The effect of equine metabolic syndrome on the ovarian follicular environment.

Obesity in many species is associated with reduced fertility and increased risk of metabolic disorders and cardiovascular dysfunction in offspring. Equine metabolic syndrome (EMS) is associated with obesity and characterized by insulin resistance, decreased adiponectin, and elevated insulin, leptin, and pro-inflammatory cytokines. These alterations can potentially disrupt follicular development and impair fertility. We hypothesized that mares with EMS have an altered follicular environment when compared to their normal counterparts, affecting gene regulation for follicle and oocyte maturation. Samples were collected from light-horse mares (11 to 27 yr) in a clinical assisted reproductive program. Mares were screened based on phenotype. Insulin sensitivity was determined by using two proxies, the reciprocal of the square root of insulin (RISQI) and the modified insulin-to-glucose ratio (MIRG). Insulin resistant mares (RISQI < 0.32 and MIRG > 5.50) were allocated to the EMS group (n = 8), and the remaining mares were considered normal controls (CON, n = 12). Follicular fluid (FF) and granulosa cells (GC) from preovulatory follicles were aspirated 24 ± 2 h after administration of a GnRH analog (SucroMate, 0.9 to 1.4 mg, i.m.) and hCG (Chorion, 1500 to 2000 IU, i.v.). After an overnight fast, blood was collected on the morning of follicle aspiration to evaluate serum concentrations of insulin, leptin, adiponectin, and inflammatory cytokines. Expression of 32 genes related to metabolism, follicle maturation, and oocyte maturation were assessed in GC. Concentrations of insulin, leptin, adiponectin, and cytokines were highly correlated between serum and FF (P < 0.001). Insulin was lower (P < 0.001) in serum and FF of CON compared to EMS, but leptin and IL1ß tended (P = 0.07 and P = 0.10, respectively) to be lower in FF of CON than EMS. Tumor necrosis factor-α in serum and FF was lower (P < 0.07 and P < 0.05, respectively) in CON than EMS. Conversely, adiponectin was higher (P < 0.05) in serum and FF in CON versus EMS. In GC from CON when compared to EMS, gene expression for epiregulin was elevated (P < 0.05) and tissue inhibitor of matrix metalloproteinase-2 tended to be lower (P = 0.09). Our findings demonstrate that the intrafollicular environment in the mare is influenced by metabolic disease, consistent with findings in other species. Influences on follicular development, oocyte maturation, and subsequent offspring by perturbations due to metabolic disease need further study.

VURD Syndrome in a Female.

VURD syndrome has been repeatedly described as unilateral reflux into a nonfunctioning renal moiety. This syndrome is considered a pop-off mechanism dissipating pressure in lower urinary tract obstruction: it may be found in association with other protective mechanisms occurring in utero, such as ascites and/or urinomas, and has been exclusively described in male patients. A premature female baby with signs and symptoms of outflow obstruction underwent diagnostic workup revealing congenital urethral hypoplasia with unilateral reflux into a dysplastic kidney. Obstetrical history was positive for early onset, serologically negative ascites without cardiomegaly, which required serial aspirations. Reconstructive surgery was carried out with good results: ascites and VURD syndrome were both deemed to be perinatal protective mechanism against excess pressure in the urinary tract. Although rare, lower urinary tract obstruction in the female can lead to the same protective mechanisms seen in male fetuses/newborns. VURD syndrome and ascites should be interpreted as such and require perinatal specialist counselling.

Temporal gene expression in equine corpora lutea based on serial biopsies in vivo.

A biopsy procedure was developed to enable repeated sampling of a single equine corpus luteum (CL) over the course of an estrous cycle. The tissue collected was utilized in characterizing mRNA abundance for genes involved in luteal formation, function, and regression in the cyclic mare. Serial biopsies of CL in cyclic mares (2.7 to 27.5 mg per biopsy) were collected using an ultrasound-guided transvaginal technique. Biopsies were collected from each mare on d 2 and 5 (d 0 = ovulation) of the estrous cycle, and every other day from d 12 through luteolysis. Samples were obtained from 4 mares with normal estrous cycles and 1 mare with a retained CL. The biopsy procedure did not adversely affect luteal size or function, as measured by luteal area and serum concentrations of progesterone. Real-time reverse-transcription PCR was used to quantify steady state mRNA concentrations in each tissue sample obtained. Mean abundance of steroidogenic acute regulatory protein (StAR) mRNA was not different (P = 0.102 to 0.964) on any of the sampling dates, but a trend for mRNA encoding StAR to decrease between d 12 and 14 (P = 0.10) was observed. Values for mRNA encoding StAR were positively correlated to serum concentrations of progesterone on d 5 (R = 0.95; P = 0.05) and 14 (R > 0.99; P < 0.01). Steady-state abundance of mRNA for 3ß-hydroxysteroid dehydrogenase, Δ 5-Δ 4 isomerase (3ß-HSD) declined between d 12 and 14 (P = 0.15). There were positive correlations between mRNA for 3ß-HSD and concentrations of progesterone on d 5 (R = 0.94; P = 0.06) and 12 (R > 0.99; P = 0.05). No difference was detected in abundance of mRNA encoding cyclooxygenase-2 (cox-2; P = 0.340 to 0.840) or caspase-3 (P = 0.517 to 0.882) between any of the sampling dates. A successful luteal biopsy procedure was developed that did not negatively affect luteal function, and abundance of mRNA encoding StAR, 3ß-HSD, cox-2, and caspase-3 was characterized in luteal biopsy tissue collected on d 2, 5, 12, and 14 of the estrous cycle in the mare.

Potential involvement of EGF-like growth factors and phosphodiesterases in initiation of equine oocyte maturation.

Human chorionic gonadotropin (hCG) was administered to mares in estrus with large, dominant ovarian follicles to initiate follicular and oocyte maturation. Follicular contents were collected at 0, 2, 4 and 6 h after hCG. Epiregulin, amphiregulin and phosphodiesterase (PDE) mRNA contents of granulosa cells (PDE 4D) were determined by reverse transcription and real-time PCR; PDE 3A mRNA content of single oocytes was determined similarly. Copy numbers of mRNA did not increase for PDE 3A or 4D over the time interval studied. Amounts of epiregulin and amphiregulin mRNA were correlated (r=0.98) when log transformed. Epiregulin and amphiregulin mRNA increased (P<0.01) from controls by 4 h after hCG administration, with amphiregulin increasing (P<0.01) by 2 h after hCG administration. Epiregulin and amphiregulin mRNA levels remained elevated (P<0.01) at 6h after hCG. These results indicate that EGF-like growth factors are likely paracrine mediators of the LH signal in the horse.

Removal of deslorelin (Ovuplant) implant 48 h after administration results in normal interovulatory intervals in mares.

Deslorelin implants, approved for use in inducing ovulation in mares, have been associated with prolonged interovulatory intervals in some mares. Administration of prostaglandins in the diestrous period, following a deslorelin-induced ovulation, has been reported to increase the incidence of delayed ovulations. The goals of the present study were: (1) to determine the percentage of mares given deslorelin that experience delayed ovulations with or without subsequent prostaglandin treatment, and (2) to determine if removal of the implant 48 h after administration would effect the interval to subsequent ovulation. We considered interovulatory intervals to be prolonged if they were greater than the mean +/- 2 standard deviation (S.D.) of the control group in study 1 and the hCG group in study 2. In study 1, we retrospectively reviewed reproduction records for 278 mares. We either allowed the mare to ovulate spontaneously or induced ovulation using deslorelin acetate implants or hCG. We administered prostaglandin intramuscularly, 5-9 days after ovulation in selected mares in each group. A higher percentage of mares which were induced to ovulate with deslorelin and given prostaglandins had a prolonged interovulatory interval (23.5%; n = 16), as compared to deslorelin-treated mares that did not receive prostaglandins (11.1%; n = 5). In study 2, we induced ovulation in mares with hCG (n = 47), a subcutaneous deslorelin implant via an implanting device provided by the manufacturer (n = 28), or a deslorelin implant via an incision in the neck (n = 43) and we removed the implant 48 h after administration. We administered prostaglandin to all mares 5-9 days after ovulation. In study 2, mares from which the implant was removed had a normal ovulation rate and none had a prolonged interval to ovulation. Administration of prostaglandin after deslorelin treatment was associated with a longer interval from luteolysis to ovulation than that found in mares not treated with deslorelin. Prostaglandin administration during diestrus may have exacerbated the increased interval to ovulation in deslorelin-treated mares. We hypothesize that prolonged secretion of deslorelin from the implant was responsible for the extended interovulatory intervals.

Deslorelin acetate (Ovuplant) therapy in cycling mares: effect of implant removal on FSH secretion and ovarian function.

Following induction of ovulation with deslorelin acetate (Ovuplant), gonadotrophin concentrations are reduced in the subsequent cycle, leading to increased interovulatory intervals in some mares. This study determined whether implant removal after 2 days prevented the decrease in gonadotrophin concentrations and follicular growth during the ensuing cycle. Twenty-four mares were randomised equally into 3 groups. Group 1 ovulated spontaneously, Groups 2 and 3 received the deslorelin implant to induce ovulation. Two days after treatment, the implant was removed from Group 3. On Day 10 postovulation, FSH was lower (P = 0.009) in Group 2, but not different between Groups 1 and 3. Follicular diameter on Day 14 was less (P<0.05) in Group 2 (19.0 +/- 2.1 mm) than in Groups 1 and 3 (36.6 +/- 2.5 and 30.5 +/- 2.0 mm, respectively). Interovulatory interval was longer (P<0.05) for Group 2 (25.8 +/- 2.9 days) compared to Groups 1 and 3 (18.5 +/- 0.7 and 19.4 +/- 0.3 days, respectively). Removal of the deslorelin implant eliminated the decreased FSH secretion and the increased interovulatory interval associated with implant administration. Therefore, it is recommended that the implant be removed after ovulation is detected to prevent the occurrence of a prolonged interovulatory interval.

Use of oocyte transfer in a commercial breeding program for mares with reproductive abnormalities.

In some mares with lesions of the reproductive tract, embryo collection and survival rates are low, or collection of embryos is not feasible. For these mares, oocyte transfer has been proposed as a method to induce pregnancies. In this report, a method for oocyte transfer in mares and results of oocyte transfer performed over 2 breeding seasons, using mares with long histories of subfertility and various reproductive lesions, are described. Human chorionic gonadotropin or an implant containing a gonadotropin-releasing hormone analog was used to initiate follicular and oocyte maturation. Oocytes were collected by means of transvaginal ultrasound-guided follicular aspiration. Following follicular aspiration, cumulus oocyte complexes were evaluated for cumulus expansion and signs of atresia; immature oocytes were cultured in vitro to allow maturation. The recipient's ovary and uterine tube (oviduct) were exposed through a flank laparotomy with the horse standing, and the oocyte was slowly deposited within the oviduct. Oocyte transfer was attempted in 38 mares between 9 and 30 years old during 2 successive breeding seasons. All mares had a history of reproductive failure while in breeding and embryo transfer programs. Twenty pregnancies were induced. Fourteen of the pregnant mares delivered live foals. Results suggest that oocyte transfer can be a successful method for inducing pregnancy in subfertile mares in a commercial setting.

Use of specific sugars to inhibit bacterial adherence to equine endometrium in vitro.

OBJECTIVE: To determine whether specific sugars inhibit adhesion of Streptococcus zooepidemicus, Pseudomonas aeruginosa, and Escherichia coli to equine endometrial epithelial cells in vitro. SAMPLE POPULATION: Endometrial biopsy specimens collected during estrus from 7 healthy mares. PROCEDURE: Endometrial specimens on glass slides were incubated for 30 minutes at 4 C with suspensions of S. zooepidemicus, P. aeruginosa, or E. coli in phosphate-buffered saline solution (PBSS) alone or with various concentrations of D-(+)-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-(+)-glucose, galactose, or N-acetyl-neuraminic acid. Inhibition of bacterial adherence was determined by comparing adhesion of bacteria (i.e., percentage of glandular epithelial cells with adherent bacteria) suspended in each sugar solution with that of bacteria suspended in PBSS. RESULTS: Mannose and N-acetyl-D-galactosamine inhibited adhesion of E. coli and P. aeruginosa to epithelial cells, whereas only mannose inhibited adhesion of S. zooepidemicus. The other sugars did not affect bacterial adherence. CONCLUSIONS AND CLINICAL RELEVANCE: Mannose and N-acetyl-D-galactosamine appear to play a role in adhesion of S. zooepidemicus, P. aeruginosa, and E. coli to equine endometrium. In horses with uterine infections, use of sugars to competitively displace bacteria from attachment sites on cells may provide an adjunct to antibiotic treatment.

Uncoupling of the equine reproductive axes during transition into anoestrus.

The reproductive activity of light horse mares (n=12) was monitored each day from 3 September until 29 January, or until the mares entered anoestrus, by behaviour evaluation, ultrasonography and blood sampling. Follicles, corpora lutea and ovulation, as well as oestradiol, progesterone, and LH and FSH concentrations, were analysed to determine a reproductive profile for the transition into anoestrus. The results of the present study indicate that light horse mares progress through four phases during the autumn transition into anoestrus: (i) normal cycles; (ii) aberrant cycles; (iii) anovulation, with significant follicular activity; and (iv) anoestrus. One of the first changes observed was a progressive decrease in mean progesterone concentrations during normal cycles (summer: 7.1 +/- 0.4 ng ml(-1); last cycle: 3.6 +/- 0.2). Regression analysis indicates that large follicles progressively lose their ability to produce oestradiol in autumn (third last cycle: 8.5 +/- 1.3 pg ml(-1); last cycle: 5.7 +/- 0.5). During the last ovulatory surge, LH concentrations decreased approximately 50% in 6 of 8 mares. The duration of the follicular phase increased with each cycle approaching anovulation. However, the diameters of the follicles ovulated did not differ. The duration of the luteal phase during ovulatory cycles did not change. Four of the 12 mares developed spontaneously prolonged corpora lutea and were eliminated from the analysis. An anovulatory follicular growth phase occurred immediately before anoestrus in 7 of 8 mares. FSH preceded follicular growth during all cycles and persisted throughout the anovulatory period. During anoestrus, plasma oestradiol, progesterone and LH concentrations remained at basal concentrations. FSH concentrations remained high in 3 of 8 mares but did not cause follicular growth. It is concluded that ovarian and pituitary events become uncoupled during the transition into anoestrus.

Vulvo-vaginitis and reproduction.

The main micro-organisms able to interfere with the reproductive function have been considered. In particular, the problems concerning the vaginal environment and its interactions with spermatozoa, immunological aspects and contraception. Lastly, reference has been made to methods of prevention and study in the field of diagnostics and of clinical management.

Determination of a new collagen type I alpha 2 gene point mutation which causes a Gly640 Cys substitution in osteogenesis imperfecta and prenatal diagnosis by DNA hybridisation.

The molecular defect responsible for a sporadic case of extremely severe (type II/III) osteogenesis imperfecta was investigated. The mutation site was localised in the collagen type I pro alpha 2 mRNA molecules produced by the proband's skin fibroblasts by chemical cleavage of mismatch in heteroduplex nucleic acids. Reverse transcription-polymerase chain reaction DNA amplification, followed by cloning and sequencing, showed heterozygosity for a G to T transversion in the first nucleotide of exon 37 of the COL1A2 gene, which led to a cysteine for glycine substitution at position 640 of the triple helical domain. This newly characterised mutation is localised in a domain which contains several milder mutations, confirming that glycine substitutions within the alpha 2(I) chain do not follow a linear gradient pattern for genotype to phenotype correlations. In a subsequent pregnancy, absence of the G2327T mutation in the fetus was shown by allele specific oligonucleotide hybridisation to the trophoblast derived fibroblast mRNA after reverse transcription and in vitro amplification. (The nucleotide number assigned to the mutant base was inferred from the numbering system devised by the Osteogenesis Imperfecta Analysis Consortium (The OIAC Newsletter, 1 April 1994).)

Post-operative ileo-ileal intussusception: sonographic approach.

Two children developed ileo-ileal intussusception a few days after major abdominal surgery. Sonographic study was of primary importance in the evaluation of this lesion which was apparent from clinical and radiographic signs of intestinal obstruction but not from barium enema.