HOW TO INCORPORATE ASSISTED REPRODUCTIVE TECHNOLOGIES


inated with valuable semen) to perform frequent evaluations of follicle development in the mare. Thus, most equine veterinarians are familiar with the basic ovarian changes seen on ultrasonogra- phy throughout the estrus cycle. In general terms, these can be described as waves of follicle growth that occur over the cycle. Mares typically have only one follicle wave through the cycle, although about 30% of mares have an additional follicle wave in diestrus.2 If a follicle wave occurs in diestrus, a cohort of follicles grow to a diameter of 20 mm, then the follicles regress; they eventually become atretic (die). Alternatively, during diestrus, folli- cles can grow to 10–20 mm and then become static, staying this size over the entire cycle.2 During es- trus, as follicles in a growing wave reach about 20–25 mm diameter, one follicle deviates from the rest, and goes on to grow to a diameter of 40 mm, becoming the dominant preovulatory follicle. The remaining follicles slow their growth and eventually regress. The dominant preovulatory follicle ovu- lates in response to luteinizing hormone (LH). This may be endogenous LH from the mare’s pituitary, released either spontaneously or after adminis- tration of exogenous agents (GnRH analogs); or exogenous LH analogs, such as human chorionic gonadotropin (hCG). Ovulation of a dominant preovulatory follicle occurs about 36–42 hours after the LH stimulus is received by the follicle. Logically, as has been shown in other species, it


could be thought that the best time to recover oocytes for ICSI use would be as follicle waves are growing, to ensure that oocytes are collected from healthy, viable follicles. However, this is not the case in the horse, as it is complicated by several factors:


1. In young growing follicles, the oocyte can be considered to be “juvenile”—it does not have the competence to complete meiosis (i.e., it lacks meiotic competence) or to make a normal embryo (it lacks developmental competence).


2. In horses, it appears that this “juvenile” period of the oocyte lasts until the growing follicle reaches about 20 mm in diameter. Oocytes recovered from viable growing follicles less than 20mmin diameter have a low maturation rate when cultured in vitro.3


3. As a follicle starts to become atretic, the oocyte within the follicle actually gains meiotic com- petence.4 Whether it gains developmental competence (has a greater ability to make a normal embryo after fertilization) is unclear, but these oocytes are capable of making blas- tocysts after fertilization at rates at least equal to that for competent oocytes from viable follicles.5


Because of these factors, recovery of oocytes in mares from growing follicles  20 mm in diameter will provide oocytes that may not mature in vitro


because they are juvenile. In contrast, recovery of oocytes from follicles that are undergoing regres- sion/atresia, even in follicles  20 mm, may provide oocytes that have a high maturation rate in vitro and can yield blastocysts. There is evidence of em- bryo production from oocytes recovered from folli- cles  10 mm diameter. Because individual mares have different patterns of follicle development, at any one examination, a specific visualized follicle could be growing, static, or regressing. In evaluating factors associated with timing of


oocyte collection that could be related to enhanced performance of the oocytes in an IVP system, few meaningful associations have been found. There is some evidence that oocytes recovered from subordi- nate follicles 24 hours after gonadotropin stimula- tion, at the time that a dominant preovulatory follicle is being aspirated for recovery of a mature oocyte (see below), appear to have slightly increased rates of in vitro maturation and blastocyst formation after ICSI compared to oocytes recovered from sub- ordinate follicles from mares in diestrus. However, the author’s practice also find that the number of oocytes recovered is lower when subordinate follicles are aspirated during estrus, vs aspiration of imma- ture follicles in diestrus, so the blastocyst production per aspiration is not different. Currently, there is no time of the cycle that is known to be the “best” for recovering oocytes for in vitro embryo production. Many people want to avoid large follicles or corpora hemorrhagica, as they make the ovary difficult to manipulate. In addition, people may wish not to, or cannot, perform the monitoring and timing needed for aspiration of a dominant stimulated follicle. In these cases, oocyte recovery for collection of im- mature oocytes is done during diestrus. Others like the chance to recover an in vivo–matured oocyte (which typically has a higher potential to form a blastocyst after ICSI) from the dominant stimulated follicle, as well as immature oocytes from the subor- dinate follicles, and so perform oocyte recovery in estrus, 20–35 hours after gonadotropin stimulation of the dominant preovulatory follicle. A related aspect of this is the influence of follicle- stimulating hormone (FSH) on follicle aspiration. The author is often asked whether administering FSH to amare would increase the number of follicles available for aspiration. While it appears no one has yet evaluated this, theoretically it should not. In the mare, the follicle does not appear to be re- sponsive to gonadotropins until it is over 15 mm in diameter. This is visible in anestrus, when gonad- otropin levels are low, as follicles will grow to about 15 mm, then regress. Additionally, during develop- ment of superovulation regimens in the mare, ad- ministration of FSH was relatively ineffective unless given to mares when the largest follicle was at least 20 mm. Since for TVA, the oocyte recov- ery rate decreases as follicle diameter increases, the optimum diameter follicle for TVA is  20 mm. Thus, in the mare, administration of FSH theoreti-


AAEP PROCEEDINGS  Vol. 65  2019 347


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