move down red when too tight) from spermatic cord → two ○ Push testicles as far into scrotum testicles as possible → place tape around ○ Look for symmetry and tone widest portion → tighten (swellings, dilations) (looking for “muffin top” when ○ Testicles should both be freely it’s tight enough) → measure movable (palpate epididymis as from straight part of buckle you’re pushing up) Measure here  Ram BSE Examine internal genitalia Ruminant semen collection ○ Accessory sex glands (prostate, ○ Electroejaculation (preferred ampulla, bulbourethral, and for rams on farms) seminal vesicles) ○ Artificial vagina Digital palpation ○ Manual massage in bulls ○ Clean out rectum ○ Can only palpate prostate (“ring on a fat finger”) and seminal vesicles (grape cluster feeling) Small ruminant AV: 1. Tube/collection device to attach to internal liner 2. Internal liner 3. Cover/sleeve 4. Black knob is where you can fill the AV with hot water (at least body temp) Helpful to have teaser ewe  Ram BSE Exteriorize penis Semen sample ○ Place on rump → hold still near ○ Gross motility= overall scrotum/sigmoid flexure area → movement push penis out Very good, good, fair, poor ○ Should be able to see glans penis ○ Progressive motility= individual and urethral process sperm movement ○ Purple coloration normal Extend out with sterile saline and watch individual sperm move ○ Morphology  Ram BSE Ultrasound ○ Place on rump/dock → push testicles into inguinal region to ultrasound ○ Proximally= pampiniform plexus ○ Moving distally → head of the epididymis → mediastinum (hyperechoic line in the center, where semen would collect) ○ Bright white line= calcifications, abnormal  Ram BSE Should be performed at least 1 month ○ Check teeth for sound bite/wear prior to the breeding season on incisors 1) physical examination ○ Check for any defects that could ○ assess the ability of a male to impair vision locate, move to, and physically 2) inspection of the reproductive mount a female in heat organs ○ Body condition of the male (ideal ○ Scrotum (scrotal circumference), 3-3.5/5 in rams) testicles, epididymis (ex: ○ Structural soundness of the feet enlarged with epididymitis from ○ Review history (ex: recent Brucella) disease, previous breeding success)  Ram BSE 3) semen collection and evaluation of ○ WBC can indicate infection sperm Males failing the BSE can be rechecked ○ Grossly evaluate for volume, after 4 to 8 weeks to see if fertility has color, contamination (urine, improved or if they have recovered blood, pus, dirt) from any existing conditions ○ Motility (gross and individual) ○ Morphology ○ All storage vessels should be warmed to prevent cold-shocking the sperm, which can kill sperm and reduce motility estimates Ram BSE Ram BSE  Sheep Pregnancy Pregnancy can be detected as early as ○ Reliable from 50-120 days of 25 days with real time B-scan US gestation in sheep and goats A-scan ultrasonic technique ○ FP from extended urinary ○ Detection of fluid filled uterus bladder, hydrometra, or ○ Transducer placed on lower right pyometra flank in front of udder in ○ FN from early or late gestation standing ewe/doe because of decrease in ratio of ○ Hair should be clipped to uterine fluid to fetal tissue facilitate optimal contact ○ Can’t detect fetal viability or ○ Use ultrasound gel to eliminate fetal numbers air space between skin and probe ○ Helpful in areas where transport or electricity may not be available  Sheep Pregnancy Doppler ultrasonics technique ○ Intrarectal doppler ○ Detection of movements such as Can be used early in second fetal heartbeat, fetal circulation, trimester and fetal movements Best to use 35-40 days of ○ Detected maternal fetal tissue gestation interfaces Fetal viability can be ○ 100% accuracy reported in ewes detected, but accurate 66-122 days of gestation detection of multiple ○ Fetal heart beat/pulse which is fetuses is difficult faster than the maternal pulse or Greater accuracy and fetal movement are positive earlier detection than A- criteria of pregnancy scan technique  Sheep Pregnancy B-scan technique ○ Fetal viability can be assessed by ○ Transabdominal scanning ideal visualizing fetal movement or time 40-75 days (when pregnant fetal heart beating uterus is lying against the right ○ Placentomes found by day 26-28 body wall) post-breeding ○ Reliable to determine pregnancy ○ Accuracy counting fetal and fetal numbers about 50 days numbers advantage over other after breeding US techniques ○ Reported accuracy transrectal ○ Optimal time to count numbers= days 25-30 between 45-90 days of gestation ○ Fetus and fetal heartbeat visible ○ +90 days= fetuses become too after day 25 large to be differentiated from each other  Sheep Pregnancy B-scan technique cont. Recommendations when using US ○ Twins can be more accurately diagnosis diagnosed than triplets Withhold feed and water 12 hours ○ Can distinguish pregnancy from prior to diagnosis hydrometra, pyometra and fetal Rectal scanning should be avoided mummification unless early diagnosis is essential → ○ Fetal age can be determined at use 5 MHz head from day 25 40-100 days of gestation by Any ewe/doe diagnosed as non- measuring width of the fetal pregnant should also be tested high in skull (helpful for predicting the groin to avoid FN parturition date when actual Late term diagnosis recommended to breeding date not known) use 3 MHz head on ventral abdomen Fetus appears as an echogenic structure inside a non-echogenic structure  = bladder = non-pregnant uterus Non-pregnant sheep: filled urinary bladder Non-pregnant uterus cranial to non-echogenic fluid-filled bladder  Sheep Pregnancy Hormone assay Progesterone test ○ Tests can detect hormones in ○ Measured in blood and milk blood, milk, and urine ○ Expensive ○ Estrone sulfate= produced by the ○ Determine plasma concentration placenta in sheep and goats at 18 days post-breeding in ewes Detected in sheep plasma and 19-23 days in does 70 days after conception ○ Concentrations in milk generally Detected in does 40-50 reflect plasma concentration but days post-breeding can be higher, can vary day to day positive= viable fetus and can also vary with type of milk sample obtained ○ Plasma concentrations more accurate than milk  Sheep Pregnancy Progesterone test cont. Rectal abdominal palpation ○ Good test for non-pregnancy ○ Hold off feed overnight ○ Only fair test for pregnancy ○ Placed on laparotomy cradle for ○ Elevated progesterone only exam indicates presence of functional ○ Enema with soapy solution CL ○ Lubricated hollow plastic rod ○ FP from conditions that can with rounded tip inserted into extend luteal lifespan rectum → free hand placed on Ex: hydrometra, pyometra, posterior abdomen while rod is EED manipulated -> move rod until obstruction is encountered/palpated or decide ewe/doe is not pregnant  Sheep Pregnancy Rectal abdominal palpation cont. Radiography ○ 97% accurate at 60 days post- ○ Can be used to detect pregnancy mating and multiple birth with +90% ○ Accuracy is greater for single vs accuracy at +90 days gestation multiple fetuses ○ More accurate in smaller ewes ○ Simple, quick and inexpensive ○ Can be used in dairy goats 58 ○ Risk of rectal trauma, abortion, days after breeding and death → more hazardous → ○ Fetal skeleton is radio-opaque generally not recommended after 65 days gestation ○ Sedation may be required ○ Uterine enlargement may be detected earlier but can’t be ddx from hydrometra or pyometra  Sheep Pregnancy Radiography cont. ○ Vaginal mucosal cells and nuclei ○ 70 days after breeding suitable half the size of those in non- time to expect 100% accuracy pregnant animals ○ Technique not practical for ○ Vaginal epithelium has fewer examining a large number of layers of cells, usually columnar, animals in the field cuboidal, and prismoidal ○ May be useful for individual ○ Samples must be taken from animal when US not available anterior vagina Vaginal biopsy ○ Gives no indication of multiple ○ 97% accuracy in ewes pregnant pregnancy +40 days ○ Accuracy high but not practical for field use because of time and expense involved  Sheep Pregnancy Palpation of uterus via laparotomy ○ Small ventral paramedian incision ○ Gravid uterus palpated directly made just cranial to the udder through small incision in ○ Thin-walled uterus containing abdominal wall fluid= positive for pregnancy ○ +92% accuracy in diagnosing ○ Aseptic technique important to ewes 4-5 weeks pregnant, almost prevent infection 100% in does +42 days gestation ○ 4-5 weeks post breeding uterine horns appear distended ○ 6 weeks post-breeding cotyledons become obvious and horns 5-10cm in diameter  Sheep Pregnancy Abdominal palpation and ballottement ○ Gravid uterus can be palpated ○ ewes/does in late stages of through relaxed abdominal wall pregnancy by placing hand on either side of ○ Becomes easier and more the abdomen and reliable as pregnancy advances squeezing/lifting upwards ○ Easier in thin animals than fat ○ Fetus can sometimes be balloted animals low in the right flank during last ○ Accuracy 80-90% in ewes 90-130 month of gestation days gestation ○ Withhold feed/water 12h before examination  Sheep Pregnancy Pregnancy-specific antigen Palpation of the cervix ○ Chorionic somatotropin in serum ○ Digital palpation of the external ○ Test can be used after day 55 cervical os per vaginum at +50 gestation days post-breeding ○ Concentration measured with ○ Very soft, blunted cervix or radioimmunoassay inability to each cervix is ○ Ewes with levels greater than suggestive of pregnancy minimum detectable level of 5 ng ○ Firm, conical-shaped cervix mL^-1 → pregnant projecting into vagina is suggestive of non-pregnancy  Sheep Pregnancy Mammary secretion Increase in body weight ○ Ewes carrying their first lambs ○ BW increase of 13-16% in ewes produced a sticky honey-like carrying twins compared to pre- mammary secretion after third breeding weights month of gestation ○ Ewes carrying single lambs had ○ Multiparous ewes produced a weight gain of 6-12% more water secretion ○ Weight changes were too ○ Honey-like secretion was variable to provide a reliable sometimes found in pregnant, means of diagnosing pregnancy non-pregnant, uniparous, or multiparous ewes (doesn’t seem very reliable)  Sheep Pregnancy Objectives Be able to give vaccines to ○ Early identification of open prevent abortion and females → ensure passive transfer of Manage flock fertility immunity Efficacy of AI or Optimum time for tansabdominal synchronization protocols ultrasound= 45-90 days gestation Possible underlying ○ Use US with 3.5-5 MHz probe diseases ○ Withhold food and water for 12h ○ Knowing pregnancy status → ○ Restrain in standing position Adjust nutrition to provide ○ Scan in inguinal region for fetal demands  Sheep Pregnancy Embryonic development ○ Organogenesis 0-40 days of gestation ○ 25 days Gestational sac Amnion Enlarged uterus Centrally located embryo Multiple fetuses difficult to positively ID  Sheep Pregnancy ○ 30-35 days Embryonic vesicles Embryo with no differentiated structures Number of embryos Heart beat (no freeze mode)  Sheep Pregnancy fetus ○ Fetal period= days 45-150 gestation → growth, development and differentiation ○ 40-45 days Placentomes (80% cases) Differentiation Head-trunk Heart beat Optimal time to determine number of fetuses head Heart beat Multiple fetuses  Sheep Pregnancy Clear heart beat ○ 50-60 days Placentomes visible in 100% cases Leg, head, spine Heart contractility between ribs Clear heart beat Bone appears highly echonic (white)  Sheep Pregnancy ○ 75-90 days Placentomes grow in size Fetus organs: vertebral column, ribs, internal organs, head, legs, heart Internal organs: lungs, liver, stomach Clear heart beat Fetuses become too large to be consistently visualized (multiple fetuses difficult to distinguish) Placentomes grow in size  Sheep Pregnancy Cartilaginous cervix Most bred by natural service in pasture Small uterine body, long horns with US: Embryonic vesicles as fluid-filled curved/curled appearance, long broad dilatations in uterine lumen from 12 ligament days gestation → visualization of Ewe cervix more tortuous than cow or conceptus at 16d in goats and 19d in doe → makes AI difficult (use sheep laparoscopic insemination) Optimal time for transrectal US to Ewe estrous cycle= 17 days, does= 21 determine single vs multiple days pregnancies= 27-30 days gestation Seasonally polyestrous, short day Ultrasonography most reliable breeders (fall) method Luteolysis similar to cows Estrone sulfate nearly 100% sensitive and specific after 50 days Gestation length 152 days  Sheep Pregnancy Placentome: Maternal → caruncle Fetal → cotyledon  Small Animal Breeding Bitch ○ Estrus reflexes ○ When was she last bred or when “Tail flagging”= tickle was her last heat? perineum and should raise ○ Use powder free gloves tail in opposite direction ○ Mammary glands should be Vulva will be swollen and she examined with external genitalia will raise it in response to the ○ Monoestrus, non-seasonal stimulation ○ Signs of estrus: softening of Reddish discharge= vulvar swelling, standing to be proestrus or early estrus mounted, stiffening of back legs, ○ Ultrasound also accurate way to skin rolled on back determine timing of ovulation (ultrasound ovaries)  Small Animal Breeding Bitch estrus= falling estrogen, ○ Can obtain blood samples to rising progesterone send out for hormonal assays diestrus= progesterone If pregnant that peaks progesterone, prolactin, ○ Length of estrus= 7-9 days and relaxin elevated anestrus= progesterone baseline proestrus= rising estrogen, rising progesterone  Small Animal Breeding Bitch cont. Use light source (ex: pen ○ Vaginoscopic exam light) to visualize vaginal Don’t use first “squirt” of mucosa lube since it may be While vaginoscope still in contaminated place obtain vaginal swab: Hold the tail out of the way take cotton swab → dip in Part the vulva open → 90% saline → insert place speculum direct 90° through vaginoscope as far towards the tail → “roll” it cranially as possible → across to enter vagina rotate in one direction in order to layer cells onto swab → roll swab onto glass slide → stain → examine under microscope  Canine Vaginal Cytology Proestrus Estrus Diestrus Anestrus **See speaker notes** Proestrus Estrus Diestrus Anestrus **Credit to Kaitlyn Bergeron for the chart** Reddish discharge (scant in estrus, Crenulation more profound in proestrus)  Small Animal Breeding Dog ○ Collect semen sample by manual ○ Start by examining external massage (may require teaser genitalia: mammary glands, bitch) prepuce, penis, testes, and Exteriorize penis from scrotum prepuce → massage caudal ○ Ultrasound examination to bulbus glandis in front of ○ Examination of internal genitalia scrotum/testes by digital palpation 3 semen fractions: Assess the prostate per 1. Prostate and rectum (“barbie butt”) urethra 2. tail of epididymis (sperm rich fraction) 3. prostate  Small Animal Breeding Dog ○ After collection apply lubricant to penis ○ Semen evaluation Volume (measured with graduated test tube) Motility  Small Animal Breeding Breeding soundness exam Palpate prostate by digital ○ Breeding history (when last rectal exam successful breeding took place, Check epididymis Brucella testing, possibility of Most common method for collection in hereditary problems) dogs is digital manipulation ○ Physical exam ○ Grasp the penis and prepuce General PE + examine behind (proximal to) the bulbus scrotum and spermatic cord glandis (bulbus) in a tourniquet- Prostate the only accessory like manner with the thumb and sex gland (responsible for forefinger maintaining moderate the volume of canine but constant digital pressure. By ejaculate) doing this, the collector is trying to mimic the copulatory lock/tie  Small Animal Breeding Progesterone analysis ○ Two of the key components of ○ Biomarker for estimating the most immunoassays are the time of ovulation and fertile primary antibody (polyclonal period for insemination antisera or monoclonal, whatever ○ Most assays for the clinical the case may be for that assay) determination of progesterone and the assay standard curve. are still immunoassays (vary ○ frequent or repeated sampling is primarily in the method of signal required so that significant detection, from the competition trends can be recognized between progesterone in the ○ LH peak= 2ng/ml sample and the radiolabeled, ○ Ovulation starting= 5ng/ml enzyme-linked, or otherwise ○ Confirm ovulation >15ng/ml conjugated progesterone reagent added in the assay)  Small Animal Breeding Progesterone cont. ○ radioimmunoassay= technique of choice for accuracy and reproducibility, but expensive and long turnaround time ○ ELISA= inexpensive and simple, quick turnaround, less reliable ○ Chemoluminescent (Immulite)= safe, fast, accurate, repeatable ○ Should collect baseline samples to compare future tests to  Semen Analysis Motility Don’t use coverslip ○ Examine ASAP Examine cells under 10x ○ Motility is the most influenced Swirling motion= cells alive parameter of semen analysis ○ Use wooden stick (thermo- neutral) ○ Examine gross motility first Motile sperm cells will try to swim upward Dead cells will settle to the bottom  Semen Analysis ○ Individual motility ○ motility= very subjective Check for progressive measurement movement ○ Computerized sperm motility Place drop of dilutent computers possibly more (saline or Na citrate) on objective warm slide goal= 10 cells/high power field Place warm cover slip on top → examine sample under high dry 40x power  Semen Analysis Morphology ○ Count 100 cells → differentiate ○ Examined with eosin-nigrosin normal from abnormal stain to highlight cells ○ Abnormalities ○ Place drop of stain on warm slide Head → then place semen → mix stain Midpiece and semen → slowly push second tail slide through the stain and across the first slide while pressing firmly down ○ goal= dark background, cells close but not overlapping ○ Examine cells under 1000x (oil)  Semen Analysis Color Red or brown discoloration of ○ F1 should be clear to slightly cloudy. F2 indicates presence of fresh ○ F2 should be some variant of milk-white or hemolyzed blood; this may and homogeneous. be indicative either of If F2 is clear or only slightly reproductive tract disease or cloudy, this may indicate a trauma at collection. decreased number of ○ F3 should be clear. spermatozoa or complete Red or brown color of F3 azoospermia. strongly suggests the presence Yellow discoloration of F2 may of a disease process such as indicate urine contamination or prostatic disease, trauma or presence of a purulent exudate. injury, or neoplasia of the genitourinary tract.  Semen Analysis Volume Cytology ○ Measure the volume of the ○ The presence of white blood cells collected ejaculate before (WBCs), red blood cells (RBCs), removing semen for anything epithelial cells, and bacteria is else. noted with a designation of 0 to pH 4+ to indicate relative amounts. ○ To measure pH, a drop of semen Longevity should be applied to pH paper. ○ In some circumstances it is ○ Normal values range from 6.5- necessary to evaluate the 7.0. longevity of a given semen ○ Changes in pH may indicate that sample that has been extended prostatic infection. and cooled.  Semen Analysis Sperm count Making a hand dilution: ○ Estimated by measuring scrotal dilute 1 part semen with 9 circumference in bulls/rams parts formal-buffered ○ Hemactyometer method saline → get 1:10 dilution Load with 1:100 dilution of → take 1 part of 1:10 semen → hemacytometer dilution and add 9 parts coverslip placed over formal-buffered saline → chambers → chambers get 1:100 dilution filled → allow sample to Cells less likely to clump settle → all sperm heads in with dilution= more middle big square counted accurate sperm count → multiply # by 10^6 to get cells/cc 25 squares 16 squares 29 sperm counted  Semen Analysis Sperm count Blank tube loaded with ○ Estimated by measuring scrotal 3.42 mL of formal-buffered circumference in bulls/rams saline → insert into ○ Spectrophotometer method machine with clear sides Can be used to count left and right → creates stallion and dog semen “baseline” from clear Machine measure amount sample of light that passes through 180 ul of semen is added to sample → calculates tube → sperm concentration of cells concentration calculated based on density reflected based on % of light transmitted through sample Contamination can result in erroneous readings  Semen Analysis Total sperm number ○ Manual ○ NucleoCounter Multiply concentration x Cell membrane disrupted volume= total number of → dye stains DNA → digital cells in the ejaculate image created → cells Total number x % counted progressively motile cells= Can also be used to count total number of intact membranes by not progressively motile cells disrupting cells first Total number progressively motile cells x % normal cells= total number of normal, motile cells Used in stallions and dogs  Stallion Analysis Wash penis before semen collection Calculating sperm concentration: Analyze total volume, color, odor → ○ Newbauer chamber (manual) remove gel fraction → evaluate gel ○ Densimeter (based on free volume density/cellularity of the semen) Normal: ~50mL gel free volume, white ○ Nucleocounter (sperm concentration to gray with milk appearance, odor based on DNA, very accurate) “suis generis” Motility- total motility, progressive Sperm concentration: total sperm 5-15 motility, sperm vigor, CASA (computer x10^9 assisted sperm analysis, objective analysis) >60% normal morphology and motility Morphology- differential interference pH 6.8-7.2 contrast, stains (Karras, Diff-quick, eosin- nigrosin)  Stallion Analysis Sperm viability Normal: 1 x 10^9 progressively motile, ○ plasma membrane integrity morphologically normal spermatozoa ○ Hypoosmotic test- coiled tail if in second of two ejaculates 1 hour viable apart, after 1 week of sexual rest ○ Eosin-nigrosin- white if viable Processing semen for AI ○ Fluorescent microscopy- green if ○ Fresh semen- 500 million viable progressively motile sperm ○ Flow cytometry analysis ○ Cooled semen- 1 billion Seminal plasma ALK Phos progressively motile sperm ○