Corpus Luteum Function Research Articles

Effect of deslorelin on the induction of estrus in anestrous bitches

The efficacy of one or multiple doses of an injectable formulation of deslorelin (a GnRH agonist) was evaluated to induce estrus in anestrous bitches. Thirteen animals composed three groups: group 1 (n=5, single IM injection of 2mg deslorelin), group 2 (n=5, four IM injections of 2mg deslorelin in alternate days), and control group (n=3, four IM saline injections in alternate days). Daily clinical evaluations, sexual behavior, vaginal cytology, plasma progesterone concentration, ovaryhysterectomy and macroscopic evaluation of the uterus and ovaries were done. In group 1, none of the bitches showed signs of estrus, while two developed clinical signs and vaginal cytology of proestrus. In group 2, all animals presented proestrus, four presented estrus, and three ovulated; resulting in a functional corpus luteum and high progesterone concentration until day 25 of diestrus, when ovaryhysterectomy was performed. The duration of the stages of deslorelin induced cycles and the progesterone profile were similar to those described in the literature, and no side effects were observed. In conclusion, injectable formulation of deslorelin in multiple injections was effective to induce fertile estrus in anestrous bitches.

Tumor necrosis factor-α and acute-phase proteins in early pregnant ewes after challenge with peptidoglycan-polysaccharide

Bacterial infection shortly after mating interferes with establishment of pregnancy. Injection of peptidoglycan-polysaccharide (PG-PS), a component of gram-positive bacteria, into sheep on day 5 after mating reduces pregnancy rate. Experiments were designed to evaluate the acute-phase response (APR) in ewes to injection of PG-PS on day 5 after mating (day 0). Catheters were inserted into the jugular and posterior vena cava on day 4. On day 5, ewes were challenged with saline or 30 μg/kg body weight (BW) PG-PS (Exp 1) or 60 μg/kg BW PG-PS (Exp 2). Blood samples were collected every 15 min for 6 h (Exp 1) and every 15 min for 2 h, hourly for 12 h, and at 24, 36, and 48 h (Exp 2). Body temperature and clinical signs of infection were monitored in Exp 2. Plasma was assayed for concentrations of a pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α); 2 APR proteins, serum amyloid A (SAA) and haptoglobin (Hp); and progesterone (P 4). Ewes injected with 60 μg/kg BW PG-PS exhibited fever, vaginal discharge, loss of appetite, and lethargy. After challenge with either 30 μg/kg or 60 μg/kg BW PG-PS, TNF-α increased in the posterior vena cava. Concentrations of SAA and Hp in the jugular increased after challenge with 60 μg/kg BW PG-PS. Only half (5/10) of the ewes treated with 60 μg/kg BW PG-PS had ultrasonically visible embryos, and none of them had functional corpora lutea (CL) (<1 ng/mL of P 4) on day 21. On the other hand, 8/9 (88.9%) control ewes had visible embryos and all had functional CL on day 21. Using logistic regression, pregnancy on day 21 was predicted to depend on concentrations of TNF-α and Hp on day 5 and concentration of P 4 on day 14. In summary, injection of PG-PS on day 5 after mating resulted in fever; increased concentrations of TNF-α, Hp, and SAA on the day of and the day after the PG-PS challenge; and decreased concentrations of P 4 on days 14 and 21. These factors were related to failure to establish pregnancy.

Changes in Ovarian Follistatin Levels During the Oestrous Cycle in Sheep may Serve as an Intraovarian Regulator

The expression and concentration of follistatin and activin change during oestrous cycle suggesting their involvement in the regulation of follicular development. The aim of this study was to determine the level, source and potential role of follistatin in the sheep ovary. Follistatin in ovarian venous blood, measured by radioimmunoassay, remained at its low level from follicular phase (day -1 and 0) to mid-luteal phase (days 11-13) phase but were significantly elevated during the late luteal phase (days 14 and 15) when corpora lutea underwent regression. Western blot analyses of follicular fluid at day 15 of the cycle showed two strong bands at 42 and 45 kDa and weakly stained bands at 39 and 31 kDa. At day 0, these bands became weaker and the 39 kDa band became undetectable. However, there were no differences in follistatin concentrations between ovaries with and without functional corpus luteum (CL) during the whole luteal phase. In addition, although the ovaries of Booroola ewes normally contain more corpora lutea than those of normal merino ewes, follistatin concentrations in both jugular and ovarian venous blood were similar in Booroola and normal merino ewes. It is concluded that the secretion of follistatin from the ovary is not related to the formation of CL or high ovulation rate of Booroola ewes. The elevation in follistatin concentration in follicular fluid and ovarian blood during late luteal phase may indicate a dual role of follistatin in the luteolysis of existing CL and development of new follicle cohort.

Differential Cellular Localization of Galectin-1 and Galectin-3 in the Regressing Corpus Luteum of Mice and Their Possible Contribution to Luteal Cell Elimination

Galectin-1 and galectin-3, beta-galactoside-binding lectins, are predominantly expressed in the regressing corpus luteum (CL) of mouse ovary. This study revealed the expression patterns and cellular localizations of galectins during CL formation and regression by ISH and IHC. Galectin-1 mRNA expression temporarily increased in active CL, preceding the expression of progesterone degradation enzyme 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which represents functional luteolysis. The expressions of both galectin-1 and galectin-3 remarkably increased in the structurally regressing CL, which vigorously expressed 20alpha-HSD and contained abundant apoptotic luteal cells. Ultrastructurally, galectin-1- and galectin-3-immunoreactive cells were identified as fibroblasts and infiltrating macrophages, respectively. In addition, some populations of luteal cells themselves expressed galectin-3 in regressing CL and formed unique demarcation membranes in the cytoplasm, showing a non-typical apoptotic feature. Ovary of adult mice with repeated estrus cycles contained CL of three different generations. Among them, the old CL formed during previous estrus cycles consisted of galectin-3-positive luteal cells. The galectin-3-positive old CL was resistant to apoptosis and seemed to be eliminated by a mechanism different from apoptosis. The stage- and cell-specific expression of galectin in CL suggests its differential contribution to luteolysis, and this expression may be mediated by major regulatory molecules of CL function, prolactin and/or prostaglandin F2alpha.

The Effect of PGF2α-Induction of Estrus on Pregnancy Rates in Mares

In mares, the onset of estrus is routinely induced after a luteolytic dose of a prostaglandin F2alpha analogue. Mares in diestrus with a mature, functional corpus luteum will respond by coming into estrus 3 to 4 days after induction 1 . Shortening the interestrous interval in mares has an important economic impact on the equine breeding industry. Because the breeding season of the mare is short, timing of insemination and appropriate coordination of endocrine events is critical to pregnancy success and ultimate foaling rates. Several recent studies have reported that the use of prostaglandin analogues is associated with lower pregnancy rates. In this study the induction of estrus with cloprostonol (125-250 ug IM) did not affect pregnancy rates in mares that have ovulatory cycles (n = 461).

Transcriptional Profiling of Equine Endometrium During the Time of Maternal Recognition of Pregnancy1

Establishment and maintenance of pregnancy are critically dependent on embryo-maternal communication during the preimplantation period. To gain new insights into this complex process in the horse, transcriptional profiling of Day 13.5 pregnant and cyclic endometrial tissue samples was carried out using custom-designed microarrays. Selected array data were validated using quantitative RT-PCR, and proteins of interest were localized using immunohistochemistry. One hundred and six transcripts were up-regulated, whereas 47 transcripts showed lower expression levels in pregnant mares, that is, were down-regulated in pregnant mares. Half of the genes with known or inferred function are classically regulated by estrogens. Elevated transcript levels were found for genes involved in cell-cell signaling, heat shock response, and secretory proteins, among others. Solute carrier family 36 (proton/amino acid symporter), member 2, SLC36A2, was one of the most highly up-regulated genes, potentially reflecting the nutritional needs of the rapidly developing embryo. Among the genes showing lower expression in pregnant mares, estrogen receptor 1 was of particular interest because of its potential involvement in the initiation of luteolysis in cyclic mares. We hypothesize that either conceptus' estrogens or luteinizing hormone of uterine origin is involved in the observed down-regulation of estrogen receptor 1. Several of the genes identified in the current study are known to play a role in early pregnancy in species other than the horse. Thus, products of these commonly expressed genes likely contain universal activities for controlling endometrial receptivity to the conceptus, whereas other factors play unique roles within specific species in ensuring ongoing corpus luteum function. This is the first systematic study of endometrial transcriptome changes in response to the presence of an embryo during maternal recognition of pregnancy and an important step toward deciphering the embryo-maternal dialogue in equids.

Rates of luteolysis and pregnancy in dairy cows after treatment with cloprostenol or dinoprost

Our objective was to determine whether rates of luteolysis or pregnancy differed in lactating dairy cows of known progesterone status and either known or unknown luteal status after either cloprostenol or dinoprost was injected as part of a timed-insemination program. In Experiment 1, 2358 lactating dairy cows in six herds were given two injections of PGF 2α 14 d apart (Presynch), with the second injection given 12 to 14 d before the onset of a timed AI protocol (Ovsynch). Cows (n = 1094) were inseminated when detected in estrus after the Presynch PGF 2α injections. Cows not inseminated (n = 1264) were enrolled in the Ovsynch protocol and assigned randomly to be treated with either cloprostenol or dinoprost as part of the timed-AI protocol. In cows having pretreatment concentrations of progesterone ≥ 1 ng/mL and potentially having a functional corpus luteum (CL) responsive to cloprostenol (n = 558) or dinoprost (n = 519), dinoprost increased (P < 0.05) luteal regression from 86.6 to 91.3%. Despite a significant increase in luteolysis, pregnancies per AI did not differ between luteolytic agents (dinoprost = 37.8% and cloprostenol = 36.7%). Fertility was improved in cows of both treatments having reduced concentrations of progesterone at 72 h and in cows showing signs of estrus. In Experiment 2, an ovulation-resynchronization program was initiated with GnRH or saline in 427 previously inseminated lactating dairy cows of unknown pregnancy status in one herd. Seven days later, pregnancy was diagnosed and nonpregnant cows were blocked by number of CL and assigned randomly to be treated with cloprostenol or dinoprost. Compared with cloprostenol, dinoprost increased (P < 0.05) luteal regression from 69.1 to 78.5%, regardless of the number of CL present or the total luteal volume per cow. Pregnancies per AI did not differ between dinoprost (32.8%) and cloprostenol (31.3%). Although dinoprost was more effective than cloprostenol at inducing luteolysis in lactating dairy cows exposed to an Ovsynch or ovulation-resynchronization protocol, resulting fertility did not differ between products.

Possible Actions of Corticotropin-Releasing Hormone in Regulating Porcine Corpus Luteum Function

The objective of the present study was to determine whether corticotropin-releasing hormone (CRH) influences porcine corpus luteum function. The gene expressions of CRH receptors (CRH-R) were determined in the CLs of Chinese Meishan pigs during the estrous cycle. The effects of CRH on progesterone (P), estradiol-17β (E) and prostaglandin (PG) F(2α) secretion by cultured luteal cells were also investigated. Messenger RNAs of the CRH-R were clearly expressed in the CL throughout the estrous cycle, and the mRNA level was higher at the regressed stage than at the other stages (P<0.05). When the cultured luteal cells obtained from the mid-luteal stage CL (days 8-11) were exposed to CRH (50-5000 ng/ml), P secretion by the cells was significantly reduced at the highest dose (P<0.05), whereas CRH had no effect on E and PGF(2α) secretion by the cells. The overall results suggest that CRH inhibits local P production from luteal cells via its specific CRH-R, implying that CRH plays some roles in regulating porcine CL function during the estrous cycle, especially in the period of luteolysis.

Luteal function induced by transvaginal ultrasonic-guided follicular aspiration in mares

Ultrasonic-guided transvaginal follicle aspiration was performed in 58 crossbreed mares in order to determine whether aspiration of various dominant follicle diameters resulted in luteal tissue capable of producing progesterone (P 4). The mares were randomly assigned to three groups according to follicular diameter (25–29 mm; 30–35 mm and >35 mm). Mares that had ovulations naturally served as controls. The serum progesterone (P 4) concentrations in the aspirated mares were greater ( P < 0.0001; r 2 = 0.6687; CV = 21.52) in mares with natural ovulation compared to mares with aspirated follicles regardless of groups. Serum P 4 concentration in aspired mares with follicular diameter of 25–29 mm declined 0.365 ng/ml/day ( P = 0.0065) from the day of aspiration (D0) up to D8. In mares with follicle diameter of 30–35 mm, serum P 4 concentration increased (0.258 ng/ml/day; P = 0.001), as well as in the mares with follicles >35 mm diameter (0.481 ng/ml/day; P < 0.0001), and in mares with natural ovulation (1.236 ng/ml/day; P < 0.0001). Out of the 25 mares with follicular aspirations that formed Corpora hemorragica (P 4 >1 ng/ml), 23 (92%) had greater (>2 ng/ml) serum P 4 concentrations on Day 8 after aspiration. Of these 23 mares, 75% were in the 25–29 mm group, 9/10 (90%) in the 30–35 mm group, and 11/11 (100%) of the mares in the >35 mm follicular diameter group had luteinization (P 4 >2 ng/ml). These results suggest that a functional Corpus luteum can be induced in mares using follicular aspiration and that a minimum 35 mm follicular diameter is needed to reach a progesterone serum concentration compatible with that of a Corpus luteum produced by natural ovulation.

Changes in the Levels of Progesterone Receptor mRNA and Protein in the Bovine Corpus Luteum During the Estrous Cycle

Progesterone (P4) is synthesized in the luteal cells of many species. The objective of the present study was to determine the expression pattern of P4 receptor (PR) mRNA and the distribution of PR protein in the bovine corpus luteum (CL) during the estrous cycle. The gene expression of PR in the bovine CL throughout the estrous cycle was determined by real-time PCR analysis, and the PR protein expression was evaluated by immunohistochemistry. Messenger RNA of PR was clearly expressed in the CL throughout the estrous cycle. The level of PR mRNA in the CL was highest at the early stage of the estrous cycle and was higher at the mid and late stages than at the regressed stage (P<0.01). In regard to the distribution of PR, the protein was expressed in both small and large luteal cells and in vascular endothelial cells throughout the estrous cycle. These results suggest that P4 has a role in regulating luteal and endothelial cell function in the bovine CL, especially at the early luteal stage as an autocrine/paracrine regulator.

141 TRANSCRIPTIONAL PROFILING OF EQUINE ENDOMETRIUM DURING THE TIME OF MATERNAL RECOGNITION OF PREGNANCY

Early embryonic development, implantation, and maintenance of a pregnancy are critically dependent upon a precisely orchestrated embryo-maternal interaction leading to a receptive uterine environment. The embryo has to signal its presence to the dam in order for estrous cyclicity to subside; however, the horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. To gain new insights into molecular mechanisms underlying this complex process in the horse, transcriptional profiling of Day 13.5 pregnant and cyclic endometrial tissue samples was carried using custom-designed 2-color microarrays. Endometrial tissue samples collected from 4 pregnant and cyclic mares each were labeled with either Cy3 or Cy5, paired, and analyzed on 4 individual arrays. Transcripts showing a difference in signal intensity of at least 1.3 and a P-value of 0.01 or less in a mixed model analysis were considered differentially expressed. Array data were validated performing quantitative RT-PCR and changes in relative gene expression between pregnant and non-pregnant mares were determined using the delta delta cycle threshold method. Proteins of interest were localized using immunohistochemistry. One hundred six transcripts were up-regulated, whereas 47 transcripts showed lower expression levels in pregnant mares. Quantitative RT-PCR for five up- and down-regulated transcripts each confirmed expression data obtained by microarray with a validation rate of 100%. Half of the genes with known or inferred function have previously been described as regulated by estrogens. Given the large quantities of estrogen synthesized by the equine conceptus, this finding confirms validity of the data. Elevated transcript levels were found for genes involved in cell-cell signaling, heat shock response, and secretory proteins among others. Solute carrier 36 member 2, SLC36A2, was the most highly up-regulated gene, reflecting the nutritional needs of the rapidly developing embryo. The beta subunit of luteinizing hormone (LHB) showed higher expression levels in pregnant mares. It is likely that the observed up-regulation of LHB leads to increased release of bioactive LH/CG into the uterine environment during early pregnancy where we hypothesize it to exert paracrine effects preparing the uterus for conceptus implantation. Among the down-regulated genes, estrogen receptor 1 was of particular interest because of its potential involvement in the initiation of luteolysis in cyclic mares. We hypothesize that either embryonic estrogen or uterine-derived choriogonadotropin is involved in the observed down-regulation of estrogen receptor 1, which may be the key to maternal recognition of pregnancy. Several of the genes identified in the current study are known to play a role in early pregnancy in species other than the horse. We thus hypothesize that a subset of genes crucial to endometrial receptivity does not differ between species, representing a common pattern during early pregnancy, whereas each species has a distinct mechanism ensuring ongoing corpus luteum function.

31 PREGNANCY RATE OF CROSSBRED COWS SUBMITTED TO FIXED-TIME AI AND ADMINISTRATION OF GONADOTROPIN-RELEASING HORMONE ANALOGUE TWELVE DAYS AFTER AI

The importance of serum progesterone (P4) during the first weeks of pregnancy to reduce the embryo mortality in cattle herds has been demonstrated (Mann and Lamming 2001 Reproduction 121, 175-180). Studies show that a P4 peak at the beginning of pregnancy (about 5 days after the natural breeding or AI) helps development of the concept via secretion of interferon-τ (Mann 2002 XXII World Buiatrics Congress 18-23, 300-306). Studies have shown that the administration of gonadotropin-releasing hormone (GnRH) or its agonists after AI may stimulate corpus luteum function, induce the formation of an accessory corpus luteum, or increase of P4 serum concentrations and, therefore, improve embryonic survival (Bartolome et al. 2005 Theriogenology 63, 1026-1037). This work aims to evaluate the effect of GnRH injection 12 d after fixed-time AI(FTAI) on P4 serum levels and pregnancy rate of beef crossbred cows. The cows (Bos taurus indicus × Bos taurus taurus, n = 59), range in body condition (BCS) from 2.5 to 3.5 (scale of 1 to 5), were not previously pregnant. The animals were allocated to 2 treatments: TEB treatment (n = 30) consisted of Day 0 insertion of progesterone intravaginal device (ID) plus 2 mg of estradiol benzoate (EB), i.m. followed on Day 8 by removal of the ID and 300 IU of eCG and 0.15 mg of PGF2α, i.m. on Day 9, 1 mg injection of EB (i.m.) was given, followed by FTAI at 48 to 56 h after the withdrawal of ID. The TEBGnRH12 treatment (n = 29) protocol was similar to TEB, but with administration of 25 μg of GnRH analogue (Lecirelina) 12 d after the FTAI. Transrectal ultrasound was used for pregnancy diagnosis on Day 35 after AI. Blood samples for P4 serum assay were collected from 30% of animals in each treatment, at the time of AI and on Days 5, 12, and 20 after FTAI. For all statistical analysis, we used the program SAS 9.0 (2002; SAS Institute Inc., Cary, NC, USA) at 5% probability. The pregnancy rate was analyzed by logistic regression. The effects of treatment and day in P4 concentration were analyzed in a split plot, with the effect of day in plot, in mixed model, considering the error and repetition as a random effect, and comparison of means by Tukey-Kramer. Pregnancy rate and the concentration of serum P4 on different days was analyzed by Spearman correlation. The pregnancy rate for TEB cows was 53.33% (16/30) for the first service compared with 37.93% (11/29) for TEBGnRH12 cows. The protocol used did not affect the pregnancy rate of crossbred cows (P &gt; 0.05). There were no treatment differences (P &gt; 0.05) between the concentrations of P4 (TEB = 3.88 ng mL-1; TEBGnRH12 = 3.12 ng mL-1). In conclusion, the administration of the analogue of GnRH 12 days after FTAI does not affect the rates of pregnancy or the concentration of P4. Supported by grants from CNPq, FAPEMIG and DZO-UFV.

439 OVARY ULTRASOUND EVALUATION OF Santa Inês EWES SUBMITTED TO SHORT DURATION PROTOCOLS WITH PROGESTOGENS ASSOCIATED WITH LECIRELIN AND/OR ESTRADIOL BENZOATE

In order to enhance the use of biotechnology in females it is necessary to have good knowledge of the ovarian events, and thus enable the manipulation of the estrus cycle to concentrate ovulations in an established period of time. In this experiment, we evaluated the ovary response and corpus luteum (CL) parameters in a short-duration protocol associated or not to lecirelin (L)(GnRH) and/or estradiol benzoate (EB). Twenty-four cyclic Santa InÊs ewes were divided into 4 groups: Control animals (G1; n = 6) received 45 μg of d-cloprostenol (Prolise®, Tecnopec, Sao Paulo, Brazil) on Day 0. Intravaginal sponges containing medroxyprogesterone acetate (MAP60®, Tecnopec) were inserted on Day 3. Then on Day 7 sponges were removed and 400 IU of eCG and 45 μg of d-cloprostenol were administered. G2 (n = 6) animals underwent the same protocol as G1 but with administration of 1 mg of EB on Day 1; G3 (n = 6) animals underwent the same protocol as G1 but with administration of 25 μg of L (Gestran plus®, Tecnopec) 30 h after sponge withdrawal; G4 (n = 6) animals underwent the same protocol as G1 but with administration of 1 mg of EB on Day 1 and 25 μg of L 30 h after sponge withdrawal. Ewe ovaries were assessed via transrectal ultrasound (US), and CL were measured 4 days after ovulation. On the same day blood samples were taken to measure plasma progesterone levels via radioimmunoassay. Twenty-three out of 24 animals ovulated. One ewe from G4 that did not ovulate was taken out of the experiment. The average size of the largest pre-ovulatory follicle was 7.3 mm (G1), 7.0 mm (G2), 6.8 mm (G3), and 7.4 mm (G4) (P &gt; 0.05). There were 1.8, 1.2, 2.0, and 1.4 ovulated follicles per animal in G1, G2, G3, and G4, respectively. The results for estrus start and duration after sponge withdrawal were G1: 32/38.6; G2: 37/69.3; G3: 29.3/37.3; G4: 44/68 h, respectively. Ovulation time span was G1: 65.3; G2: 88; G3: 53.3; G4: 82.4 h after sponge withdrawal. There was a high correlation of ovary mensuration and progesterone level (r = 0.64; P &lt; 0.0001) on the fourth day after ovulation. Progesterone levels after ovulation were 2.37 ng mL-1 (G1); 1.5 ng mL-1 (G2); 3.22 ng mL-1 (G3) and 1.99 ng mL-1 (G4), being higher in G3 than in G2 (P &lt; 0.05). It was seen that the EB is prejudicial to the CL function. Despite the fact that there was no significant difference of progesterone levels within G1, G3, and G4, animals in G3 displayed higher levels of progesterone; hence, there is a need for further studies using a larger number of animals and fertility test. CNPq e Tecnopec.

The efficiency of progesterone vaginal gel versus intramuscular progesterone for luteal phase supplementation in gonadotropin-releasing hormone antagonist cycles: a prospective clinical trial

We compared the efficiency of progesterone vaginal gel (PVG) with intramuscular progesterone (IMP) supplementation for luteal phase support after in vitro fertilization and embryo transfer in gonadotropin-releasing hormone (GnRH) antagonist cycles. The treatment outcomes were similar for PVG and IMP for luteal support in GnRH-antagonist protocols. With its ease of use, high tolerability by patients, and fewer side effects, PVG can be a successful alternative to IMP.

Luteal support: Progestogens for pregnancy protection

Following ovulation, the granulosa cells undergo luteinization and form part of the corpus luteum; this then secretes progesterone that causes secretory transformation of the endometrium so that implantation can occur. The ideal time for implantation is 6–10 days after the luteinizing hormone (LH) surge; implantation occurring outside this optimal window is associated with a higher likelihood of miscarriage. Before the placenta takes over progesterone production, the progesterone produced by the corpus luteum also provides the necessary support to early pregnancy. A defect in corpus luteum function is not only associated with implantation failure but also with miscarriage. In assisted reproduction, both the use of gonadotropin-releasing hormone analogues to prevent the LH surge and aspiration of granulosa cells during the oocyte retrieval may impair the ability of the corpus luteum to produce sufficient progesterone. This may be treated effectively with progestational agents such as progesterone or dydrogesterone, which have a very similar pharmacological profile. Studies indicate that an estrogen may be given during the luteal phase to optimise the estrogen:progestogen ratio to facilitate implantation, although the available evidence is inconsistent in its strength for this hypothesis. In addition to assisted reproduction, progestational agents have shown beneficial effects in the management of patients with recurrent spontaneous miscarriage of unknown cause. In conclusion, despite the wide-spread use and many years of clinical experience, the amount of data from well-controlled clinical trials is currently limited. Further studies are therefore required to establish the optimal treatment situation and type and dose of progestational agent.

Equine chorionic gonadotropin improves the efficacy of a progestin-based fixed-time artificial insemination protocol in Nelore ( Bos indicus) heifers

A total of 177 Nelore heifers were examined by ultrasonography to determine the presence or absence of a corpus luteum (CL) and received a 3 mg norgestomet ear implant plus 2 mg of estradiol benzoate i.m. On Day 8, implants were removed and 150 μg of d-cloprostenol i.m. was administered. At the time of norgestomet implant removal, heifers with or without CL at the time of initiating treatment were assigned equally and by replicate to be treated with 0 IU ( n = 87) or 400 IU ( n = 90) eCG i.m. All heifers received 1 mg of EB i.m. on Day 9 and were submitted to fixed-time artificial insemination (FTAI) 30–34 h later. The addition of eCG increased the diameter of the largest follicle (LF) at FTAI (10.6 ± 0.2 mm vs. 9.5 ± 0.2 mm; P = 0.003; mean ± SEM), the final growth rate of the LF (1.14 ± 0.1 mm/day vs. 0.64 ± 0.1 mm/day; P = 0.0009), ovulation rate [94.4% (85/90) vs. 73.6% (64/87); P = 0.0006], the diameter of the CL at Day 15 (15.5 ± 0.3 mm vs. 13.8 ± 0.3 mm; P = 0.0002), serum concentrations of progesterone 5 days after FTAI (6.6 ± 1.0 ng/ml vs. 3.6 ± 0.7 ng/ml; P = 0.0009), and pregnancy per AI [P/AI; 50.0% (45/90) vs. 36.8% (32/87); P = 0.04]. The absence of a CL at the beginning of the treatment negatively influenced the P/AI [30.2% (16/53) vs. 49.2% (61/124); P = 0.01]. Therefore, the presence of a CL (and/or onset of puberty) must be considered in setting up FTAI programs in heifers. In addition, eCG may be an important tool for the enhancement of follicular growth, ovulation, size and function of the subsequent CL, and pregnancy rates in progestin-based FTAI protocols in Bos indicus heifers.

Molecular Mechanisms Associated with Conceptus-Endometrium Interactions During the Peri-Implantation Period in Ruminants

Abstract: In mammals, the establishment of pregnancy is dependent upon coordinated biochemical signaling and physical interactions between the developing conceptus and uterine endometrium. These essential forms of communication between the conceptus and its maternal environment result in continued production of progesterone from the corpus luteum (CL) and the initiation of implantation/placentation. During the peri-implantation period, conceptuses in ruminant ungulates secrete interferon-tau (IFNT), which acts on uterine endometrium and attenuates endometrial production of the luteolysin, prostaglandin F2α, resulting in the maintenance of CL function. Expression of the ovine IFNT (olFNT) genes is restricted to the mononuclear cells of the trophoblast and the protein is produced for only a relatively short and discrete window of time during early pregnancy. This review deals with identification, characterization and regulation of IFNT gene transcription, and uterine responses associated with pregnancy esta...

Size of ovulatory follicles in cattle expressing multiple ovulations naturally and its influence on corpus luteum development and fertility12

Long-term genetic selection of cattle for fraternal twins has increased the frequency of twin and triplet ovulations. In contrast, the ratio of fetal numbers to ovulation sites in pregnant females with twin (0.83) or triplet (0.73) ovulations is <1.0 and the number of calves per parturition is 1.6 and 2.0, respectively. Failure of individual twin or triplet ovulations to yield a conceptus in fertile females indicates a significant contribution of ovulation or oocyte anomalies to increased fertilization failure or early embryonic mortality. The present objective was to identify physiological traits affecting conception in cyclic cattle expressing multiple ovulations naturally, including the effect of ovulation rate on follicle or corpus luteum (CL) size, and their relationship to conception. Diameter of the individual ovulatory follicles was measured by transrectal ultrasonography at AI and ranged from 8 to 30 mm, with a trend for diameter of the individual follicles, and associated CL, to decrease with increasing ovulation rate. Independent of ovulation rate, ovulatory follicles were smaller (P < 0.05) for nulliparous heifers (1.5 yr) compared with parous cows (> or =2.5 yr). Pregnancy and fetal status were diagnosed by transrectal ultrasonography between 42 and 72 d after AI. Fertility was reduced (P < 0.01) for small twin or triplet ovulatory follicles (8 to 8.9 mm vs. 10 to 17.9 mm diam.), whereas fertility in monovular females was reduced (P < 0.01) for large ovulatory follicles (> or =22 vs. 14 to 17.9 mm). Plasma progesterone concentrations increased with ovulation rate and were correlated positively with total CL or ovulatory follicle volume per female, indicating that CL size and function were influenced by the size of the follicle of origin. Progesterone was greater (P < 0.05) in the blood of nulliparous heifers compared with parous cows. The increased proportion of small ovulatory follicles associated with twin and triplet ovulations indicates that some ovulatory follicles were either selected to ovulate at a lesser stage of maturity or rescued while undergoing atresia, thus compromising oocyte competency or ovulation. Of greatest importance for reduced fertility was the greater incidence of pregnancy losses occurring in the middle of gestation in females gestating 2 or more fetuses as an apparent effect of uterine crowding, especially when 2 or more fetuses were contained within 1 uterine horn.

Ovarian endocrine profile and long-term vascular patency following heterotopic autotransplantation of cryopreserved whole ovine ovaries

This study examined the ability of cryopreserved whole ovine ovaries to resume function in vivo following autotransplantation. Swaledale ewes had their left ovaries removed and either perfused but not cryopreserved (n = 4; control), or perfused and cryopreserved (n = 8; cryopreserved) before autotransplantation sub-cutaneously to the neck by microvascular anastomosis. Right ovaries were removed and fixed as non-grafted controls. Weekly jugular venous blood samples were analysed for plasma FSH, LH, inhibin A and progesterone levels, grafts were scanned transdermally and oestrus was detected. Vascular patency was assessed post-mortem and follicle populations were measured in recovered tissue. Immediate vascular patency was achieved in all ewes and maintained in 7/8 cryopreserved and 3/4 control grafts. Functional corpora lutea were identified in three ewes (one control; two cryopreserved) 18-25 weeks after grafting. Inhibin A levels indicated resumption of follicular development in four cryopreserved and one control ewes, however, castrate gonadotrophin levels persisted in five cryopreserved and two control ewes. Primordial follicle density was reduced following grafting in both cryopreserved and non-frozen ovaries (P < 0.001). In conclusion, these results demonstrate successful partial restoration of ovarian function following cryopreservation of the whole ovary and vascular pedicle in a large monovulatory species. The inability to restore full ovarian function was related to loss of primordial follicles rather than vascular patency in both frozen and fresh tissue, suggesting that factors associated with cannulation and perfusion may contribute to this depletion. Further work is therefore needed to elucidate these factors before the procedure could be considered a viable option for fertility preservation.

The effect of a single midcycle administration of 0.5 or 2.0 mg dienogest (17 alpha-cyanomethyl-17 beta-hydroxy-estra-4,9-dien-3-one) on pituitary and ovarian function--investigation for the use as a postcoital contraceptive.

Estradiol-17 beta, progesterone, LH and FSH levels in plasma were measured simultaneously by radioimmunoassay and BBT was recorded in order to investigate the effect of a single midcycle oral dose of 0.5 or 2.0 mg dienogest (17 alpha-cyanomethyl-17 beta-hydroxy-estra-4,9-dien-3-one, VEB Jenapharm, Jena/GDR) on pituitary and ovarian function in 18 healthy fertile females. After application of 0.5 mg dienogest in the follicular phase the cycles appeared to be anovulatory. Administration of 2.0 mg two days prior to the expected LH-surge produced a delay of the LH-peak combined with an absence of ovulation as well as the absence of the normal subsequent increase of progesterone. With 0.5 mg the delay of the LH-surge was followed by an ovulation with normal corpus luteum function. The application of 0.5 or 2.0 mg one day before or during the rising LH-peak lowered the LH-surge but ovulation and luteal phase were not altered. Administration of both dosages during the LH-peak could neither prevent ovulation nor disturb corpus luteum function. Postovulatory ingestion of 0.5 or 2.0 mg dienogest during the BBT-rise produced no alteration of the further cycle. These results demonstrate that dienogest in a single-dose-administration in midcycle can alter pituitary and ovarian function depending on the time interval between application and the day of LH-surge.