Corpus Hemorrhagicum Research Articles

A retrospective analysis of sheep generated by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) is one of the primary methods for production of genetically engineered sheep, which allows for gene editing or transgene introduction in somatic cells. The use of SCNT eliminates the risk of genetic mosaicism in embryos and animals that is commonly observed after zygote micromanipulations. This retrospective analysis of SCNT in sheep performed at Utah State University, spanning from 2016 to 2021, examined parameters that may impact pregnancy and full-term development, including donor oocytes (donor age), donor cell lines, SCNT parameters (time of oocyte activation following SCNT, number of transferred embryos, in vitro maturation and culture conditions), and recipients (surgical number and ovulatory status), as well as factors that may correlate with large offspring syndrome or abnormal offspring syndrome (LOS/AOS) in the fetuses and lambs. Our findings indicated that compared to prepubertal oocytes, the SCNT embryos produced from adult sheep oocytes had comparable in vitro maturation rates, pregnancy and full-term development rates, as well as SCNT efficiency. In addition, earlier activation time of SCNT embryos (e.g. 24–26 h post maturation) was correlated to the early pregnancy loss rate, full-term rate, and SCNT efficiency. Compared to our standard serum-containing medium, commercial serum-free culture medium showed a positive correlation with the full-term development of sheep SCNT embryos. Transferring 15–30 embryos per recipient resulted in consistently good pregnancy rates. Surgical numbers and ovulatory status (having at least one follicle between 6 and 12 mm in size or a corpus hemorrhagicum (CH)) of recipients did not affect pregnancy and full-term development rates. In summary, this retrospective analysis identified parameters for improving pregnancy and full-term development of SCNT embryos in sheep.

Macroscopic morphological analysis of different stages of Cyclic Corpus Luteum in Indian buffalo (Bubalus bubalis)

The current study was undertaken with the aim to characterize the macroscopic morphological and morphometrical features of cyclic corpus luteum (CL; n=40) and CL of pregnancy (n=10) in buffalo. The four stages of cyclic CL were interpreted after ovarian analysis i.e., c olor, consistency, vasculature of CL, number and size of follicles into early (Stage I, 1 to 5 days, n=10), mid (Stage II, 6 to 11 days, n=10), late luteal phase (Stage III, 12 to 16 days, n=10) and follicular phase (Stage IV, 17 to 20 days, n=10). In Stage I, it was slightly protruded from the surface of ovary, bloody in appearance due to increased blood congestion, soft in consistency and termed as corpus haemorrhagicum. In Stage II, initially CL was bright red in color, later fleshy in color and soft in consistency. In Stage III, it was shrunken to great extent and pale yellow to creamish in color due to reduced vascularity. At Stage IV it was shrunken and rigid; texture became firmer, completely condensed into small whitish in color due to complete loss of vascularity. It varied in size and weight as well during the varying stages of estrus cycle depicting changes in its morphology. Therefore, by recording the macroscopic observations on cyclic CL and CL of pregnancy, it was further characterized into different stages.

Seasonal influence on the luteal growth and regression characteristics of crossbred dairy cows of Kerala

The study was aimed at investigating the effect of seasonal changes on luteal growth and regression characteristics in crossbred (CB) cows of Kerala. The seasons of the year were classified as rainy (June-September), post-monsoon (October-January) and summer (February- May). In each season, nine healthy regularly cycling CB lactating pluriparous cows were selected for the study. Luteal growth and regression pattern were studied at 24 h intervals from one ovulation to the next ovulation, by transrectal ultrasound (TRUS) examination of the ovaries. During the study period, parameters like the day of earliest detection of corpus hemorrhagicum (CH), size at detection, maximum size of corpus luteum (CL), the day of CL with maximum diameter, day of onset and completion of CL regression were recorded and compared between seasons. Corpus hemorrhagicum was detected one day after ovulation in 55.60, 66.70 and 80 per cent of animals during rainy, post-monsoon and summer seasons, respectively. There existed no statistically significant difference in the size of CL, either on the day of first detection or on the day of maximum size attainment. However, CL during the rainy season needed a significantly (P<0.05) longer period to attain maximum size (10.33 ± 0.58), compared to the post-monsoon (8.33 ± 0.62) and summer seasons (8.40 ± 0.56). The season did not affect the day of onset of luteal regression or its completion neither in animals with two-wave cycle nor with three- wave. The study concluded that the season had less or no impact on the growth and regression pattern of CL in CB cattle of Kerala.

PSXIV-14 Selenium and Copper Deficiency Associated Decline in Reproductive Performance in Summer Bred, Rotationally Grazed, Forage fed Beef Cattle

Abstract This case study reports on selenium and copper deficiency associated decline in reproductive performance in summer bred, rotationally grazed, forage fed beef cattle at the Berea College Educational Farm over a 6-year period (2016 to 2021). The Angus-Hereford crossbred breeding herd (N=45/year) received a significantly lower selenium and copper free fed mineral supplement between 2014 and early 2021 compared with what was provided beginning in mid-2021, in response to veterinary diagnosed selenium and copper deficiency. Supplement was also provided intramuscularly January 20, 2022. Cattle liver biopsies (n=5) and postmortem necropsies (n=3) were performed to determine a mineral panel. Annually, ultrasound guided pregnancy diagnosis and BCS were conducted each fall after a 60-day summer breeding season. Moderate BCS (avg=5.4) were maintained during the first 4 years, including 2021 (5.8) where visibly noticeable weight loss was evident in primiparous heifers during summer. Declining annual pregnancy rates from 2016 (y = -0.0153x + 0.08731) also declined (9.32%) to 73.68% (Fig. 1) in 2021. Manual palpation (n=12) during the Fall 2021 pregnancy examinations and dissection (n=2) of the reproductive tracts of nulliparous heifers 104 days later determined abnormal morphology. In Fig. 2, a cystic corpus hemorrhagicum (1.7 x 1.4 cm) and bilateral oviduct aplasia was discovered in one heifer. In the other heifer, a cystic corpus luteum (0.7 x 0.6 cm), initially detected at the pregnancy examination was discovered (Fig. 3), which resulted in a 53% weight difference between the right and left ovary (6.1, 4.0 g). Mineral panels taken at the end of 2021 confirmed a selenium and copper deficiency with the liver biopsy averaging 2.38 ppm Cu (Table 1). Hence, annual declines in reproductive performance of summer bred, rotationally grazed, forage finished beef cattle at Berea College Educational Farm was associated with selenium and copper deficiency.

Regulation of cellular communication network factor 1 by Ras homolog family member A in bovine steroidogenic luteal cells.

Development of the corpus luteum (CL) requires the growth of a new capillary network from preexisting vasculature, a process known as angiogenesis. Successful building of this capillary network occurs through a sequence of cellular events-differentiation, proliferation, migration, and adhesion-which are regulated by a suite of angiogenic proteins that includes cellular communication network factor 1 (CCN1). We previously reported that the expression of CCN1 was highest in luteal tissue obtained from the early-cycle, 4-d-old bovine CL (i.e., corpus hemorrhagicum) compared to the mid- and late-cycle CL. In the present study, we treated steroidogenic bovine luteal cells from early-cycle CL with luteinizing hormone (LH), but it had no effect on CCN1 expression. Direct stimulation of the canonical LH pathway with forskolin and dibutyryl-cyclic adenosine monophosphate (cAMP), however, inhibited CCN1 mRNA expression. In endothelial cells, stimulation of Ras homolog family member A (RhoA) induces CCN1 expression, whereas RhoA inactivation inhibits it. Yet, it is unknown if regulation of CCN1 in steroidogenic luteal cells works likewise. We hypothesized that a similar mechanism of CCN1 regulation exists in bovine luteal cells and that thrombin, a known RhoA activator, may be a physiologic trigger for this mechanism in the early-cycle CL. To test this hypothesis, ovaries were collected from lactating dairy cows on days 3 or 4 of the estrous cycle, and corpora lutea were dissected and dissociated. Steroidogenic luteal cells were suspended in defined Ham's F12 medium, supplemented with insulin/transferrin/selenium and gentamicin, and seeded into 6-well plates. After 24 h, spent medium was replaced with fresh Ham's F12, and the cells were cultured for 24 to 48 h. Cells were treated for 2 h with defined medium, 10% fetal bovine serum (FBS), thrombin (1, 5, 10 U/mL), or Rho Activator II (0.25, 1, 2 μg/mL). Cells were then lysed for RNA extraction, followed by cDNA generation, and quantitative polymerase chain reaction (qPCR). Thrombin (1, 5, 10 U/mL; n = 3) and Rho Activator II (0.25, 1, 2 μg/mL; n = 6) increased (P < 0.05) CCN1 mRNA expression. In summary, CCN1 in bovine steroidogenic luteal cells was induced by thrombin and appeared to be regulated in a Rho-dependent manner. Future work will elucidate the signaling partners downstream of Rho which leads to CCN1 gene expression.

Dynamic changes in mitochondrial 3D structure during folliculogenesis and luteal formation in the goat large luteal cell lineage

In mammalian ovaries, mitochondria are integral sites of energy production and steroidogenesis. While shifts in cellular activities and steroidogenesis are well characterized during the differentiation of large luteal cells in folliculogenesis and luteal formation, mitochondrial dynamics during this process have not been previously evaluated. In this study, we collected ovaries containing primordial follicles, mature follicles, corpus hemorrhagicum, or corpus luteum from goats at specific times in the estrous cycle. Enzyme histochemistry, ultrastructural observations, and 3D structural analysis of serial sections of mitochondria revealed that branched mitochondrial networks were predominant in follicles, while spherical and tubular mitochondria were typical in large luteal cells. Furthermore, the average mitochondrial diameter and volume increased from folliculogenesis to luteal formation. In primordial follicles, the signals of cytochrome c oxidase and ATP synthase were undetectable in most cells, and the large luteal cells from the corpus hemorrhagicum also showed low enzyme signals and content when compared with granulosa cells in mature follicles or large luteal cells from the corpus luteum. Our findings suggest that the mitochondrial enlargement could be an event during folliculogenesis and luteal formation, while the modulation of mitochondrial morphology and respiratory enzyme expressions may be related to tissue remodeling during luteal formation.

Scanning electron microscopic study of ovary and oviduct of crossbred dairy cows with ovarian hypoplasia

Hypoplasia of ovary is one of the major causes of infertility in dairy cattle, which is characterized by absence of oestrus cycle, affecting livestock productivity and economics to a great extent. This study was conducted on the female genitalia collected from 100 dairy cows/heifers from the Meat Technology Unit, Mannuthy which included six animals culled on account of factors other than infertility with normal genitalia (control group) and remaining animals with a known history of infertility. Two animals of 22 months and 24 months of age showed bilateral ovarian hypoplasia. The history revealed that the animals were healthy, but had not shown oestrus. Grossly ovaries of the first animal appeared as pink-coloured, small, wrinkled, flattened, elongated structures without any follicles or CL. Second animal showed inactive, small, flat, streak-like left ovary without any cyclical structures. But the right ovary showed a single large corpus haemorrhagicum on the caudal end. Then ovarian tissue was fixed in 2 percent gluteraldehyde in 0.1 M phosphate buffer at pH 7.3. Under scanning electron microscopy, the ovary exhibited an uneven surface with clefts and grooves. The surface cells lost their normal appearance without any microvilli. Large round smooth germ cells were also observed on the ovarian surface. In the oviduct, the mucosa was lined by nonciliated cells having rounded surface interspersed among shrunken cells. The epithelial cell surface was covered with bulbous processes. Ciliated cells were not observed in the present study. Ovarian abnormality is reported to be the main cause of altered morphology of the surface epithelial cells and change in the ultrastructure of oviductal mucosa.

86 Ultrasonographic evaluation of preovulatory follicle and endometrial echo texture for ovulation prediction in Marwari mares

The objective of the present study was to evaluate the application of ultrasonographic changes in the preovulatory follicle and uterus for ovulation prediction in Marwari mares. Mares (n=20) were teased daily by a teaser pony and, from the day of first detected oestrus, the ovaries and uterus were examined by transrectal ultrasonography using Exago ECM ultrasound equipped with linear rectal probe (10MHz) until ovulation. Recorded data were analysed by one-way ANOVA and correlation coefficients. The duration of the oestrus period ranged from 5 to 7 days in all mares, with an average of 6.35±0.13 days, consistent with rectal findings. The last day of oestrus was marked by disappearance of the preovulatory follicle and presence of a corpus haemorrhagicum. The mean diameter of the follicles increased significantly (P&amp;lt;0.01) from Day 1 to Day 7, and the diameter of follicles before ovulation was 44.88±0.46 mm; follicles changed from round to oval or irregular before ovulation. In 65% (13/20) of the mares, ovulation occurred on the left ovary. The daily mean growth rate of size of ovulating follicles from the first day of oestrus until ovulation was 3.51±0.42 mm/d. The mean average growth rate of follicles was significantly different (P ≤ 0.01) on different days of oestrus. The mean endometrial echo texture score was 1.30±0.10 on Day 1, 2.10±0.16 on Day 2, 3.20±0.15 on Day 3, and highest 3.85±0.08 on Day 4, declining thereafter to reach a score of 1.36±0.11 on Day 7 of the oestrous cycle. The mean slope values of pixel intensity for the granulosa and anechoic layers of the preovulatory follicles determined by Image-Pro software (Media Cybernetics) from Days −3 to −1 was 13.8±0.7 on Day −3, 15.7±0.9 on Day −2, and 20.1±1.3 on Day −1. None of the follicles ovulated before granulosa layer echogenicity reached a score of ≥2.5 and prominence of anechoic layer reached a score of ≥2. Significant positive correlations were found between the cross-sectional diameter of the uterine horn and estrual endometrial echotexture score (P&amp;lt;0.01; r=0.95), estrual endometrial echo texture score and preovulatory follicle diameter (P&amp;lt;0.01; r=0.25), and preovulatory follicle diameter and cross-sectional diameter of uterine horn (P&amp;lt;0.01; r=0.36) during the oestrous cycle. We conclude that various ultrasonographic characteristics of preovulatory follicle and uterus can be used to predict ovulation in Marwari mares.

10 Segmental cervical aplasia in a mare with mosaic X-chromosome aneuploidy

Segmental cervical aplasia is a congenital Müllerian abnormality characterised by the complete or partial failure of cervical development resulting from abnormal fusion of the Müllerian ducts to the urogenital sinus. In the present case report, we describe a congenital segmental cervical aplasia in a Colombian creole mare. The mare was presented to the Animal Reproduction Clinic of Universidad Nacional de Colombia for diagnosis because the external orifice of her cervix was not detected when a uterine lavage was attempted as a therapy for detectable uterine fluid accumulation. The mare had a history of 20-day oestrous cycles confirmed by receptivity to a mature stallion and had no history of natural service or artificial insemination. A complete breeding soundness evaluation of the mare including transrectal palpation, ultrasonography, vaginoscopy, endoscopy, transvaginal aspiration of the uterine fluid and cytogenetic analysis, and an oestrous cycle follow-up were performed. Clinical and ultrasonographic evaluation of the genital tract revealed normal-size ovaries with structures suggestive of regular ovarian activity. Ovulation was confirmed by the formation of a corpus haemorrhagicum followed by a mature corpus luteum in diestrus. In addition, granular free-floating fluid material distending the uterus was detected. Upon vaginal examination, the organ ended in a blind bag with a small papilla with no evident external os cervix. Cytology of the uterine fluid obtained by transvaginal aspiration showed predominant neutrophils with diplococcus bacteria and inflammatory cells compatible with inflammatory content. Cytogenetic analysis of 134 metaphase lymphocytes showed that the mare had an abnormal karyotype [64,XX]/[63,XO]/[65,XXX] with a ratio of 45%, 45%, and 10%, respectively. G- and C-banded analysis was conducted for the X chromosome. Mosaicism of the X chromosome was diagnosed, and the observed congenital segmental cervical aplasia was proposed as the clinical consequence of the mosaicism detected. To our knowledge, this is the first case of this reproductive pathology in a Colombian mare with regular ovarian activity and X chromosome aneuploidy in mosaic form. The cause of the persistently contaminated uterine content in this mare was not clear; it is possible that via the systemic or transcervical route, bacterial contamination could have colonized the uterus, resulting in chronic inflammation and fluid accumulation. This case report demonstrates the importance of performing an adequate routine gynaecological examination in mares to determine their reproductive health. In most cases, the diagnosis of congenital pathologies of the cervix is an incidental finding when performing breeding programs or therapeutic strategies for managing uterine inflammatory conditions. Furthermore, cytogenetic analysis is an important complementary tool for clinical reproductive examination, to accurately identify causes of congenital malformations, and to determine additional causes of reproductive failure in mares.

Hypothalamic Astrocyte Development and Physiology for Neuroprogesterone Induction of the Luteinizing Hormone Surge.

Neural circuits in female rats sequentially exposed to estradiol and progesterone underlie so-called estrogen positive feedback that induce the surge release of pituitary luteinizing hormone (LH) leading to ovulation and luteinization of the corpus hemorrhagicum. It is now well-established that gonadotropin releasing hormone (GnRH) neurons express neither the reproductively critical estrogen receptor-α (ERα) nor classical progesterone receptor (PGR). Estradiol from developing ovarian follicles acts on ERα-expressing kisspeptin neurons in the rostral periventricular region of the third ventricle (RP3V) to induce PGR expression, and kisspeptin release. Circulating estradiol levels that induce positive feedback also induce neuroprogesterone (neuroP) synthesis in hypothalamic astrocytes. This local neuroP acts on kisspeptin neurons that express PGR to augment kisspeptin expression and release needed to stimulate GnRH release, triggering the LH surge. In vitro and in vivo studies demonstrate that neuroP signaling in kisspeptin neurons occurs through membrane PGR activation of Src family kinase (Src). This signaling cascade has been also implicated in PGR signaling in the arcuate nucleus of the hypothalamus, suggesting that Src may be a common mode of membrane PGR signaling. Sexual maturation requires that signaling between neuroP synthesizing astrocytes, kisspeptin and GnRH neurons be established. Prior to puberty, estradiol does not facilitate the synthesis of neuroP in hypothalamic astrocytes. During pubertal development, levels of membrane ERα increase in astrocytes coincident with an increase of PKA phosphorylation needed for neuroP synthesis. Currently, it is not clear whether these developmental changes occur in existing astrocytes or are due to a new population of astrocytes born during puberty. However, strong evidence suggests that it is the former. Blocking new cell addition during puberty attenuates the LH surge. Together these results demonstrate the importance of pubertal maturation involving hypothalamic astrocytes, estradiol-induced neuroP synthesis and membrane-initiated progesterone signaling for the CNS control of ovulation and reproduction.

A preliminary study of the heterogeneity in endometrial morphology and glycosylation in the uterine horns of the non-pregnant impala (Aepyceros melampus)

Placentation commences only in the right uterine horn in impala (Aepyceros melampus). To investigate possible differences in morphology or glycosylation between the two horns, right and left uterine horns from six non-pregnant, wild impala were examined morphometrically and histochemically using a panel of 23 lectins and an avidin-biotin revealing system. The presence of ovarian 3ß hydroxysteroid dehydrogenase (3ßHSD) and aromatase was also investigated using immunocytochemistry. There were few detectable differences in morphology and glycosylation between right and left uterine horns in five of the specimens, but the sixth had deep clefts and plentiful exocrine secretions in the right horn, and not the left. Heavily glycosylated clusters of supranuclear granules were present in the epithelial cells, which had many classes of O-linked glycans. The serum progestagen was not markedly different, however, from that of the other specimens. In five of the six specimens, the height of luminal epithelium was greater on the right than that on the left, and the height of the gland epithelium was also greater on the right side in four of these. The 3ßHSD and aromatase activities were present in the ovaries and were similar in impala with or without progestagen concentrations >1 ng/ml in peripheral blood. No corpus haemorrhagicum or corpus luteum could be discerned. These findings indicate there are morphological and biochemical differences between right and left uterine horns in the impala and further studies are needed on both impala and other species in which placentation commences only in one uterine horn, to establish the cyclical hormone changes which induce them.

Immunohistochemical localization of progesterone receptors alpha (PRA) in ovary of the pseudopregnant rabbit.

Progesterone plays an important role in the reproductive function and follicular development in mammals. The aim of the present study was to examine the localization of progesterone receptor alpha (PRA) in ovary of pseudopregnant rabbit by immunohistochemical methods. Samples were collected from 14 h. to 18 days of pseudopregnancy. At the first stage of pseudopregnancy (14 h.), the rabbit ovary showed moderate immunostaining of PRA in the granulosa cells and theca interna cells of preovulatory follicle and in the stroma cells. At the middle stage of pseudopregnancy (3-7 days), the rabbit ovary showed strong immunostaining of PRA in ovarian surface epithelial cells, follicular cells of the primary follicle, granulosa cells and theca interna cells of the growing and antral follicles. Moderate immunoexpression of PRA were observed in the large lutein cells and endothelial cells of the corpus haemorrhagicum and corpus luteum and in the stroma cells. At the end of pseudopregnancy (18 days) strong PRA reactions were detected in the small lutein cells of the regressed corpus luteum. Moderate to strong PRA immuno-expression were observed in the proliferated theca interna cells of the atretic antral follicles. The atretic large lutein cells of the regressed corpus luteum showed negative immunostaining for PRA. This study showed that the PRA positive small lutein cells of the regressed corpus luteum and the PRA positive proliferated theca interna cells of the atretic antral follicles were transformed into PRA positive interstitial gland cells. In conclusion, the present study had described the distribution of PRA in the ovary of pseudopregnant rabbit, which is not discussed before in the available literature. It also gives more information about follicular dynamic, formation and origin of interstitial glands, mechanism of ovulation, formation and regression of the corpus luteum.

165 In vitro maturation of ovine and caprine oocytes during breeding and nonbreeding seasons

The current study investigated the in vitro meiotic competence of ovine and caprine oocytes that underwent nuclear maturation during the breeding and nonbreeding seasons. We hypothesised that maturation rates of ovine and caprine oocyte would be significantly lower during the nonbreeding season. Ovine (Katahdin crossbred) and caprine (mainly Spanish crossbred) ovaries were collected from a local abattoir in the southern United States. Age of the animals was not determined. Cumulus-oocyte complexes (COC) were harvested by slicing the ovaries and searching using a stereomicroscope. Oocytes with more than 3 layers of unexpanded cumulus cells and with evenly granulated cytoplasm were selected for in vitro maturation (IVM). A commercial bovine IVM media (IVF Bioscience, Falmouth, United Kingdom) was used throughout the study. After 24h of IVM, ovine and caprine oocytes were denuded and oocytes with an extruded polar body (meiotic metaphase II oocytes) were considered to have reached nuclear maturation. The seasons in this study were defined as follows: breeding season=September to April and nonbreeding season=May to July. The presence of corpus hemorrhagicum or corpus luteum in at least 70% of the ovaries indicated the breeding season for the animals. Proportions of oocytes undergoing nuclear maturation were analysed using a two-tailed Chi-squared test. Statistical significance was set at P ≤ 0.05. The ovine maturation rate was 59% (65/111) and 49% (254/519) and the caprine maturation rate was 70% (39/56) and 40% (64/162) during the breeding and nonbreeding seasons, respectively. These results show a significant difference in nuclear maturation for caprine oocytes (P&amp;lt;0.001) during the breeding and nonbreeding seasons; however, there was no significant difference in nuclear maturation for ovine oocytes (P=0.06) during the breeding and nonbreeding seasons. High environmental temperatures during the nonbreeding season may have had detrimental effects on oocyte nuclear maturation in caprine but not in ovine oocytes. Why oocytes from these 2 species differ on how they are adversely affected by season remains to be elucidated.

Characterizing estrus by trans-abdominal ultrasounds, fecal estrone-3-glucuronide, and vaginal cytology in the Steller sea lion (Eumetopias jubatus)

The ability to monitor the estrus cycle in wild and captive marine species is important for identifying reproductive failures, ensuring a successful breeding program, and monitoring animal welfare. Minimally invasive sampling methods to monitor estrus in captive populations have been developed, but results suggest these tools can be species-specific in their precision and accuracy. Therefore, the minimally invasive sampling methods of trans-abdominal ultrasounds, a fecal steroid analysis (estrone-3-glucuronide, E1G), and vaginal cytology, were evaluated for their efficacy to characterize and monitor estrus in a captive breeding population of Steller sea lions (Eumetopias jubatus). Three adult females were sampled over five breeding seasons, resulting in six estrus profiles characterized by trans-abdominal ultrasounds, five by fecal E1G, and four by vaginal cytology. Animals were trained to allow trans-abdominal ultrasounds, fecal samples, and vaginal swabs to be collected approximately daily. Of the 76 trans-abdominal ultrasound sessions attempted, 8 successfully visualized both ovaries. From these scans, the chronology of ovarian changes during proestrus and estrus was estimated. The time from the detection of developing follicles to the identification of a dominate follicle occurred in 2–5 days and a corpus hemorrhagicum formed approximately 4 days later. However, because visualization of the ovaries was prevented by the gastrointestinal system in 88% of scans, this tool was overall unreliable for monitoring changes associated with estrus. To detect fine scale physiological changes associated with estrus, we analyzed changes in fecal E1G (n = 62) and vaginal cytology (n = 157) 15 days before and after each female's single copulation event (Day = 0). Changes in fecal E1G had the highest accuracy at detecting Day = 0. Fecal E1G increased leading up to estrus, peaked at Day = 0, and then declined. Although we did observe the characteristic increase in superficial cells associated with impending estrus, the type of cell which peaked closest to Day = 0 was intermediate. The uncertainty around the peak in intermediate cells, indicating estrus, was greater than the uncertainty associated with detecting estrus from fecal E1G. Collectively, these results suggest that changes in fecal E1G and vaginal cytology are viable tools to detect estrus in Steller sea lions, but require daily sampling to detect gradual changes, limiting their applicability to studies of wild populations.

161 EFFECT OF OVIDUCTAL FLUID ON BOVINE OOCYTE ZONA PELLUCIDA HARDENING AND SPERM BINDING, AND ON EARLY EMBRYO DEVELOPMENT

This study was designed to evaluate the effects of oviductal fluid at different periovulatory times on oocyte maturation, modification of the zona pellucida (ZP), fertilization and embryo development. Bovine oviducts were collected at a slaughterhouse and classified as preovulatory (pre-ov: 1 pre-ov follicle and a regressing corpus luteum) or post-ovulatory (post-ov: a corpus haemorrhagicum or recent corpus luteum; n = 10 cows/stage). Both oviducts were flushed with 1 mL of sterile TCM-199, and oviductal flushes (OF) were aliquoted and stored at –80°C. Abattoir-derived bovine ovaries were aspirated and cumulus‐oocyte complexes (COC) with at least 3 cumulus layers and homogeneous oocyte cytoplasm were in vitro matured for 22 h in standard maturation medium (control group, n = 319) or in standard medium with 2× concentrated additives supplemented (50% v/v) with pre-ov OF (n = 255) or post-ov OF (n = 248). After in vitro maturation (IVM), subgroups of COC were denuded, and the time of digestion of the ZP by pronase 0.1% (v/v in TCM-199) was determined to evaluate ZP hardening. After IVM, COC were fertilised in vitro for 18–20 h at a final concentration of 1.106 million spermatozoa (spz)/mL. After in vitro fertilization (IVF), COC were denuded, washed twice and cultured for 8 days more under standard conditions. After IVM, IVF, and embryo culture, oocytes/embryos were fixed with ethanol, stained with Hoescht, and examined under fluorescence microscopy for determination of (1) maturation and developmental stages, (2) numbers of fertilised and polyspermic oocytes, and (3) spz bound to the ZP. Percentages were compared between groups by chi-square. Times of ZP digestion were compared by Kruskal‐Wallis test. Numbers of spz bound to the ZP were compared by ANOVA on normalised data followed by Newman-Keuls tests. Data are presented as mean ± SEM. A P &lt; 0.05 was considered significant. Addition of OF during IVM had no effect on maturation rates compared with the control. However, the digestion time of the ZP by pronase was reduced after IVM with pre-ov OF (313 ± 21 s; n = 26) compared with post-ov OF (459 ± 23 s; n = 23) but not with the control (416 ± 30 s; n = 25). After IVF, the number of spermatozoa bound to the ZP was increased after IVM with pre-ov OF (57 ± 5 spz/oocyte; n = 67) and decreased after IVM with post-ov OF (34 ± 3 spz/oocyte; n = 76) compared with the control (42 ± 5 spz/oocyte; n = 60). Addition of OF during IVM had no effect on rates of IVF and polyspermia. However, the rate of development to the blastocyst stage was less after IVM with post-ov OF (10%, n = 97 cleaved oocytes) compared with control (24%, n = 130) and pre-ov OF (29%, n = 101). In conclusion, the OF collected before ovulation decreased the resistance of the ZP to protease digestion and increased its ability to bind spz, whereas it was the opposite for the post-ov OF. Furthermore, the post-ov OF decreased the developmental competence of fertilised oocytes.

Sequential ovulation and fertility of polyoestrus in American black bears (Ursus americanus).

American black bears (Ursus americanus) are seasonally polyoestrous and exhibit delayed implantation, which may allow equal and independent fertility of recurrent oestruses of a mating season. We postulated that the luteal inactivity during delayed implantation allows bears to have sequential ovulation during a polyoestrous mating season such that each oestrus of a polyoestrous female will have equivalent fertility, and pregnancy would not preclude subsequent ovulation and superfetation. Controlled mating experiments were conducted on semi-free-ranging female American black bears during three mating seasons, wherein females were bred by different male cohorts in each oestrus. Behavioural observation, vulva score ranking, genetic paternity analysis, gross morphology of ovaries and microscopic morphology of diapaused embryos were used to evaluate the fertility of each subsequent oestrus in polyoestrous females. Oestrus duration, number of successful mounts and median vulva scores were similar between first and subsequent oestruses of the season. Polyoestrus occurred in 81.3% of oestrous females, with a 9.7 ± 5.5 day (mean ± SD) inter-oestrous interval. Sequential ovulation was documented in three polyoestrous females, including one that possessed both a corpus haemorrhagicum and a developed corpus luteum. Among polyoestrous dams, four of nine embryos were conceived in the first oestrus and five of nine in the second oestrus. These results indicate that each oestrus of polyoestrous females is capable of fertility, even if the female is already pregnant from a prior oestrus. Although superfetation was not directly observed in the present study, our results strongly suggest the potential of superfetation in the American black bear and provide novel insight into the complex behavioural and physiological breeding mechanisms of bears. Given that most endangered bear species share similar reproductive traits with American black bears, captive breeding programmes could take advantage of superfetation by mating females with different males at each subsequent oestrus of the season in order to increase the genetic diversity of captive endangered bears.

Noncitrated blood transfusions used as adjunctive treatment in a 7‐year‐oldShetlandPony with haemoperitoneum due to a rupturedcorpus haemorrhagicum

SummaryChronic bleeding due to extensive haemorrhage from a rupturedcorpus haemorrhagicumdeveloped 2–4 weeks after parturition and was identified as the cause for a haemoperitoneum in a 7‐year‐old Shetland Pony mare, leading to anaemia. Diagnosis was made upon exploratory coeliotomy and a unilateral ovariectomy was performed. Intra‐ and post operatively, the mare received autologous blood transfusions of noncitrated blood. To our knowledge, this is the first report of an autologous blood transfusion accomplished using noncitrated blood collected from the abdominal cavity.

Acute right lower abdominal pain in women of reproductive age: Clinical clues

To study possible gynecological organ pathologies in the differential diagnosis of acute right lower abdominal pain in patients of reproductive age. Following Clinical Trials Ethical Committee approval, the retrospective data consisting of physical examination and laboratory findings in 290 patients with sudden onset right lower abdominal pain who used the emergency surgery service between April 2009 and September 2013, and underwent surgery and general anesthesia with a diagnosis of acute appendicitis were collated. Total data on 290 patients were obtained. Two hundred and twenty-four (77.2%) patients had acute appendicitis, whereas 29 (10%) had perforated appendicitis and 37 (12.8%) had gynecological organ pathologies. Of the latter, 21 (7.2%) had ovarian cyst rupture, 12 (4.2%) had corpus hemorrhagicum cyst rupture and 4 (1.4%) had adnexal torsion. Defense, Rovsing's sign, increased body temperature and increased leukocyte count were found to be statistically significant in the differential diagnosis of acute appendicitis and gynecological organ pathologies. Gynecological pathologies in women of reproductive age are misleading in the diagnosis of acute appendicitis.

Ovary-sparing surgery for teratomas in children

To share our experience in ovary-sparing surgery for teratomas in children. The medical records of nine patients (mean age of 11.2 years, r 6-15 years) who had undergone ovary-sparing surgery for teratoma were analyzed retrospectively. Mean duration for follow-up was 29.5 months (r 15-75 months). Five patients suffered from chronic abdominal pain; two had acute colicky abdominal pain. In two patients, there was no presenting clinical symptom. Two patients were operated on emergency basis due to symptoms related with acute abdomen. On the other hand, seven were operated electively. Ultrasonography was performed in all patients. Additionally, MRI and tumor markers were performed in all but two. Main radiologic findings consisted of heterogenous cystic and solid ovarian masses predictive of teratoma. The definitive diagnosis in emergency cases were as follows: perforated appendicitis plus teratoma [1]; torsion of the ovarian mass with teratoma [1]. The final diagnosis in electively treated seven patients were: unilateral ovarian teratoma [4], bilateral ovarian teratoma [1], bilateral teratoma plus appendiceal inflammatory mass [1], unilateral teratoma and contralateral corpus hemorrhagicum cyst [1]. The operations were performed by open conventional surgery in six and laparoscopy in three patients. The procedures were ovary-sparing surgery in 12 ovaries, appendectomy in 2 patients and detorsion of ovary in 1 patient. The main indication for ovary-sparing surgery was the "existence of a perfect dissection plane between the tumor margins and healthy ovarian tissue". The remaining ovarian tissue was evaluated macroscopically for residual lesions. Frozen section was performed in three suspected patients and the ovarian margins were free of any tumor cell. The pathologic diagnosis was: mature cystic teratoma in 10, immature teratoma in 1 and corpus hemorrhagicum cyst in 1. The postoperative outcome and follow-up was uneventful. Heterogenous ovary mass containing solid and cystic portions with echogenic areas on ultrasound imaging is highly suggestive of ovarian teratomas. Emergent surgical intervention is indicated if there is any suspicion of ovarian torsion. Otherwise, MRI is performed for further radiological evaluation. Based on radiologic findings, ovary-sparing surgery can be safely performed if the preoperative diagnosis is teratoma and there is always a plane of dissection between the normal ovary and cyst wall.

Interferon stimulated gene 15 (ISG15): Molecular characterization and expression profile in endometrium of buffalo (Bubalus bubalis)

Interferon stimulated gene 15 (ISG15), one of the several proteins induced by conceptus derived Type I and/or a Type II interferon (IFN), is implicated as an important factor in determining the uterine receptivity and conceptus development. However, presence as well as specific role of the ISG15 in buffalo (Bubalus bubalis) reproduction is yet to be elucidated. In the present study, both genomic and cDNA sequences of bubaline (bu) ISG15 were cloned and investigated for its expression in different tissues of female reproductive tract of buffalo. Sequence analysis revealed 100% identity among the genomic sequences (1014bp) of buISG15 from three different breeds of buffalo (viz., Murrah: Acc. No. DQ118137, Mehsana: Acc. No. DQ118138, and Nagpuri: Acc. No. DQ118136) and cDNAs (Acc. Nos. HM543268–HM543270HM543268HM543269HM543270). As in cattle, the buISG15 was comprised of two exons of 57bp and 520bp encoding a peptide of 154 amino acids. Moreover, the buISG15 cDNA sequence exhibited 98.3% and 98.5% identity with that of taurine and indicine cattle, respectively. Subsequent reverse transcription PCR analysis revealed expression of the buISG15 in the uterine endometrium, corpus luteum (CL), corpus hemorrhagicum and oviduct. Quantitative Real Time PCR (RTqPCR) analysis also confirmed the constitutive expression of the buISG15 in the uterine endometrium during different stages (i.e. estrus, diestrus and proestrus) of estrous cycle and also during early (∼d 30–40) pregnancy. Western blot analysis of the endometrial extract from both estrous cyclic as well as pregnant buffalo demonstrated the presence of only conjugated ISG15 which was >40kDa. ISG15 mRNA and immune-reactive proteins were localized in the stromal as well as glandular epithelial cells of the uterine endometrium of estrous cyclic as well as pregnant buffalo. However, there was no significant difference in amount of ISG15 mRNA across the different reproductive phases. To conclude, this study will be helpful for the further understanding of the roles of the ISG15 in pregnancy of buffalo cows.