A Retrospective Analysis of 2,253 Cases Submitted for Endocrine Diagnosis of Possible Granulosa Cell Tumors in Mares

Endocrine diagnoses of granulosa cell tumors (GCTs) in the mare are frequently based upon determination of serum concentrations of inhibin, testosterone, and progesterone (GCT panel). In the present study, we examined results from 2,253 samples submitted for determination of GCT panel. In an additional subset of samples (n = 75), diagnosis of GCT was confirmed based upon surgery or necropsy. The objective of the current study was to examine the agreement between serum inhibin and testosterone concentrations in mares with serum progesterone concentrations less than or ≥1 ng/mL. Across all samples, elevations in serum inhibin or testosterone were noted in 15.2% or 17.3% of samples, respectively. For samples with progesterone <1 ng/mL, more (P = .001) samples had elevated inhibin than elevated testosterone, whereas for samples with progesterone ≥1 ng/mL, more (P < .0001) samples had elevated testosterone than elevated inhibin. For samples with progesterone <1 ng/mL, the agreement between elevated inhibin and testosterone was greater than that for samples with progesterone ≥1 ng/mL. In a subset of 53 samples with progesterone ≥1 ng/mL, 28 samples had endocrine evidence of a granulosa cell tumors based upon elevations of serum anti-Müllerian hormone, and 4 of these samples had endocrine evidence of pregnancy based upon estrone sulfate concentrations. For samples from mares with confirmed GCTs, 85% and 55% had elevated inhibin or testosterone, respectively. Based upon endocrine diagnosis, GCTs occur in mares with elevated progesterone and/or estrone sulfate, albeit at a low rate.     

Granulosa cell tumours in the mare: A review of 52 cases

Granulosa cell tumour (GCT) is reported to be the most common ovarian neoplasm in the mare. A multicentre retrospective study evaluating the medical records of horses undergoing ovariectomy for treatment of a GCT was undertaken, documenting the presenting clinicopathological features, treatment and outcome. Follow‐up information was obtained by telephone questionnaire. Fifty‐two mares were included. Most presented with behavioural/reproductive abnormalities, including stallion‐like behaviour (50%), aggression (31%), prolonged oestrus or nymphomania (19%), and persistent anoestrus (8%). Most (93%) affected ovaries had a multilocular appearance when examined ultrasonographically. Only 60% of horses with histopathologically confirmed GCTs demonstrated elevations in serum inhibin concentrations. Twenty‐five percent of horses had histopathologically confirmed bilateral GCTs. Ninety‐four percent of horses that underwent ovariectomy were discharged from the hospital. Standing laparoscopic ovariectomies had a lower complication rate (34%) than other surgical methods. Long‐term complications were uncommon (11%). In conclusion, histopathologically confirmed GCTs can occur in the presence of normal serum inhibin concentrations. The high prevalence of bilateral GCTs indicates that thorough evaluation of the contralateral ovary is necessary. Surgical ovariectomy for treatment of GCTs has favourable short‐ and long‐term outcomes. Standing laparoscopic ovariectomy may be associated with a lower complication rate than other surgical methods.     

Changes of serum anti‐Müllerian hormone in a mare with granulosa cell tumour following surgery and reinitiation of follicular activity

Anti‐Müllerian hormone (AMH) has been reported to be elevated in mares with granulosa cell tumour (GCT). An 8‐year‐old Thoroughbred mare was presented for not being observed in oestrus after the beginning of the breeding season. Rectal palpation and ultrasonography revealed enlargement and cystic appearance of the left ovary while the right ovary was small with an anoestrous‐like appearance. The AMH concentration was 694.9 ng/ml. Presumptively diagnosed with GCT, the mare was subjected to tumour removal. Histopathology confirmed GCT. To evaluate changes of AMH concentration following surgery, blood samples were collected immediately prior to surgery, and on Days 1, 2, 4, 8, 16 and 32 after surgery. Thereafter, blood samples were collected monthly and also at the time the mare was observed in oestrus (148 days after tumour removal). The AMH concentrations decreased over the first 2 months after surgery (from 721.2 ng/ml to 0.1 ng/ml). Subsequently, AMH concentration increased and peaked at the time of oestrus expression (0.7 ng/ml). The mare then became anoestrous, and AMH concentration decreased and reached 0.2 ng/ml, which was not significantly different from the mean concentration of AMH in normal anoestrous mares (n = 5; 0.26 ± 0.07 ng/ml). In conclusion, the present report implies the potential use of AMH for determination of the time of follicular resumption in mares after GCT removal.     

Elevated Serum Anti-Müllerian Hormone in an Arabian Mare with Granulosa Cell Tumor

Anti-Müllerian hormone (AMH), produced by granulosa cells, has been reported to be elevated in mares with granulosa cell tumor. A 13-year-old Arabian mare was referred after 3 years of infertility. Rectal and ultrasonographic examination revealed enlargement and multicystic appearance of the left ovary. The mare had substantially higher concentration of AMH (21.6 ng/mL) compared with normal cyclic mares (n = 5; 0.36 ± 0.02 ng/mL). Testosterone (50 pg/mL) concentration was also elevated, but progesterone concentration (0.4 ng/mL) was lowered. Accordingly, the mare was presumptively diagnosed with granulosa-theca cell tumor and a left ovariectomy implemented using ventral midline approach under general anesthesia. Histopathological examination confirmed the diagnosis of granulosa-theca cell tumor. Serum concentrations of AMH decreased to 2 ng/mL and 0.5 ng/mL 2 and 4 weeks after tumor removal, respectively. In conclusion, the current report presents confirmatory evidence that evaluation of serum AMH could be useful for diagnosis of granulosa cell tumor in mare.     

Review of seven cases of granulosa cell tumour of the equine ovary

This paper reviews the equine granulosa cell tumour (GCT) and describes the clinicopathological features, treatment and outcome in seven cases of GCT in mares. Mares were presented with unilateral ovarian enlargement during the 2007 to 2010 breeding seasons. The mean (sd) age of the mares was 11.7 (5.96) years. Three mares were multiparous barren, three were nulliparous and one was primigravida. Behaviour at presentation was 57 per cent anoestrus, 28 per cent with stallion-like behaviour and 14 per cent with persistent oestrus. All mares had unilateral ovarian enlargement. Six non-pregnant mares had a small and inactive contralateral ovary; the pregnant mare had a single small corpus luteum on the contralateral ovary and was at three-and-a-half months' gestation. Enlarged ovaries measured 7 cm to an estimated 30 cm in diameter. 28 per cent had a multicystic ultrasound appearance, 57 per cent were dense structures and 14 per cent were of mixed appearance. Mean concentrations of progesterone were <1 ng/ml, oestrone sulphate 3.06 (2.32) ng/ml and testosterone 0.58 (0.64) nmol/l in non-pregnant mares. Inhibin was elevated in all non-pregnant cases at 7.6 (12.45) ng/ml. The pregnant mare had concentrations of progesterone 2.5 ng/ml, oestrone sulphate 81.0 ng/ml, testosterone 1.9 nmol/l and inhibin 1.31 ng/ml. Mares demonstrating stallion-like behaviour had a significantly higher (P<0.001) testosterone concentration (1.85 [0.07] nmol/l) than those that did not (0.34 [0.26] nmol/l). Three mares underwent unilateral ovariectomy and resumed cyclic ovarian activity within nine months of surgery.     

Equine nonneoplastic abnormal ovary in a draft mare with high serum anti-Müllerian hormone: a case study

We performed a standing hand-assisted laparoscopic ovariectomy in a draft mare that presented with high serum anti-Müllerian hormone (AMH) level and had an enlarged single cystic ovary. Histopathological examination revealed no tumor cell proliferation in the ovary, but the presence of a large ovarian cyst was confirmed. In the diagnosis of abnormal ovaries in mares, a comprehensive assessment should be performed, including the monitoring of ovarian morphology and biomarkers over time, to determine the disease prognosis and treatment plan. The case of this mare with a nonneoplastic abnormal ovary and increased serum AMH level was rare. We suggest that standing hand-assisted laparoscopic ovariectomy is useful for the removal of large ovaries in draft mares.     

Luteoma in two mares treated by ovariectomy

A 15-year-old Paint mare (Case 1) and a 15-year-old Arabian mare (Case 2) were presented for aggressive and undesired behaviour. Reproductive evaluation revealed, initially, an enlarged ovary with a smaller/normal sized contralateral ovary in each case. Granulosa cell tumour (GCT) panel testing revealed elevated inhibin B in both cases and elevated anti-Mullerian hormone (AMH) levels only in Case 1 determined from currently established reference ranges. Based on a presumptive diagnosis of GCT, bilateral standing ovariectomy was elected for both mares. In each case neoplastic tissue consistent with luteoma was detected only on histological examination in the smaller/normal sized left ovary; the right ovary appearing normal. Long-term follow-up was performed 1 year and 5 years post-operatively by telephone. The owners were satisfied with the outcome and each patient demonstrated resolution of aggressive stallion-like behaviour. Each case returned to the owners' intended use. In conclusion, luteoma should be considered as a differential diagnosis for ovarian neoplasms in horses related to behavioural abnormalities, even in normal-sized ovaries.     

Stallion-like Behavior in Mares: Review of Incidence, Characteristics, Ovarian Activity, and Role of Testosterone

Stallion-like sexual behavior in mares is rare, except in association with ovarian tumors or hormonal treatments. The rarity of the phenomenon was confirmed in a recent 3-year study. The mean number of mares with detected stallion-like behavior, including mounting with thrusts, during an entire ovulatory season was 5.7 (17/3 years) in a herd averaging 105 mares (5% incidence/mare/season). From a total of 17 mountings of an estrous mare by another mare, 15 occurred when the mounting mare was in the follicular phase and two when in the early luteal phase. Plasma testosterone concentration on the day of mounting was higher (P < 0.01) in the mounting mares (17.7 ± 2.3 pg/ml) than in the standing mares (10.9 ± 0.5 pg/ml). No other deviation in the endocrine, behavioral, or morphologic aspects of the estrous cycle was observed. In another study, testosterone was assayed daily from 7 days before to 4 days after ovulation in seven mares during estrous cycles with no detected mare-on-mare mountings. Concentrations during the follicular phase were highest on the days corresponding to when mare-on-mare mounting was detected in the previous study. It is concluded that the rare occurrence of stallion behavior by untreated mares with no detected ovarian tumors is a consequence of an unusually high, apparently transient fluctuation in circulating testosterone at the time of mounting.     

Immunohistochemical and qPCR determination of the expression and serum level of anti‐Müllerian hormone in pre‐pubertal,intact and ovarian remnant syndrome detected bitches

The aim of this study was to determine the serum concentrations, ovarian presence and expression of anti‐Müllerian hormone (AMH) in pre‐pubertal, bitches with signs of ovarian remnant syndrome (ORS) and intact bitches. In addition, we aimed to verify the suitability of serum AMH concentrations for diagnostic purposes in sterilized bitches and/or in suspected cases of ORS in the field of veterinary medicine. For this purpose, 36 healthy female dogs divided into six groups: proestrus, oestrus, dioestrus, anoestrus, pre‐pubertal and ORS. Serum AMH concentrations were determined by electrochemiluminescence immunoassay, and ovarian presence and distribution of AMH was confirmed by immunohistochemical and qPCR techniques. According to the results of qPCR, while the expression values of AMH were at the highest concentrations in the proestrus and oestrus, there was a statistically significant decrease in these values at the later stages of the cycle (p < 0.05). According to hormone analysis, the serum AMH values of the ORS group had decreased significantly compared with the proestrus and oestrus (p < 0.05). Although serum AMH levels of ORS group were increased compared with anestrus and pre‐pubertal groups, this increase was statistically non‐significant (p > 0.05). Immunohistochemically, AMH expression was first observed in the granulosa cells of primordial follicles in folliculogenesis. Expression values were the highest in the proestrous and oestrus groups, but values from bitches in later stages of the cycle were statistically significant decrease in comparison with these groups (p < 0.05). As a result, AMH concentration and expression were found to be higher in proestrus and oestrus than in other periods (p < 0.05). In addition, the measurable level of AMH concentration in bitches with ORS is an indication that it can be used in the diagnosis of ORS.     

Anti‐Muellerian hormone concentration in bitches with histopathologically diagnosed ovarian tumours and cysts

Increased concentrations of Anti‐Muellerian hormone (AMH) can indicate a granulosa cell tumour as shown in women, mares and cows. To investigate AMH to differentiate canine granulosa cell tumour from other ovarian pathologies, we evaluated the ovaries of 63 bitches. Blood serum samples were collected before surgery for AMH analysis. Ovaries were submitted for histopathological examination. Fourteen bitches showed normal ovaries. These bitches had AMH values between 0.12 and 0.99 ng/ml. In 20 bitches ovarian cysts i.e., follicular cysts (n = 8), corpora lutea cysts (n = 7), subsurface cysts (n = 5) were diagnosed. These dogs had AMH values of 0.11–2.09 ng/ml. Bitches with small luteinized follicular cysts had slightly higher AMH values than those without ovarian alteration. In 29 cases ovarian neoplasms i.e., granulosa cell tumour (n = 9), epithelial tumours (n = 16), dysgerminomas (n = 3) and one sarcoma were identified. Anti‐Muellerian hormone values of bitches with an ovarian neoplasm except granulosa cell tumour ranged from 0.18 to 1.18 ng/ml. The AMH values of bitches with granulosa cell tumour ranged from 1.12 to ≤23 ng/ml and were significantly higher (p < .05) than in all of the other bitches. The cut‐off of 0.99 ng/ml gave a sensitivity of 100% and a specificity of 94.44% to diagnose granulosa cell tumour. In conclusion, markedly elevated AMH concentrations in bitches are indicative for a granulosa cell tumour. However, negative testing does not rule out the existence of small one. Differentiation of GCT from luteinized follicular cysts may especially be difficult.     

Experimental reactivation of equine herpesvirus‐3 following corticosteroid treatment

State of latency, well known for several herpesviruses, has been proposed for equine herpesvirus‐3 (EHV‐3) and supported by epidemiological observations. No detailed assessment about reactivation, patterns of excretion and re‐excretion has been formally reported. An experimental reactivation study by corticosteroid treatment in previously naturally infected horses was therefore carried out. Two polo mares with clinical and virologically confirmed history of equine coital exanthema were injected with dexamethasone and prednisolone on 3 successive days. Clinical signs, body temperature and clinical samples for virological and serological studies were obtained daily. Mares did not show any systemic clinical signs or hyperthermia. EHV‐3 shedding, seroconversion and the presence of a small lesion were observed in one of the mares under study 2 weeks after corticosteroid treatment. The results demonstrate that this virus exhibits a latency‐reactivation behaviour similar to that of other alpha herpesviruses. Reactivation of latency may have an important bearing on the appearance of clinical signs in mares and/or stallions during the breeding season without the actual evidence of transfer from mare to stallion or vice versa.     

Treatment of haemoperitoneum secondary to ruptured granulosa cell tumours in two mares

This report describes two cases of successful surgical management of granulosa cell tumours (GCT) in mares presenting with haemoperitoneum (HP). Controlled abdominal drainage was initially attempted in Case 1 but was not successful. A ventral midline exploratory laparotomy allowed removal of a haemorrhaging 13 kg GCT. The mare made a full recovery and returned to normal work as a driving pony 11 months post-operatively. In Case 2 controlled abdominal drainage was followed by standing left flank laparoscopic visualisation of the bleeding ovary and transection of the ovarian pedicle by electrocautery. The GCT was then removed via a ventral midline incision due to its large size. Haemoperitoneum can be associated with GCTs and in some cases is severe enough to prompt emergency treatment. Stabilisation of the patient and removal of the haemorrhaging GCT can lead to a successful outcome.     

Passive immunization of cyclic mares against androgen: gonadotropin and progesterone concentrations and estrous characteristics

Antiserum generated in a horse against testosterone conjugated to bovine serum albumin (BSA) was administered to six lighthorse mares (androgen-immunized mares) 1 to 3 d before a prostaglandin-induced estrus and twice again at 2-d intervals. Six control mares were administered antiserum generated against BSA on the same schedule. Relative to testosterone, cross-reactivities of other steroids with the testosterone antiserum were (%): dihydrotestosterone, 52; 5 alpha-androstane-3 alpha,17 beta-diol, 8.6; androst-4-ene-3,17-dione, 1.2; and all others tested less than .1. Tritiated testosterone binding in plasma increased (P less than .01) in androgen-immunized mares within 1 h and remained elevated (P less than .01) relative to controls for greater than 21 d. There was no effect (P greater than .10) of passive immunization against androgen on interval to estrus after prostaglandin injection, duration of estrus, ovarian volume, number of palpable follicles or follicular volume during estrus. In contrast, concentrations of luteinizing hormone (LH) were higher (P less than .05) in androgen-immunized mares than in control mares during estrus and early diestrus. Concentrations of follicle stimulating hormone (FSH) and progesterone at those times were not affected (P greater than .10). From these data, we conclude that androgens in the mare during estrus may be involved with the regulation of LH secretion. In contrast, no involvement with FSH secretion was apparent under these short-term conditions.     

Evaluation of a Motorized Morcellator for Laparoscopic Removal of Granulosa‐Theca Cell Tumors in Standing Mares

Objective: To describe a motorized morcellator technique for laparoscopic removal of granulosa‐theca cell tumors (GCT) in standing mares and to evaluate long‐term outcome. Study Design: Case series. Animals: Mares (n=7) aged 4–15 years, with unilateral GCT. Methods: Tumor size was determined by transrectal palpation and ultrasonography. Standing sedated mares had 3 laparoscopic portal sites in the paralumbar fossa. After laparoscopic observation of the GCT, the mesovarium was desensitized, the ovarian pedicle transected with a LigaSure^® device, and the ovary grasped with forceps and cut in cylindrical tissue blocks using a motorized morcellator. Tissue blocks were removed through the laparoscopic sleeve. Outcome was determined by telephone interview of owners 6–40 months after surgery. Results: Estimated ultrasonographic median GCT diameter was 17 cm (range, 10–22 cm). Surgical time was 2–4.5 hours. There were no surgical complications. Two mares had mild subcutaneous emphysema at the portals after surgery. Convalescence was short, owners were satisfied with cosmetic outcome, and clinical signs associated with GCT did not recur. Conclusion: The motorized morcellator allows piecemeal removal of large GCT through a relatively small laparoscopic portal. Surgical complications were rare and the cosmetic outcome is favorable. Clinical Relevance: A motorized morcellator is a safe and minimally invasive technique for laparoscopic removal of GCT in mares.     

Equine granulosa cell tumors.

Unilateral ovariectomy was performed on 3 mares affected with granulosa cell tumors. Tumor fluid in each mare was found to contain estrogen, testosterone, and progesterone. In 2 mares, preoperative blood plasma concentrations of these hormones were comparable to those of a series of clinically normal mares. The other mare, which had a history of aggressive, masculine behavior, had higher testosterone content in the tumor fluid and in the preoperative blood sample. After surgical removal of the tumors, each mare developed follicles and ovulated with the remaining ovary. Each was eventually bred and 2 conceived. The probability of metastasis of these tumors in mares appears uncertain. Data from other species suggests a guarded long-term prognosis may be justified.     

Endometrial nitric oxide synthase activity in mares susceptible or resistant to persistent breeding‐induced endometritis and the effect of a specific iNOS inhibitor in vitro

Emerging research suggests that the nitric oxide system may play a role in persistent breeding‐induced endometritis (PBIE) in the mare. Differences in uterine nitric oxide (NO) levels between mares susceptible or resistant to PBIE and a dose‐dependent inhibitory effect of NO on uterine contractility have been demonstrated. The objectives of this study were to investigate the difference in total nitric oxide synthase (NOS) activity of the endometrium between susceptible and resistant mares and the effect of a specific inducible nitric oxide synthase (iNOS) inhibitor on the endometrial NOS activity in vitro. Six susceptible and six resistant mares were selected based on preset criteria and the results of an intrauterine challenge with killed spermatozoa during oestrus. Endometrial biopsy samples were collected 24 hr post‐challenge and cultured at 37°C for 24 hr in L‐arginine supplemented minimum essential medium with or without a specific iNOS inhibitor (1,400 W dihydrochloride, 1 mM). The medium and the cultured endometrial tissue were collected after 24 hr of culture and assayed for NO and total protein, respectively. Total NO content of the medium, normalized to endometrial tissue wet weight or total protein, was used as a measure of endometrial NOS activity. Non‐parametric tests were applied for statistical analysis. Susceptible mares had significantly greater endometrial NOS activity than resistant mares. The iNOS inhibitor treatment significantly reduced NOS activity in endometrial samples derived from susceptible and resistant mares. These findings provide a basis for in vivo testing of specific iNOS inhibitors as preventative or therapeutic options for PBIE in mares.     

Diagnosis and surgical removal of a granulosa-theca cell tumor in a mare

An 8-year-old mare showed stallion-like behavior and aggressiveness. A granulosa-theca cell tumor was tentatively diagnosed based on history, transrectal palpation, and ultrasonography, and surgically removed. Serological levels of testosterone and inhibin were elevated. Histopathological examination confirmed the diagnosis. The mare recovered and by day 45 could be turned out with other horses.     

Administration of RRR‐α‐tocopherol to pregnant mares stimulates maternal IgG and IgM production in colostrum and enhances vitamin E and IgM status in foals

This study assessed the effect of a vitamin E supplement given to pregnant mares on immunoglobulins (Ig) levels in foals. In addition, the fatty acid (FA) content and composition of the mares’ milk was assessed. Milk α‐tocopherol concentrations were compared between pregnant Danish Warmblood mares (n = 17) given a daily oral supplement of 2500 international units (IU) RRR‐α‐tocopherol in the last 4 weeks of pregnancy and a group of unsupplemented mares (n = 17) receiving 170–320 IU vitamin E daily originating from the feed. Milk α‐tocopherol was higher in supplemented mares (36.7, 12.4 and 9.8 μmol/l respectively) in relation to control mares (13.1, 6.4 and 5.8 μmol/l on days 1, 2 and 3 respectively; p < 0.001). Milk IgG was higher on days 2 and 3 post‐partum (PP) in supplemented mares (1.03 and 0.73 mg/ml respectively) in relation to control mares (0.79 and 0.56 mg/ml respectively; p < 0.05). Milk IgM was higher on days 2 and 3 post‐partum (PP) in supplemented mares (0.19 and 0.17 mg/ml) in relation to control mares (0.13 and 0.11 mg/ml respectively; p < 0.05). Plasma α‐tocopherol in foals was higher from supplemented mares on days 1, 2 and 3 (5.7, 14.8 and 19.2 μmol/l respectively) in relation to foals from control mares (3.6, 6.1 and 7.6 respectively; p < 0.001). Foal plasma IgM was higher from supplemented mares on day 3 (0.50 mg/ml) in relation to foals from control mares (0.32 mg/ml; p < 0.001). The total FA content in milk was highest on day 1 (21.6 g FA/kg milk) in relation to days 2 and 3 (13.6 and 13.5 g FA/kg milk respectively; p < 0.001). In conclusion, a daily oral supplement of 2500 IU RRR‐α‐tocopherol increased α‐tocopherol content in mare milk and foal plasma, IgG and IgM in mare milk and IgM in foal plasma.     

Bilateral ovarian leiomyoma treated by standing laparoscopic ovariectomy

An 11‐year‐old Warmblood mare presented to a referral facility due to behavioural problems. A hormonal profile performed prior to referral included increased systemic concentrations of testosterone and progesterone but a low concentration of inhibin. Transrectal ultrasound revealed ovaries consistent with a normal cycling mare. At the request of the owner, standing bilateral laparoscopic ovariectomy was performed without complication. Both ovaries were submitted for histopathological examination which revealed bilateral ovarian leiomyoma.     

Effects of the dam on equine chorionic gonadotropin concentrations during pregnancy

Breeding trials were designed to determine the influence of the mare on serum concentrations of equine chorionic gonadotropin (eCG) from Day 39 to Day 104 of gestation. Sires were ranked according to mean eCG concentrations found in the groups of randomly selected mares to which they were mated in 1983. Mares were ranked according to their mean eCG concentrations on Days 55, 71 and 85 of gestation (Day 0 = mating), in 1983 and 1985. In the 1986 breeding season, mares that had pregnancies characterized by high eCG levels were mated to sires previously associated with low eCG concentration pregnancies and low producing mares were mated to sires associated with high eCG concentration pregnancies. The highest eCG concentrations (12.8 ug/ml serum) were detected on Day 55 of gestation in mares mated to the low-ranked sire (P<0.01), indicating an influence of the mare on serum eCG concentration. A comparison of eCG production by individual mares in 1985, 1986 and 1987 showed that mares retained the same rank (P<0.001), regardless of the rank of the sire to which they were mated. It was concluded that the influence of the mare predominates in determining eCG concentration. In 1987, the highest ranked sire was mated to the highest eCG producing mares (ranked 1–20) and the second highest ranked sire was mated to mares ranked 21–40. Two distinct subgroups resulted, suggesting that the selection of sires and mares could be used to maximize the production of eCG.