Ultrasonography of the bovine ovary

A linear-array ultrasound scanner with a 5-MHz transducer was evaluated for the study of follicular and luteal status in heifers. The ovaries of five heifers were monitored daily until all heifers were examined for a period from three days before an ovulation to three days after the next ovulation. There was a significant difference among days for diameter of the largest follicle and second largest follicle and for the number of follicles 4-6 mm and >10 mm. Differences seemed to be caused by the presence of several 4- to 6-mm follicles in early diestrus, growth to an ostensibly ovulatory size and subsequent regression of a follicle during mid-cycle, and selective accelerated growth of the ovulatory follicle four days before ovulation. The corpus luteum became visible approximately three days after ovulation and was identifiable throughout the rest of the interovulatory interval. In two of the five heifers, the corresponding corpus albicans was identified for three days after the second ovulation. Two heifers were induced to superovulate and follicular growth was monitored. The results indicated that the follicles which ovulated originated from the population present when the superovulation treatment was initiated. The ultrasound instrument was judged effective for monitoring and evaluating ovarian follicles and corpora lutea in normal and superovulated heifers.     

Spontaneous embryonic death on Days 20 to 40 in heifers

Transrectal ultrasound examinations were used in nulliparous Holstein heifers to study the association between time of spontaneous embryonic death (cessation of heartbeat) and luteal regression, and to determine the fate of the conceptus after embryonic death. There was no significant difference between nonbred heifers (n = 135) and bred, nonpregnant heifers (embryonic heartbeat never detected, n = 40) for day of onset of luteal regression (means, 17.6 and 17.9, respectively) or for length of interovulatory interval (means, 20.6 and 20.9 days, respectively). Pregnancy was confirmed by detection of an embryonic heartbeat on Day 24 (ovulation = Day 0) or later, or on two consecutive days prior to Day 24; on average, an embryonic heartbeat was detected on Day 22.0 (n = 104). Pregnancy rate on Day 24 was higher (P<0.02) in heifers bred on Day -1 (116/149, 77.8%) than in heifers bred on Day -2 (51/79, 64.6%), and was higher (P<0.05) in heifers with an embryo transferred ipsilateral to the corpus luteum than in heifers with an embryo transferred contralateral to the corpus luteum (3/4 vs 0/5). Embryonic death (lack of embryonic heartbeat following confirmation of pregnancy) and presumptive embryonic death (embryonic heartbeat detected on one day only, prior to Day 24) were detected prior to Day 25 in one and two bred heifers, respectively, and in one and two heifers with an embryo transferred contralateral to the corpus luteum, respectively. In these six heifers, luteal regression preceded, and apparently caused, embryonic death. In seven of eight heifers in which embryonic death was detected between Days 25 and 40, the onset of luteal regression was detected at least 3 d (range, 3 to 42 d) after detection of embryonic death. The incidence of embryonic death on Days 29 to 32 was lower (P<0.02) in heifers bred on Day -1 than in heifers bred on Day -2 (0 of 96 vs 3 of 40, respectively). In heifers in which luteal regression preceded embryonic death, the conceptus was lost rapidly, with minimal evidence of degeneration. In heifers in which embryonic death preceded luteal regression, there was ultrasonic evidence of conceptus degeneration, but conceptus fluid and tissue appeared to be maintained. In all heifers with embryonic death, the conceptus and its breakdown products apparently were eliminated by expulsion through the cervix rather than by resorption.     

Role of luteal regression in embryo death in cattle

Inseminated crossbred beef cows and heifers were used in 2 experiments to investigate embryo survival after prostaglandin-induced or spontaneous luteal regression. In Experiment 1, luteal regression was induced by an intramuscular (im) injection of cloprostenol (500 mug) on Day 15 (day of ovulation = Day 0). Progestagen was replaced 24 or 36 h later either by one-and-a-half Syncro-Mate-B (SMB) ear implants (9 mg of norgestomet) and norgestomet solution (2.25 mg, im) containing no estradiol in 1 replicate or by 2 SMB implants (12 mg of norgestomet) and progesterone (100 mg, im) in the second replicate. Combined for both replicates, the Day-24 pregnancy rate in an untreated control group (Group 1, 16/19; 84%) was higher (P < 0.01) than in the 24-h group (Group 2, 9/20; 45%), which also was higher (P < 0.02) than in the 36-h group (Group 3, 3/23; 13%). In Experiment 2, 15 or 16 d after breeding, cattle with a corpus luteum at least 16 x 16 mm were given either 2 SMB implants or no treatment. At 24 to 26 d after breeding, pregnancy rates (48/65, 74% versus 49/68, 72%) were not significantly different, and all but 1 of the pregnant progestagen-supplemented cattle had a functional corpus luteum.     

Concentrations of oestradiol-17β and progesterone in the plasma of dairy heifers before and after cloprostenol-induced and natural luteolysis and during early pregnancy

Plasma oestradiol-17β and progesterone levels were measured in seven nulliparous, dairy heifers (British Friesian breed) that were administered cloprostenol (a synthetic analogue of prostaglandin F_2α) between days 8 and 14 of the oestrous cycle and inseminated (AI) 72 and 96 h later, and in seven heifers inseminated (AI) at natural oestrus.In both treated and untreated heifers, the beginning of the progesterone fall and the oestradiol-17β rise associated with luteolysis appeared to be synchronous but, whereas the rate of fall in progesterone level was greater for the treated heifers, that of the oestradiol-17β rise did not differ between treated and untreated heifers. Mean pre-ovulatory peaks of oestradiol-17β were 8 pg/ml and 10 pg/ml for treated and untreated heifers respectively.A post-ovulatory peak of oestradiol-17β in plasma 5–6 days after the pre-ovulatory peak occurred in all heifers whether or not conception had taken place. It is suggested that 7 days after the initiation of oestradiol-17β secretion by the pre-ovulatory follicle, another follicle begins to mature and secrete oestradiol-17β and that the progress of the latter towards full maturation and potential ovulation is stopped by rising progesterone levels from the corpus luteum; as a result in normal, non-pregnant cattle an interval of about 21 days elapses before another ovulation (of another follicle) takes place. In the event of premature luteolysis (in the present study induced between the 8th and 14th day) there is no evidence that the timing of this luteolysis influences the time taken for a follicle to enter the final stages of pre-ovulatory maturation, when increasing amounts of oestradiol-17β are secreted. Thus the interval between ovulations may not be less than 7 days but, depending on corpus luteum survival, may vary between 7 and 21 days.In one heifer after natural luteolysis a normal plasma oestradiol-17β peak followed but this was not associated with ovulation and corpus luteum formation. The second oestradiol-17β peak 6 days after the first, however, evidently assumed the ovulatory role; presumably the secreting follicle concerned, not being subject to inhibition by progesterone rising to luteal levels, matured fully and ovulated. Thus the second, normally post-ovulatory, oestradiol-17β peak in cattle can, in the event of failure of ovulation at the normal time, itself assume the ovulatory function, the oestrous cycle length then being about 28 days.     

Ultrasonic anatomy of equine ovaries

A linear-array ultrasound scanner with a 5-MHz transducer was evaluated for studying follicular and luteal status in mares, and the ultrasonic properties of equine ovaries were characterized. Follicular diameters were estimated in vivo and after removing and slicing six ovaries. Correlation coefficients between the two kinds of determinations were 0.91 for number of follicles >/=2 mm in diameter and 0.95 for diameter of largest follicle. The ovaries of five mares were examined daily until all mares had been examined from three days before an ovulation to three days after the next ovulation. There was a significant difference among days for diameter of largest follicle and second largest follicle and for number of follicles 2-5 mm, 16-20 mm, and >20 mm. Differences seemed to be caused by the presence of many 2- to 5-mm follicles during early diestrus, initiation of growth of large follicles at mid-cycle, selective accelerated growth of an ovulatory follicle beginning five days before ovulation, and regression of large nonovulatory follicles a few days before ovulation. In one of the five mares, the corpus luteum was identified throughout the interovulatory interval, and the corresponding corpus albicans was identified for three days after the second ovulation. In the other four mares, the corpus luteum was last identified an average of 16 days after ovulation or five days before the next ovulation. In a blind study, the location of the corpus luteum (left or right ovary) as determined by ultrasonography agreed with a previous determination of side of ovulation by palpation in 88% of 40 mares on days 0-14. In the remaining 12% and in all of 12 estrous mares, the location was recorded as uncertain. The ultrasound instrument was judged effective for monitoring and evaluating follicles and corpora lutea.     

Transvaginal collection and ultrastructure of llama (Lama glama) oocytes

Ultrasound-guided transvaginal follicle aspiration has been described as a noninvasive and repeatable procedure for oocyte collection in several species, but its use has not been described for any of the members of the family, Camelidae. A study was designed to determine the feasibility of an ultrasound-guided transvaginal approach for oocyte collection in llamas. Fifteen non-pregnant, adult female llamas (10 non-stimulated and 5 superstimulated) were examined by transrectal ultrasonography with a 7.5-MHz linear-array transducer to determine the number and diameter of follicles available for aspiration. After caudal epidural anesthesia was induced, the 7.5-MHz linear-array transducer was fastened to a long rigid handle and inserted intravaginally. The free hand was placed into the rectum to manipulate the ovaries, one at a time, in position against the vaginal wall over the face of the transducer. A 20-gauge, 55-cm-long, single-lumen needle was advanced through the vaginal fornix and into follicles > or = 3 mm in diameter. Follicular contents were aspirated using a regulated vacuum pump (flow rate = 33 mL/min; approximately 150 mm Hg) into a tube containing 3 mL of phosphate buffered saline and 0.2% BSA. Fluid was filtered (75 microm mesh), and oocytes were located and morphologically evaluated using a stereomicroscope. Overall, 134 follicles were aspirated, and 76 oocytes were collected (collection rate = 57%). Thirty-two oocytes (42%) were surrounded by multiple layers of compacted granulosa cells and had homogenous dark ooplasm; 13 oocytes (17%) were surrounded by the corona radiata layer only and had heavily granulated ooplasm; 9 oocytes (12%) were denuded and had homogenous dark ooplasm; and 22 oocytes (29%) were denuded and displayed signs of ooplasm degeneration. The ultrastructure of llama oocytes was similar to that of cattle except for conspicuous accumulation of large lipid droplets in the cytoplasm. Twenty-four hours after follicle aspiration, the ovaries were examined by transrectal ultrasonography and intrafollicular hematomas were detected in 3 llamas (9 of 48 follicles aspirated). Results demonstrate the potential utility of a transvaginal ultrasound-guided technique for oocyte collection and in vitro embryo production in llamas. Oocytes of llamas bear an ultrustructural resemblance to those of cattle, but are distinguished by a predominance of cytoplasmic lipid.     

Ultrasonic morphology of corpora lutea and central luteal cavities during the estrous cycle and early pregnancy in heifers

Ultrasonography was used once daily to quantify corpora lutea, central luteal cavities, and luteinized tissue during interovulatory intervals (n = 66) and during Days 0 to 60 of pregnancy (n = 14) in nulliparous Holstein heifers (ovulation = Day 0). The corpus luteum of the estrous cycle was detectable by ultrasonography in most heifers from the day of ovulation (mean, Day 0.5) and extending into the regressive phase beyond the next ovulation (mean, Day 1.4 +/-0.2 after the next ovulation). During pregnancy, the corpus luteum was detected until Day 60 (end of study). Maximal central luteal cavity area detected on Days 0 to 20 was used retrospectively to group luteal glands into four cavity categories: no, small, medium, and large. These categories corresponded to approximate cavity diameters of <2 mm, 2 to 5 mm, 6 to 10 mm, and >10 mm, respectively. The incidence of each cavity category was similar between interovulatory intervals and pregnancies (combined incidence, 17 80 , 8 80 , 33 80 , and 22 80 for no, small, medium, and large cavities, respectively; total with cavities, 63 80 , 79%). Mean day of first detection of a central cavity was earliest for large cavities during interovulatory intervals (means, Days 4.7, 4.4, and 3.0 for small, medium, and large cavities, respectively; P<0.04) and during pregnancies (means, Days 5.5, 4.2, and 3.3, respectively; NS). However, the day that the cavities reached maximum size (range of means, Days 5.5 to 7.0) did not differ among categories. Mean day of last detection of the central cavity was significantly different among cavity categories during interovulatory intervals (means, Days 9.3, 11.1, and 17.4 for small, medium, and large cavities, respectively) and pregnancies (means, Days 7.0, 8.8, and 20.2, respectively). Time of loss of central cavities was similar between nonbred and pregnant heifers, and there was no significant difference among cavity categories in the length of the interovulatory interval (mean, 20.1 d). Luteal tissue area was not significantly different among cavity categories during interovulatory intervals. There were no indications that cavities were functionally important. Luteal tissue area increased linearly in pregnant heifers on Days 21 to 60 (mean slope, 2.6 mm(2)/day).     

Gamete recovery and follicular transfer (graft) using transvaginal ultrasonography in cattle

Current in vitro culture systems may not be adequate to support maturation, fertilization and embryo development of calf oocytes. Thus, we initiated a study to investigate an alternative method of assessing oocyte competence in vivo, initially using oocytes from adults. Experiment 1 was done to determine if follicle puncture would alter subsequent follicle development, ovulation and CL formation. In control (no follicle puncture, n = 3) and treated (follicle puncture, n = 3) heifers, ultrasound-guided transvaginal follicle aspiration was used to ablate all follicles > or = 5 mm at random stages of the estrous cycle to induce synchronous follicular wave emergence among heifers; PGF2 alpha was given 4 d later. Three days after PGF2 alpha, the preovulatory follicle in treated heifers was punctured with a 25-g needle between the exposed and nonexposed portions of the follicular wall, and 200 microL of PBS were infused into the antrum. There was no significant difference between control and treated heifers for mean diameter of the dominant follicle prior to ovulation, the interval to ovulation following PGF2 alpha, or first detection and diameter of the CL. Experiment 2 was designed to assess multiple embryo production following interfollicular transfer of oocytes (i.e., transfer of multiple oocytes from donor follicles to a single recipient preovulatory follicle). Follicular wave emergence was synchronized among control (no follicle puncture, n = 5), oocyte recipient (n = 7) and oocyte donor (n = 5) heifers as in Experiment 1. In control and oocyte recipient heifers, a norgestomet ear implant was placed at the time of ablation and removed 4 d later, at the second PGF2 alpha treatment. In oocyte donor heifers, FSH was given the day after ablation, and, 4 d later, oocytes were collected by transvaginal follicle aspiration, pooled and placed in holding medium. Five or 6 oocytes were loaded into the 25-g needle of the follicle infusion apparatus with < or = 200 microL of transfer medium. Puncture of the preovulatory follicle of recipient heifers was done as in Experiment 1. Immediately thereafter, LH was given to control and oocyte recipient heifers, but only the recipients were inseminated. Ovarian function was assessed by transrectal ultrasonography and control and oocyte recipient heifers were sent to the abattoir 2 or 3 d after ovulation, where excised oviducts were flushed. The interval between LH administration and ovulation (33 to 36 h) was highly synchronous within and among control and oocyte recipient heifers. Four of 5 (80%) ova were collected from controls and 16 of a potential 43 (37%) ova/embryos were recovered from oocyte recipients; 8 embryos from 3 heifers. Thus, the gamete recovery and follicular transfer procedure (GRAFT) did not alter ovulation or subsequent CL formation, and resulted in the recovery of multiple ova/embryos in which a total of 19 oocytes yielded as many as 8 early embryos, a 42% embryo production rate.     

The ability of subordinate follicles of the second follicular wave to become dominant is lost by day 15 of the estrous cycle in cattle

Generally, unilateral ovariectomy before a critical period in the latter part of the estrous cycle induces a transitory increase in plasma FSH, which causes subordinate follicles to develop and maintain ovulation rates characteristic of the species. A limiting period for subordinate follicles to assume dominance and from which ovulation occurs has not been shown for cattle. Growth and/or regression of subordinate follicles were characterized following removal of the dominant follicle at different days of the luteal phase of the estrous cycle in cattle in this study. In the mid-luteal phase (Day 13 or 15), the ovary with the dominant follicle of the second wave was ablated via unilateral ovariectomy; the corpus luteum also was removed. In the late luteal phase (Day 17 or 19), the dominant follicle was ablated with an ultrasonically guided 20 gauge needle. When the dominant follicle was removed on Day 13, the largest subordinate follicle of the second wave of follicular development became dominant and ovulation occurred from this follicle in 4 of 4 animals. However, when the dominant follicle was removed on Day 15, 17 or 19, a new wave of follicular development was induced in 14 of 15 animals. Moreover, the recovered subordinate follicle of the second wave of follicular development had similar growth characteristics to naturally occurring dominant follicles. In conclusion, the subordinate follicle in the second follicular wave in cattle retained the ability to become dominant, but this ability was lost by Day 15 of the estrous cycle. However, cattle then were able to maintain ovulation by developing a new wave of follicular growth.     

Ultrasound-guided intrafollicular treatment in mares

A technique for intrafollicular treatment with a transvaginal ultrasound-guided injection needle was developed using equine chorionic gonadotropin (eCG) as the test substance. An injection was made into one growing follicle of a wave when the follicles were 20 to 23 mm. The treated follicles were injected with 1000 iu of eCG in 0.2 ml saline solution and control follicles were injected with 0.2 ml of the saline vehicle (10 mares per group, 1 follicle per mare). The injection system used an inner 25-gauge needle and an outer 20-gauge needle inserted together through the needle-guide channel of a linear-array trans vaginal transducer. The outer needle was pushed through the vaginal wall and the inner needle was then advanced into the follicle during monitoring on the ultrasound screen. The turbulence in the follicular fluid associated with injection was observable on the screen. Seven follicles were successfully injected in each group. The follicular fluid in the control follicles remained anechoic until the follicle was no longer identifiable or ovulated. All 7 follicles in the eCG group showed ultrasonic indications of luteinization, based on the formation of an echogenic, thickened wall or area. Five of the 7 developed a central area that had the ultrasonic appearance of a blood clot similar to the appearance of a corpus hemorrhagicum. Ovulation was not detected in any of the eCG-treated follicles. The maximum post-treatment diameter of follicles was greater (P < 0.05) for the eCG group (32.7 +/- 3.8 mm) than for the control group (23.4 +/- 1.8 mm). The mean diameter for the first 5 days post-treatment (before the occurrence of an ovulation in any mare) was also greater (P < 0.002) in the eCG group (21.6 +/- 0.8 mm vs 19.6 +/- 0.8 mm). Results indicated that this novel research approach is practical and has potential for studies on folliculogenesis. The technique provides a research model between the extremes of an in vitro culture system and treatment of the whole animal.     

Follicular dynamics and temporal relationships among body temperature, oestrus, the surge of luteinizing hormone and ovulation in Holstein heifers treated with norgestomet

The effects of chronic treatment with norgestomet on follicular dynamics, corpus luteum growth and function as well as the temporal relationships among body temperature, oestrous behaviour, the luteinizing hormone (LH) surge and ovulation following implant removal were studied in 16 Holstein heifers. Oestrous cycles of the heifers were initially synchronized using 2 injections of prostaglandin F-2 alpha (PGF-2 alpha) 12 days apart. The heifers were then implanted with a norgestomet ear implant for 9 days, beginning either at the middle of the synchronized cycle (dioestrus) or at the end of the synchronized cycle (pro-oestrus). Follicular dynamics, corpus luteum growth and regression, and plasma progesterone were not affected by norgestomet treatment at dioestrus. The dominant follicle present at the time of norgestomet implantation in the pro-oestrus group was maintained during the 9-day implant period of 6 of 8 heifers and ovulated after implant removal. Time from implant removal to onset of standing oestrus and time to LH peak following implant removal were highly correlated with the time of ovulation (r = 0.92 and 0.96, respectively). Onset of standing oestrus and the LH peak and the onset of standing oestrus and peak vaginal and rectal temperatures were also highly correlated (r = 0.96, 0.82 and 0.81, respectively). It is concluded that any decrease in pregnancy rates following treatment with norgestomet is not due to asynchrony among oestrus, the LH surge and ovulation.     

Ultrasonic detection of the conceptus and characterization of intrauterine fluid on days 10 to 22 in heifers

Nonbred and pregnant heifers were examined ultrasonically on alternate days from Days 10 to 22 in three experiments. The goal was to define criteria that could be used for identification of the early conceptus. The location and size of circular and elongated nonechongenic structure (apparently free intrauterine luminal fluid) were recorded (Experiment 1). On Day 12, there was a significantly greater number of circular nonechogenic structures in the ovarian half of the uterine horn ipsilateral to the corpus luteum in pregnant heifers than in nonbred heifers (means, 5.1 vs 3.5). Therefore, the embryonic vesicle was apparently detected on Day 12, but was not distinguished from circular nonechogenic structures representing free luminal fluid. The number of circular nonechogenic structures decreased after Day 14, with a corresponding increase in elongate nonechogenic structures associated with luteal regression (nonbred heifers) or elongation of the blastocyst (pregnant heifers). On Day 16, there were more elongated than circular nonechogenic structures in pregnant heifers than in nonbred heifers. In nonbred heifers, there was no evidence of a local utero-ovarian relationship between the amount of intrauterine luminal fluid and the location of either the corpus luteum or the next ovulatory follicle. Uterine luminal fluid in each horn increased several fold between Days 16 and 18 in nonbred heifers; there was significantly more lumina fluid in nonbred than in pregnant heifers on Days 18 and 20. In pregnant heifers, intrauterine luminal fluid area increased first in the ovarian end of the uterine horn ipsilateral to the corpus luteum; this was apparently due to detection of the elongating blastocyst. The length of the longest specular reflector on the endometrial surface of the uterine lumen increased over Days 10 to 22 in nonbred and pregnant heifers, but was not a useful criterion for detection of the conceptus (Experiment 2). The accuracy of a 7.5 MHz transducer for pregnancy diagnosis was not greater than a guess (50%) before Day 16 (Experiment 3). Results indicated that early pregnancy diagnosis in heifers was confounded by the presence of intrauterine fluid.     

Follicular populations during the estrous cycle in heifers. II. Influence of right and left sides and intraovarian effect of the corpus luteum

Ovarian follicles ≥2 mm were studied in 22 Holstein heifers by daily ultrasound examinations. Data were partitioned by right vs. left ovary and corpus luteum bearing ovary vs. the contralateral ovary. There were significantly more (P < 0.03) follicles 4–6 mm, > 13mm and total ≥2 mm in the right ovary, regardless of the presence of a corpus luteum. Significantly more (P < 0.05) follicles 2–3 mm, > 13 mm and total ≥2 mm were observed in the ovary bearing the corpus luteum. Interactions between day and corpus luteum appeared to be due to a greater number of follicles in the ovary bearing the corpus luteum during the first part of the interovulatory interval. There was also a day by right side vs. left side interaction for the number of follicles > 13 mm. Interpretation of the interactions was that the presence of a corpus luteum was conducive to the development of more anovulatory diestrous follicles > 12 mm. However, as regression of the corpus luteum progressed, there was an apparent proclivity for preovulatory follicular development in the right ovary. There was no apparent pattern of alternating sides of ovulation or of alternating sides of development of anovulatory diestrous follicles and preovulatory follicles in heifers observed for more than one interovulatory interval. There was not a significant difference in the maximum diameter attained by the anovulatory diestrous follicle or preovulatory follicle between ovaries ipsilateral or contralateral to the corpus luteum; however, the maximum diameter attained by the preovulatory follicle was greater (P < 0.05) than that attained by the anovulatory diestrous follicle.     

Ovarian steroid hormones and endometrial response

As a bioassay of the steroidogenic function of the corpus luteum, endometrial biopsy has been proposed as the most efficient way of diagnosing corpus luteum insufficiency. However, analysis of our data on luteal phase evaluation in infertility shows that most cases (86%) of endometrial luteal inadequacy are associated with normal hormone (progesterone, estradiol) stimulation. This apparent lack of endometrial progestational response may be explained by an end organ defect localized to the endometrial steroid receptors.     

Ovarian synchronization following ultrasound-guided transvaginal follicle ablation in heifers

In Experiments 1 and 2, ultrasound-guided transvaginal follicle aspiration was used as a method of follicle ablation to induce and synchronize subsequent follicular wave emergence and enhance ovulation synchrony following PGF(2alpha) administration. Heifers were at unknown stages of the estrous cycle at the start of both experiments in which all follicles >/=5 mm in diameter were ablated; luteolysis was induced 4 d later with cloprostenol (500 ug/dose, im). In Experiment 1, heifers were randomly assigned to either an ablation (n=17) or a procedural control (no follicle ablation, n=17) group. Ablation-induced wave emergence was indicated by a significant increase in the total number of follicles >/=5 mm within 2 d of ablation (mean, 1.5 d), which was preceded by a significant surge in circulating FSH. Although the mean (+/-SEM) interval from PGF(2alpha) administration to ovulation did not differ between follicle-ablated heifers (5.1+/-0.5 d range, 3 to 9 d) and control heifers (5.1+/-1.0 d; range, 1 to 5 d), the variability of the interval was different (P<0.05). Inequality of variance between the 2 groups was attributed to a greater (P<0.08) degree of ovulation synchrony in the ablation group than in the control group; 13 16 (81%) versus 9 17 (53%), respectively, ovulated within 5 d of cloprostenol administration. Relative asynchrony of ovulations in control heifers was associated with the status of the follicular wave at the time of PGF(2alpha) administration and, in part, to incomplete luteolysis following a single dose of PGF(2alpha). Experiment 2 was designed to examine the efficacy of 2 doses of cloprostenol 12 h apart (n=7) versus a single dose (n=8) to induce complete luteolysis subsequent to follicle ablation-induced wave emergence. Two doses of cloprostenol potentiated ovulation synchrony; more (P<0.05) 2-dose heifers (7 7 , 100%) than single-dose heifers (4 8 , 50%) ovulated within 5 d after PGF(2alpha) administration. In summary, ultrasound-guided transvaginal follicle ablation, done at random during the estrous cycle, induced and synchronized subsequent follicular wave emergence, and resulted in a high degree of ovulation synchrony among heifers after PGF(2alpha) induced luteolysis, especially when 2 doses of PGF(2alpha) were administered 12 h apart.     

Fate of the theca interna following ovulation in the ewe

Summary The fate of the theca interna after ovulation was studied in ewes, using light and electron microscopic histology and histochemistry. At the time of ovulation the theca interna was incorporated, apparently completely, into the margin of the developing corpus luteum and into the centres of many infoldings of the follicular wall. There was no evidence of degeneration of the more highly differentiated theca interna cells at or following the time of ovulation. Within 24 h of ovulation, cells derived from the theca interna began migrating from their original sites into the deeper, granulosa-derived areas of the luteal tissue. At later stages cells derived from the theca interna remained concentrated in septa derived from the follicular infoldings, but were also widely distributed throughout the luteal tissue. Structural evidence supported the view that the small luteal cells and fibroblasts of the corpus luteum were derived from the theca interna, and the large luteal cells from the membrana granulosa.The authors wish to thank Mrs. Linda Musk and Miss Anneke Veenstra for skilled technical assistanceDeceased on May 4, 1979     

Changes in steady-state concentrations of messenger ribonucleic acids in luteal tissue during prostaglandin F2alpha induced luteolysis in mares

Transvaginal ultrasound-guided luteal biopsy was used to evaluate the effects of prostaglandin (PG)F2alpha on steady-state concentrations of mRNA for specific genes that may be involved in regression of the corpus luteum (CL). Eight days after ovulation (Hour 0), mares (n=8/group) were randomized into three groups: control (no treatment or biopsy), saline+biopsy (saline treatment at Hour 0 and luteal biopsy at Hour 12), or PGF2alpha+biopsy (5mg PGF2alpha at Hour 0 and luteal biopsy at Hour 12). The effects of biopsy on CL were compared between the controls (no biopsy) and saline+biopsy group. At Hour 24 (12h after biopsy) there was a decrease in circulating progesterone in saline group to 56% of pre-biopsy values, indicating an effect of biopsy on luteal function. Mean plasma progesterone concentrations were lower (P<0.001) at Hour 12 in the PG group compared to the other two groups. The relative concentrations of mRNA for different genes in luteal tissue at Hour 12 was quantified by real time PCR. Compared to saline-treated mares, treatment with PGF2alpha increased mRNA for cyclooxygenase-2 (Cox-2, 310%, P<0.006), but decreased mRNA for LH receptor to 44% (P<0.05), steroidogenic acute regulatory protein to 22% (P<0.001), and aromatase to 43% (P<0.1) of controls. There was no difference in mRNA levels for PGF2alpha receptor between PG and saline-treated groups. Results indicated that luteal biopsy alters subsequent luteal function. However, the biopsy approach was effective for collecting CL tissue for demonstrating dynamic changes in steady-state levels of mRNAs during PGF2alpha-induced luteolysis. Increased Cox-2 mRNA concentrations suggested that exogenous PGF2alpha induced the synthesis of intraluteal PGF2alpha. Thus, the findings are consistent with the concept that an intraluteal autocrine loop augments the luteolytic effect of uterine PGF2alpha in mares.     

Effects of hyperadrenal states on luteinizing hormone in cattle

The effect of an induced hyperadrenal state on luteinizing hormone (LH) secretion and subsequent ovarian function was examined in both intact and adrenalectomized (ADRX) heifers. Treatments were begun on Day 2 or Day 16 of an estrous cycle in order to examine their effect on corpus luteum development or ovulation, respectively. In Experiment I, continuous intravenous infusion of ACTH (1.0 mg/24 h) to intact heifers decreased LH concentrations during the early phase of the cycle (Days 3-5). Treatment of ADRX heifers with hydrocortisone succinate (HS) (100 mg/24 h) did not appear to change mean LH concentrations, although da Rosa and Wagner (1981) have reported reduced plasma concentrations of progesterone at mid-cycle in these ACTH-treated intact heifers and HS-treated ADRX heifers. ACTH treatment of ADRX heifers had no effect on LH or progesterone. In the second study, there were similar frequencies of LH surges at the anticipated time of ovulation in all treatment groups. HS (100 mg/24 h) in ADRX heifers and ACTH (0.5 mg/24 h) in intact heifers was given continuously beginning on Day 16 of an estrous cycle. Although some animals in all groups exhibited LH surges, the ACTH-treated intact and HS-treated ADRX heifers failed to show a consistent subsequent increase in progesterone concentrations in plasma, suggesting a failure of luteal development. Although no difference was seen in baseline concentrations of LH, there was a greater difference between basal and overall mean LH concentrations in control groups than was observed in ACTH- or HS-treated animals. These induced hyperadrenal states resulted in depression of ovarian function as shown by decreased plasma progesterone during the luteal phase of the cycle. It is not known if other noncorticoid steroids from the adrenal cortex are necessary for a full expression of this effect.     

Effects of testosterone and 5alpha-dihydrotestosterone on luteal lifespan in dairy heifers

Endogenous concentrations of testosterone increase approximately 7 d prior to estrus in cattle and goats. Inhibition of testosterone synthesis results in a delay of luteal regression in both species. The purpose of this experiment was to determine if treatment with testosterone or 5alpha-dihydrotestosterone (DHT), 2 to 6 d prior to the endogenous rise in testosterone, would result in premature luteal regression. Sixteen heifers were randomly assigned to one of three treatment groups: 1) Control (n = 6); 2) testosterone (100 mug, n = 5); or 3) DHT (100 mug, n = 5). Each heifer received a single injection of the appropriate steriod on Day 8, 9, 10, 11 or 12 post estrus. Jugular venous blood samples were collected at frequent intervals for 24 h to quantify testosterone, and then daily for 14 d to quantify progesterone. Concentrations of testosterone increased within 15 min of injection of testosterone, and reached a maximum at 30 min. Concentrations were maintained at > 2 ng/ml throughout the first 24 h after injection. Based on concentrations of progesterone, neither androgen had any effect on the lifespan of the corpus luteum or the level of luteal function.     

Effect of prostaglandin F2alpha dosage and stage of estrous cycle on the estrous response and corpus luteum function in beef heifers

Ninety-five normal cyclic crossbred beef heifers were used to determine if the proportions of heifers showing estrus, intervals to estrus and corpus luteum (CL) function were influenced by PGF(2alpha) dosage and (or) the stage of luteal phase when PGF(2alpha) was administered. Heifers were assigned randomly to treatments in a 4 x 3 factorial arrangement. Treatments were 5, 10, 25 or 30 mg PGF(2alpha) injected either in early (5 to 9 d), mid (10 to 14 d) or late (15 to 19 d) stages of the luteal phase. Jugular samples were taken at 0 h and at 8 h-intervals for 48 h and again at 60 h after PGF(2alpha) treatment for progesterone assay. Heifers were observed for estrus continuously for 120 h PGF(2alpha) treatment. The proportion of heifers showing estrus was dependent upon (P<0.05) both dosage of PGF(2alpha) and stage of luteal phase. Heifers given 5 mg of PGF(2alpha) showed estrus only if treated during the late stage, while those given 10 mg of PGF(2alpha) showed a progressive increase of heifers in estrus as stage of luteal phase advanced. The proportion of heifers showing estrus after 25 and 30 mg of PGF(2alpha) increased from 56% for the early stage to 100% for the mid and late stages. Interval to estrus in heifers showing estrus within 120 h after PGF(2alpha) treatment did not differ (P>0.05) among dosages but tended (P=0.10) to be longer in heifers treated during the mid luteal stage (67 h) than in heifers treated in the two other stages (56 h). A greater proportion of heifers (P<0.05) showed estrus by 60 h after PGF(2alpha) when treated during the early and late luteal stages (75.5%) than for heifers treated during the mid luteal stage (30.4%). Patterns of progesterone concentrations were influenced (P=0.08) by the three way interaction of dosage, stage and time. In heifers that showed estrus, rate of decline in progesterone tended (P=0.07) to be slower during the mid luteal stage than during the early and late stages. Progesterone did not drop below 1 ng/ml until 32 h in heifers treated during the mid luteal stage; whereas progesterone dropped below 1 ng/ml by 24 h in heifers treated during the early and late stages. These data may be useful in designing more efficient systems for using PGF(2alpha) or its analogues in estrus synchronization of beef cattle.