Hi Hank, Emma and Ashley

Topic 1: Reproductive Endocrinology and Evaluation of the Non-Pregnant Cow (Note: familiarize yourself with the graph representing the bovine estrus cycle. This can be found in the Youngquist reference, in Dr. Farin’s notes on the Therio course website, or in Kelly’s section below.)

I. The Bovine Estrous Cycle (P4=progesterone, E2=estradiol, PGF=prostaglandin F, LH=luteinizing hormone, FSH=follicle stimulating hormone) a. Length17-24 days, average 21 days, heifers usually 1-2 days shorter than cows b. The Cycle: i. Estrus (day 0) – sexual receptivity, follicular growth 1. average length of behavioral estrus 15 hrs (range 2-50 or 6-24 depending on who you ask)…dairy v. beef? 2. ovulation 24-30 hours post-estrus Luteal Phase (day 1-18) ii. Metestrus (day 1 to 3) – final follicular maturation  ovulationearly CL  secretion of P4 1. low levels of LH, E2 and P4 2. in the process of ovulation, inhibin from dominant follicle decreases causing a brief rise in FSH, which stimulates first new wave of follicular growth iii. Diestrus (day 4 to 18) – CL secretes P4 until PGF is released from non-pregnant endometrium (about day 14), causing luteolysis 1.  P4 means negative feedback to hypothalamus/pituitary  lower frequency and amplitude pulses of LH (but some needed to maintain the CL), FSH pulses correspond to waves of follicular growth 2. max P4 reached by day 8-10 (CL is biggest) 3. CL also secretes oxytocin! (from large luteal cells) – plays a role in luteolysis via interaction with the endometrium causing more PGF secretion (+ feedback) 4. E2 from dominant follicle initiates luteolysis in absence of pregnancy by inducing oxytocin receptors on endometrium, when occupied by oxytocin activate production of PGF 5. PGF transferred from uterus to ovary via countercurrent mechanism Follicular Phase (day 19 to estrus) iv. Proestrus (day 19 to estrus) 1. in general:  P4 (means  negative feedback on hypothalamus/pituitary) and E2 (positive feedback on hypothalamus/pituitary) from dominant follicle   LH, pre-ovulatory surge  estrus

II. More about Ovaries & Follicles a. pulsatile secretion of FSH causes 2-4 waves of follicular growth during each estrous cycle. i. Recruitment: a cohort of follicles increase in diameter ii. Selection & Atresia: largest of cohort becomes dominant and continues to grow, the rest degenerate and undergo atresia iii. Growth, Stasis & Regression (or ovulation): the dominant follicle goes through 3 phases (increase, no change, decrease diameter), or if P4 is decreasing, will continue to mature and ovulate. b. the dominant follicle produces inhibin, which inhibits growth of smaller follicles in the cohort. The development of 2 dominant follicles simultaneously is possible (twins), but less likely. c. theca and granulosa cells, at ovulation both differentiate into luteal cells: i. thecasmall luteal cells: LH receptors, contribute 15% P4 ii. granulosalarge luteal cells: PGF/E receptors, contribute 85% P4, also oxytocin and neurophysin d. follicle ovulatescorpus hemorrhagicumcorpus luteum(CL)corpus albicans (see drost project: female repro system: ovaries) e. at least one dominant follicle is present in the pair of ovaries at any given time in the cycle! This is why you can’t stage the cycle palpation of the ovaries alone.

f. Cysts: 2 types follicular and luteal (3rd type = cystic CL, NOT pathologic, normal in most mature CLs)) i. definition: an anovulatory follicular structure, >2.5cm, that persists over time (note: a single finding of a large cyst-like structure does not equal a cystic ovary) ii. a serious cause of anestrus/infertility in dairy cows due to additional follicular waves being inhibited by E2 and inhibin iii. 20% of cysts regress by themselves and the ovaries resume normal cycling iv. Follicular: thin-walled, may be dominant and secrete E2 (many layers of granulosa cells) or not (few granulosa cells) v. Luteal: thick walled, contain theca cells and sometimes luteinized granulosa cells, produce lots or little P4, thought to develop from luteinized follicular cysts 1. the two types cannot be distinguished by palpation, but can be distinguished by U/S vi. Treatment: GnRH or hCG induce luteinizationreturn to estrus in 21 days OR give PGF2a 9 days after GnRH to shorten interval to estrus

III. More about HORMONES QuickTime™ and a TIFF (LZW) decom pressor are needed to see this picture.

Class Hormone Source Effect (female) Peptide GnRH Hypothalamus Stimulate release of LH/FSH from Anterior Pituitary Oxytocin Posterior Uterine contractions, luteolysis Pituitary Glycoprotein FSH Anterior Stimulate follicle growth Pituitary LH Anterior Low levels in mid-cycle: maintain Pituitary CL Surge in late cycle: triggers ovulation

Steroid Progestagens Progesterone (P4) CL (-) feedback on hypothalamus secretion of GnRH = quiescent Estrogens (E2) Ovarian follicle (+) feedback on hypothalamus secretion of GnRH = estrus behavior

Prostaglandins Endometrium Luteolysis

IV. The Non-Pregnant Cow a. post-partum involution i. expulsion of lochia (uterine fluid post-partum) complete by day 14-18 (normally brownish-red, may see remnants of necrotic caruncles) ii. decrease in uterine diameter complete by day 45 (although pregnant horn may remain larger than non-pregnant horn) iii. changes in cervical diameter (slowest to complete involution) – should be less than uterine diameter until day 15 postpartum, if bigger than uterus before day 20 involution is abnormally slow and is associated with decreased fertility. If cervix is >50mm after day 45, think uterine and cervical infection or incomplete involution b. rectal palpation i. ovarian structures: follicles, CLs, cysts ii. uterine condition: involution, fluid, tone, adhesions, etc. c. clinical changes during the estrous cycle: i. Follicular phase: rapid regression of the CL beginning day 17-18 and concurrent increase in diameter of pre-ovulatory follicle 1. increase in circulating E2 2. increasing uterine tone ii. Estrus: very toned uterus, small hard CL (really a corpus albicans) from previous cycle, plus 1+ follicles palpable iii. Luteal phase (diestrus): 1. an ovulation depression may be palpated for a short time after ovulationovaries feel smooth/inactive for 3-4 days with a soft follicle-like structure sometimes palpable (corpus hemorrhagicum)by day 5 the CL with crown can be palpated (the ovary with the CL is usually larger than the opposite ovary, most of CL is within the parenchyma) 2. the uterus is tonic/edematous in the early luteal phase, becoming flaccid by the end (influence of P4) 3. many follicle develop and regress during the luteal phase d. Vaginoscopic Exam i. to identify urine pooling, vaginal tears, vaginitis, and urethral damage (post-partum) ii. evaluate the patency of the cervix and any cervical discharge iii. clear, abundant, stringy mucus = periestus iv. small amounts of blood = metestrus e. Ultrasonagraphy i. a more reliable method to identify ovarian structures and evaluate the uterus (presence and characterization of fluid, regression of caruncles, etc.) f. Plasma or Milk P4 i. P4 levels can be used to assess luteal status (how good is your heat detection? are the right proportion of cows cycling?) and for differentiating between luteal and follicular ovarian cysts (different tx) g. Uterine Culture and Biopsy V. Puberty = onset of first estrus associated with a potentially fertile ovulation, followed by a luteal phase of normal duration a. occurs at 8-19 months b. dependant on an interaction of environment, nutrition, genetics c. pre-pubertal state is maintained by inactivity of the hypothalamus: before puberty, LH and FSH secretion is low level and irregularafter puberty, LH secretion becomes more regular

VI. Bos indicus is Special a. shorter period of estrus, shorter interval from onset of estrus to ovulation, reduced magnitude of pre-ovulatory LH surge, smaller CL, lower luteal concentrations of P4 b. puberty occurs later – 17-19 months Here’s a fast a hopefully not too painful summary of the complicated stuff

FSH present------increase follicles maturing----inhibin released to stop more follicles from developing so 1 big one matures-----increased estradiol levels -----acts on the brain (hypothalamus and pituitary)-----LH surge------ovulation----- CL forms ------progesterone made, stops new waves and ovulations Progesterone: aka hormone of pregnancy (think P and P or PROgesterone + Pro-Preg.)

Produced in the adrenal glands, the brain, and mostly the CL of the ovary after ovulation and by the placenta during pregnancy. It converts the uterus to a secretory stage to prepare the uterus for implantation. At the same time progesterone affects the vaginal epithelium (surface cells) to make thick cervical mucus which makes the cervix and impermeable to sperm. If pregnancy does not occur, progesterone levels will decrease. During pregnancy and birth, it does a lot, too. If progesterone is high, estrogen is low, and so cows don’t cycle (helps maintain pregnancy). If it’s low, estrogen is high, and the animals cycle. Basically: makes uterus ready for implantation and maintains pregnancy

Estrogen: estradiol

Made by the ovaries, it acts as a growth hormone for tissue of the reproductive organs and supports the lining of the vagina, the cervical glands, the uterus and the fallopian tubes. It helps to maintain the oocytes in the ovary. During a cycle, estradiol that is produced by the growing follicle triggers, via a positive feedback system, the hypothalamic-pituitary events that lead to LH surge, inducing ovulation. In the luteal phase it helps get the uterus ready for a baby. Again, lots of roles in pregnancy and birth. It is responsible for the secondary sex characteristics of females during puberty as well. Basically: triggers LH surge, maintains oocytes.

Lutenizing Hormone (LH):

With the rise in estrogens, LH receptors are also expressed on the maturing follicle that produces an increasing amount estrogen. Eventually at the time of the maturation of the follicle, the estrogen rise causes a release of LH over a 24-48 hour period from a positive-feedback loop involving the hypothalamus. This 'LH surge triggers ovulation and causes the CL to form. The low levels of LH throughout the rest of the cycle helps maintain the CL. It’s important in pregnancy as well! Basically: causes ovulation

Follicle Stimulating Hormone (FSH):

FSH initiates follicular growth, specifically affecting granulosa cells. As the follicles grow, the largest follicle secretes inhibin, which causes the FSH levels to decline in the late follicular phase. At the end of the luteal phase, there is a slight rise in FSH that seems to be of importance to start the next ovulatory cycle. Females are born with all the eggs they’ll ever have. New follicles are constantly trying to mature. Those that do mature ovulate, those that don’t are “atretic” (fancy word for “they die”). FSH doesn’t cause follicles to mature, but it stops the number becoming atretic so that one can really mature. Basically: recruits follicles to mature to be ovulated Inhibin:

Made in cells in the follicle in the ovary from an increase in FSH. This negative- feedback loop causes a decrease in FSH. Inhibin decreases with an increase in GnRH. Stops new follicles from maturing (meaning don’t waste oocytes since only 1 can ovulate every 21 days!!!) It also helps decrease the occurrence of multiple births (less follicles maturing, less multiples being released). Basically: stops more than 1 large follicle from completing maturation

Gonadotropin-Releasing Hormone (GnRH):

Works on the pituitary to cause the secretion of the FSH and LH. Low frequency GnRH pulses lead to FSH release, whereas high frequency GnRH pulses stimulate LH release. GnRH1 controls a complex process follicular growth, ovulation and CL maintenance in the female. Basically: initiates follicular wave and ovulation.

Prostaglandin (PGF2a):

A fatty acid made in the uterus that causes uterine contractions. This causes abortion and lyses the current CL (gets rid of it). This causes a new follicular wave to occur with a new follicular maturation, and so the animal comes into heat. After PGF injections, most animals come into heat between 2-5 days after the injection if they’re responsive, with most animals around 3 days. This depends on what stage the most mature follicle is in. 2 days: a follicle is almost already mature. 5 days: need to recruit a follicle and then the follicle has to mature (takes longer). Basically: lyses the CL.