Uterine Flushings Research Articles

Progesterone and interferon tau-regulated genes in the ovine uterine endometrium: identification of periostin as a potential mediator of conceptus elongation

During early pregnancy in ruminants, progesterone (P(4)) and interferon tau (IFNT) act on the endometrium to regulate genes hypothesized to be important for conceptus development and implantation. The present study was conducted to verify several candidate genes (actin alpha-2, smooth muscle, aorta (ACTA2), collagen, type III, alpha-1 (COL3A1), periostin (POSTN), secreted protein acidic cysteine-rich (SPARC), S100 calcium-binding protein A2 (S100A2), STAT5A and transgelin (TAGLN)) regulated by pregnancy, P(4), and/or IFNT in the endometrium determined using a custom ovine cDNA array. S100A2 mRNA was detected primarily in endometrial epithelia and conceptuses. S100A2 mRNA increased in endometrial epithelia from days 10 to 16 in cyclic ewes and from days 10 to 14 in pregnant ewes and declined thereafter. The abundance of S100A2 mRNA was less in endometrial luminal epithelium of IFNT-infused ewes receiving P(4). Expression of COL3A1, SPARC, ACTA2, and TAGLN was independent of pregnancy, P(4), or IFNT. POSTN mRNA was detected primarily in compact stroma of intercaruncular and caruncular endometria, but not in the conceptus. Endometrial POSTN mRNA increased between days 12 and 14 in pregnant but not cyclic ewes, and POSTN mRNA was more abundant in uterine stroma of ewes treated with P(4). POSTN protein was detected in uterine flushings of pregnant ewes and found to mediate attachment and stimulate migration of ovine trophectoderm cells in vitro. These results support the ideas that POSTN and S100A2 are regulated by P(4) and IFNT respectively, and that POSTN is involved in conceptus elongation during early pregnancy.

Glucose and Glucose Transporters in Pig Uteri and Conceptuses.

Glucose, the main energy source for mammalian embryos during the peri-implantation period, may promote growth and development of conceptuses via the FRAP1 nutrient sensing cell signaling pathway. Therefore, mechanisms for glucose transport into the uterine lumen and uptake by conceptuses are essential. We reported significant increases in glucose in the uterine lumen during early pregnancy in sheep associated with increases in expression of both facilitative and sodium-dependent glucose transporters in the uterine endometrium that were regulated by pregnancy, progesterone (P4) and interferon tau (IFNT). However, little information is available on glucose transporters in uteri and conceptuses in other domestic animals including pigs. Experiment 1 determined effects of day of the estrous cycle and pregnancy on total recoverable glucose in uterine flushings from gilts on Days 5, 9, 12 and 15 of the estrous cycle (Cy) and Days 9, 10, 12, 13, 14 and 15 of pregnancy (Px) (n=4). Total recoverable glucose increased (P<0.05) with day of the Cy and Px. Experiment 2 determined recoverable glucose in uterine flushings of gilts ovariectomized on Day 12 and treated daily with either corn oil (OVX-CO; n=4) or 200 mg progesterone (OVX-P4; n=5) to Day 39 and hysterectomized on Day 40. Recoverable glucose in uterine flushings (mean + SEM; nmol) was greater for OVX-P4 than OVX-CO gilts (4,955+2534 vs 726+133), but this difference was not significant due to variability and the small number of gilts. Experiment 3 determined amounts of glucose in uterine flushings of gilts on Day 90 of pseudopregnancy (PP) that was induced by treatment with 5 mg/day estradiol benzoate on Days 11 to 15 after onset of estrus. The flushings (109+24 ml) contained significant amounts (nmol) of glucose (14,007+3,946). Uterine and conceptus tissues collected from experiments 1-3 were examined by in situ hybridization for expression of Facilitated Glucose Transporters SLC2A1, SLC2A2, SLC2A3 and SLC2A4. Results indicated that: 1) SLC2A3 mRNA was not detectable or very weak in conceptuses and endometria, respectively; 2) SLC2A1 mRNA was expressed by all cell types in conceptuses and was abundant in uterine luminal epithelia (LE) of Cy, Px, PP and OVX-P4 gilts; 3) SLC2A4 mRNA was moderately abundant in LE of Px, but not Cy gilts, only between Days 13 and 25 of Px and LE and GE of OVX-P4 gilts, and not detected in LE or GE of PP gilts; 4) SLC2A2 mRNA was abundant in conceptuses from Days 12 to 40 of pregnancy, decreased to Day 50 and then increased and was maintained specifically in placental areolae and basal crypts of the chorioallantois to Day 80 of gestation. SL2CA2 was also expressed in uterine LE of Px and PP gilts and LE and GE of OVX-P4 gilts. Results of these studies indicate that: 1) the availability of glucose increased in the uterine lumen of Cy and Px gilts between Days 9 to 15; 2) glucose was abundant in uterine flushings from PP and OVX-P4 gilts; 3) temporal and cell specific changes in abundance of mRNAs for specific glucose transporters in uterine LE and GE, as well as chorioallantois likely affect abundance of glucose in the uterus and conceptus; and 4) SLC2A1 expression in uterine endometrium was stimulated by P4. Thus, facilitative glucose transporters responsible for transport of glucose into the uterine lumen of pigs occurs primarily when the uterus is under the influence of progesterone and are available for transport of glucose to the conceptus. (poster)

Evidence for Estrogen-Dependent Uterine Serpin (SERPINA14) Expression During Estrus in the Bovine Endometrial Glandular Epithelium and Lumen1

Uterine secretions have a dominant impact on the environment in which embryo development takes place. The uterine serpins (SERPINA14, previously known as UTMP) are found most abundantly during pregnancy in the uterus of ruminants. Although progesterone is currently assumed to be the major regulator of SERPINA14 expression, our recent study of transcriptome changes in bovine endometrium during the estrous cycle unexpectedly detected a marked upregulation of SERPINA14 mRNA levels at estrus. The present study describes the full-length mRNA sequence, genomic organization, and putative promoter elements of the SERPINA14 gene. The SERPINA14 mRNA abundance was quantified by real-time RT-PCR in intercaruncular endometrium at several time points during the estrous cycle and early pregnancy. Highest levels were found at estrus, followed by a dramatic decrease and a moderate expression during the luteal phase. Transcript levels were higher in pregnant endometrium compared with controls at Day 18. At estrus, immunoreactive protein was localized in deep glandular epithelium, and Western blotting concomitantly showed the 52-kDa form in uterine flushings. SERPINA14 mRNA was significantly upregulated in glandular endometrial cells in vitro after stimulation with estradiol-17beta and progesterone, but not after interferon-tau treatment. Our results clearly demonstrate that SERPINA14 appears distinctly in bovine endometrium during the estrus phase. A supporting role toward providing a well-prepared endometrial environment for passing gametes, especially sperm, is assumed.

Immunisation of the equine uterus against Streptococcus equi subspecies zooepidemicus using an intranasal attenuated Salmonella vector

Attenuated Salmonella enterica serovar Typhimurium MGN707, expressing the SzP protective protein of the MB9 serovar of Streptococcus equi subspecies zooepidemicus (SzP-MB9) was tested for its safety and efficacy as a nebulised intranasal vaccine against streptococcal uterine infections in mares. In a preliminary study, vaccinated mares (n=5) displayed serum, nasal and uterine responses (P<0.05) to S. Typhimurium lipopolysaccharide (St-LPS). Subsequently, vaccinated mares (expressor group, n=7), but not mares vaccinated with the vector only (control group, n=7), displayed significant increases in SzP-MB9 antibodies in serum, nasal and uterine washes (P<0.05). Assuming the uteri of all nine mares were free of streptococci prior to challenge with 6.3 x 10(9) colony forming units of S. e. zooepidemicus MB9, significantly fewer S. e. zooepidemicus were cultured from the uterine flushings of expressor-vaccinated mares (n=4) compared to control-vaccinated mares (n=5) (P<0.001). The only adverse reaction to vaccination was nasal haemorrhage in one mare.

Effect of the conceptus on uterine prostaglandin-F2α and prostaglandin-E2 release and synthesis during the periimplantation period in the pig

The present study was conducted to evaluate the effect of the conceptus on uterine prostaglandin-F2alpha (PGF2alpha) and prostaglandin-E2 (PGE2) release and the expression of prostaglandin synthase enzymes during the periimplantation period in the pig. A surgically generated model with conceptuses developing in only one of the uterine horns was created. The highest concentration of PGF2alpha and PGE2 was found in the gravid uterine horn, compared with the non-gravid horn and the intact horn of cyclic gilts. Endometrial concentration of both PGs in pregnant gilts was elevated regardless of the conceptus in the uterine horn, whereas only myometrial PGE2 concentration increased during pregnancy. Expression of prostaglandin-E2 synthase (mPGES-1) mRNA in the endometrium was upregulated during the oestrous cycle, while protein expression presented a similar pattern to that of PGE2 concentration in the uterine flushings. Prostaglandin-F2alpha synthase (PGFS) mRNA and protein expression in the endometrium did not differ between pregnancy and oestrous cycle but PGFS mRNA in the myometrium increased during pregnancy both in the gravid and the non-gravid uterine horns. We suggest a local effect of the conceptus on PG release pathways but also a more systemic effect within the whole uterus with regard to PG synthesis and accumulation in uterine tissues.

Immunohistochemical and histochemical identification of proteins and carbohydrates in the equine endometrium: Reaction patterns in the cycling mare

Little is known about the stage of estrus cycle-dependent variations of uterine secretions of the more and most published results were based on evaluation of uterine flushings. Hence, the aim of this study was to define patterns of expression of different endometrial proteins throughout the estrus cycle using newly established immunohistochemical methods and endometrial biopsies. Therefore, endometrial biopsies were collected from three mares on defined days of the estrus cycle with known concentrations of estradiol and progesterone in serum. The proteins uteroglobin, uteroferrin, calbindin(D9k) and uterocalin were examined using polyclonal antibodies. Furthermore glycogen was identified using the PAS-reaction prior to and after a-amylase digestion. During the estrus cycle expression of the examined proteins, as well as glycogen, revealed a typical, progesterone-dependent pattern. In the early- and mid-interestrus there is a simultaneous increase in expression of uterocalin and glycogen staining intensity associated with increasing concentrations of progesterone in peripheral blood. The maximal expression of calbindin(D9k) was also in association with high levels of circulating progesterone in mid-interestrus. In contrast, secretion of both uteroglobin and uteroferrin was simultaneously associated with the decrease in circulating concentrations of progesterone in mid-to late-interestrus. However, it has to be emphasized that distinct among mare differences were obvious. This new method enables pathologists to interpret changes in the uterine secretion pattern associated with fertility reducing diseases by using a single endometrial biopsy

137 DIFFERENCES IN EMBRYO PRODUCTION BETWEEN LACTATING AND NON-LACTATING HOLSTEIN DONOR COWS

Embryo transfer is a common practice in reproductive management of large dairy herds. Data from 2 dairies in the San Joaquin Valley in California (5000 lactating Holstein producing 27 500 pounds of milk in 365 days) was used to evaluate the differences in embryo production between lactating (105.3 ± 24.6 pounds of milk/day, 161 ± 85 days in milk and 3 ± 1 lactations) and nonlactating donor cows, from July 2006 to June 2007. Cows were observed daily for estrous signs and superovulation treatment was initiated 10 d later. Cows received FSH twice a day during 4 days in decreasing doses. On the fifth treatment of FSH, cows received an injection of PGF2α and 48 h later an injection of GnRH. Cows were inseminated 12 and 24 h after the GnRH injection. Uterine flush was performed 7 d after the first AI only in cows that had 2 or more CL. The total number of ova/embryos, viable embryos (transferred fresh or frozen), degenerated, and unfertilized structures were recorded (classified according to the guidelines of IETS). A Bernoulli regression model was fitted using the R software (www.r-project.org) for the variable superovulation response and the covariates were cow condition (lactating or nonlactating), season, and interactions. From 719 superovulated cows, 668 were flushed (92.9%): 86.8% (309/356) of the lactating cows and 98.9% (359/363) of the nonlactating cows (P &lt; 0.01). There was evidence of season effect (P = 0.09). Cows tended to respond less to superovulation treatment during summer and fall. A negative binomial regression model was fitted to the response variables total number of ova/embryo and viable embryos and the covariates were cow condition, season, and interactions. Lactating cows produced fewer (6.77) ova/embryos than nonlactating cows (14.14; P &lt; 0.01). The adjusted means were 5.93 v. 12.53 for fall, 6.30 v. 13.32 for winter, 8.24 v. 17.40 for spring and 6.32 v. 13.34 for summer, for lactating and nonlactating cows, respectively. Cows produced more ova/embryos during spring (P &lt; 0.01). Less viable embryos were recovered from lactating cows (3.62) than nonlactating cows (5.44; P &lt; 0.01). The adjusted means were 2.52 v. 3.98 for fall, 3.61 v. 5.69 for winter, 4.51 v. 7.12 for spring and 3.37 v. 5.31 for summer, for lactating and non lactating cows, respectively. The production of viable embryos in fall was different from winter and spring (P &lt; 0.01). Lactating cows responded less to superovulation treatment and produced less viable embryos than nonlactating cows, probably because of the increased metabolism due to milk production. Warmer seasons were associated with lower viable embryo production. It is important to have a group of nonlactating donors to increase embryo production in large ET programs.

134 VIABILITY OF EQUINE BLASTOCYSTS SUBJECTED TO BIOPSY FOR PRE-IMPLANTATION GENETIC DIAGNOSIS

Pre-implantation genetic diagnosis of embryos is commonly performed in humans but has not been developed in the horse. To our knowledge, only one previous report of transfer of equine embryos after biopsy is available (Huhtinen et al. 1997 Theriogenology 48, 361); in that study, three pregnancies were achieved after transfer of 14 embryos. Failure of pregnancy after biopsy of equine embryos has been attributed to difficulty in penetrating the equine embryonic capsule, and possible effects of capsule damage on subsequent embryo survival. Recent developments in intracytoplasmic sperm injection (ICSI) and embryo culture make it possible to produce equine blastocysts in vitro. In vitro-produced equine embryos lack a capsule, but form one when transferred to the uterus. This study was designed to examine the viability of equine blastocysts subjected to biopsy. In Study 1, in vitro- and in vivo-produced embryos were vitrified after biopsy, as would be needed while awaiting results of genetic testing. In vitro-produced blastocysts (n = 5) were obtained from slaughterhouse-derived oocytes that were matured, fertilized by ICSI, and cultured 7 to 8 days. In vivo-recovered blastocysts (n = 9) were obtained by uterine flush of inseminated mares 6.5 days after ovulation. The biopsy procedure was conducted using a Piezo drill in modified PBS without calcium and magnesium. Two to three biopsy manipulations were conducted on each embryo to ensure that an adequate number of cells was obtained. After biopsy, embryos were vitrified as described by Eldridge-Panuska et al. (2005 Theriogenology 63, 1308). Four vitrified in vivo-produced blastocysts were warmed and cultured for 2 days. Of these, two appeared viable, showing a 50- to 100-μm increase in diameter, and two did not grow. The remaining 5 in vivo- and 5 in vitro-produced vitrified blastocysts were shipped to a transfer facility, warmed and transferred transcervically to recipient mares. None of these embryos established pregnancy. In Study 2, three Day-6 in vivo-recovered blastocysts were biopsied then shipped by air (6 h transport time) for transfer to recipient mares. All three embryos established pregnancies and showed a normal heartbeat at 25 days. These results indicate that cells may be obtained from in vitro and in vivo-produced equine blastocysts by biopsy with the Piezo drill. In vivo-produced blastocysts transferred directly after biopsy were viable (3/3). While some blastocysts vitrified after biopsy showed growth in vitro, vitrified embryos did not produce pregnancies after transfer. To our knowledge, this is the first report of normal pregnancy rate after transfer of biopsied equine embryos. Further studies are needed to improve survival of embryos vitrified after biopsy. This work was supported by the Link Equine Research Endowment Fund, Texas A&amp;M University.

Quantificação do interferon-tau durante o reconhecimento materno da gestação em fêmeas bovinas Bos taurus indicus

Durante o período crítico do reconhecimento materno, compreendido entre o 15º e 19º dias da gestação, o concepto deve sintetizar competentemente moléculas capazes de bloquear a síntese de prostaglandina F2α (PGF2α) e a luteólise. Em bovinos, a principal macromolécula protéica envolvida em tal bloqueio é o interferon-tau (IFN-τ). Durante o período crítico, falhas neste reconhecimento determinam à mortalidade embrionária em até 40% das fêmeas inseminadas. Informações sobre o IFN-τ em animais Bos taurus indicus, ainda são restritas. Este estudo objetivou uma avaliação quantitativa do IFN-τ durante o período crítico do reconhecimento materno, em lavados uterinos obtidos por sonda de Foley (dias 14, 16 e 18 pósestro) ou post-mortem (dia 18 pós-estro). Para tanto, foram utilizadas fêmeas multíparas azebuadas (Bos taurus indicus), cíclicas ou prenhes, nos dias 14, 16 e 18 pós-estro. Para a obtenção dos lavados, os úteros foram infundidos com solução de Ringer Simples. Os lavados foram concentrados por ultra-filtração e liofilizados. As macromoléculas protéicas foram separadas por Eletroforese Unidimensional SDSPAGE, em gel com 15% de poliacrilamida. A quantificação do IFN-τ nos lavados uterinos foi realizada por Western-Blotting e densitometria. Tanto nos lavados obtidos por sonda de Foley quanto nos post-mortem foi possível observar bandas de proteínas que apresentaram reação cruzada com os anticorpos utilizados no Western-Blotting. O IFN-τ foi detectado apenas nos lavados uterinos post-mortem de vacas prenhes (P&lt;0,05). A densidade óptica não foi afetada pelo dia do período crítico, estado (cíclico ou prenhe) ou interação dia x estado. Nos lavados post-mortem não houve efeito de peso do concepto ou concentração de progesterona plasmática no dia do lavado na densidade da banda protéica referente ao IFN-τ . Concluiu-se que a detecção e quantificação do IFN-τ no ambiente uterino de vacas azebuadas, nestas condições experimentais, é possível apenas em lavados uterinos obtidos post-mortem.

STUDIES REGARDING MARE EMBRYOS RECOVERY AND EVALUATION

Nowadays, embryos recovery and transfer, immediately after recovery are allowed in most horse breed. During February-May 2008 researches were made on 4 mares with age between 4 and 22, belonging to different breed, from which 2 were proposed for the embryos recovery. From the total of six uterine flushings made at the 2 mares, four (66%) had no result, no emryos being identified in the recovery medium. All four flushings were performed at mare 1. In the case of the two flushings made at mare 2, there were identified two embryos, with maximum efficiency. Embryos recovering rate was 33%.

Stanniocalcin 1 Is a Luminal Epithelial Marker for Implantation in Pigs Regulated by Progesterone and Estradiol

Stanniocalcin 1 (STC1) is a glycoprotein that decreases calcium and increases phosphate in cells/tissues. This investigation examined endocrine regulation of STC1 in endometria of pigs during the estrous cycle and pregnancy. STC1 mRNA was present exclusively in luminal epithelium (LE) between d 12 and 15 of the estrous cycle, increased between d 12 and d 20, and was not detectable by d 30 of pregnancy. STC1 protein was also detected in uterine flushings. To determine effects of estrogen and progesterone, pigs were ovariectomized and treated with these hormones alone or together. Progesterone, but not estrogen, induced STC1 in LE. Cotreatment with progesterone and estrogen further stimulated STC1 over progesterone alone. To determine effects of pseudopregnancy, nonpregnant gilts were given daily injections of estradiol benzoate from d 11 to d 14. STC1 was not expressed in LE on d 90 of pseudopregnancy, suggesting that the estradiol given to induce pseudopregnancy and/or long-term exposure to progesterone are required for down-regulation of STC1. To determine effects of long-term progesterone, without effects of estradiol, pigs were ovariectomized on d 12, given daily injections of progesterone through d 39, and hysterectomized on d 40 after estrus. STC1 was expressed in LE of progesterone-treated pigs, suggesting that estrogen is involved in down-regulation of STC1. We conclude that STC1 is induced in LE by progesterone and further stimulated by estrogen, and its down-regulation in LE by d 25 likely requires exposure of the progestinized uterus to estrogen. The temporal and cell type-specific expression of STC1 makes this gene a unique marker for implantation in pigs.

Immunohistochemical and histochemical identification of proteins and carbohydrates in the equine endometrium: Expression patterns for mares suffering from endometrosis

Although alterations in patterns of protein secretion revealed in uterine flushings from mares suffering from endometrosis have been described, little is known about alterations at the cellular level. Hence, the aim of this study was to characterize deviations in patterns of uterine gland secretion patterns using endometrial biopsies, histochemical and newly established immunohistochemical methods. Forty-eight endometrial biopsies were obtained from mares suffering from various types of endometrosis (active and inactive, destructive and non-destructive) and degree (mild to severe) were analyzed for expression of the proteins uteroglobin, uteroferrin, calbindin D9k and uterocalin as representatives of endometrial proteins detectable by immunohistochemistry using polyclonal antibodies. Glycogen was identified using the PAS-reaction and mucopolysaccharides were stained with alcian blue. Uterine glandular epithelia within fibrotic foci mostly revealed a protein and carbohydrate pattern of expression which was independent of hormonal changes during the estrous cycle. In comparison to non-affected glands, most epithelial cells within periglandular fibrosis exhibited decreased immunostaining intensity for proteins, especially when there was destructive endometrosis. However, uteroferrin staining intensity was strong within areas of severe destructive endometrosis. Moreover, only few basal glandular epithelial cells, especially those in cystic glands, stained for mucopolysaccharides that are typically seen within the luminal epithelia. Usually a single fibrotic focus caused only slight alterations in glandular proteins and carbohydrate reaction patterns, so that only more severe endometrosis lead to deviations which were detectable in uterine flushings. The highly sensitive methods used in the present study allow studies of uterine secretion patterns in the context of routine assessment of endometrial biopsies.

Select Nutrients in the Ovine Uterine Lumen. I. Amino Acids, Glucose, and Ions in Uterine Lumenal Flushings of Cyclic and Pregnant Ewes1

Nutrients in uterine secretions are essential for development and survival of conceptuses (embryo and associated extraembryonic membranes) during pregnancy; however, little is known about changes in the amounts of specific nutrients in the uterine fluids of cyclic and pregnant ruminants. This study determined quantities of glucose, amino acids, glutathione, calcium, sodium, and potassium in uterine lumenal fluid from cyclic (Days 3-16) and pregnant (Days 10-16) ewes. Total recoverable glucose, Arg, Gln, Leu, Asp, Glu, Asn, His, beta-Ala, Tyr, Trp, Met, Val, Phe, Ile, Lys, Cys, Pro, glutathione, calcium, and sodium were greater in the uterine fluid of pregnant compared with cyclic ewes between Days 10 and 16. In cyclic ewes, only modest changes in the total amounts of glucose, Asn, Cit, Tyr, Trp, Met, Val, Cys, glutathione, calcium, and potassium were detected between Days 3 and 16. However, in pregnant ewes, amounts of glucose, Arg, Gln, Glu, Gly, Cys, Leu, Pro, glutathione, calcium, and potassium in uterine fluids increased 3- to 23-fold between Days 10 and 14 and remained high to Day 16. Of particular interest were increases in glucose, Arg, Leu, and Gln in uterine flushings of pregnant ewes between Days 10 and 16 of pregnancy. Total amounts of His, ornithine, Lys, Ser, Thr, Ile, Phe, Trp, Met, and Cit in uterine fluids also increased, but to a lesser extent during early pregnancy. These novel results indicate activation of pregnancy-associated mechanisms for transport of nutrients into the uterine lumen, and they provide a framework for future studies of nutrients, including glucose, amino acids, and glutathione, required to activate nutrient-sensing cell signaling pathways for growth, development, and survival of conceptuses, as well as for optimization of culture media for in vitro studies of conceptus development.

Gossypol Disrupts Embryo Development in Heifers

Our objectives were to determine the effects of dietary free gossypol (FG) intake on plasma and uterine gossypol concentrations and embryo development and viability before and after culture with gossypol. Fifty postpubertal Holstein heifers weighing (±SD) 406±34.5kg at 11.5 mo of age were blocked by age and body weight (BW) and randomly assigned to 1 of 3 isocaloric and isonitrogenous diets differing in their FG content: control (0mg of FG/kg of BW), moderate (17.8mg of FG/kg of BW), and high (36.8mg of FG/kg of BW). Heifers were fed the diets for 70 d before superovulation and embryo collection. Superovulated heifers were flushed on d 5 after induction of ovulation, and early morulae were either stained, to determine the number and proportion of live and dead cells, or randomly assigned to an in vitro culture for 96h in media containing either 0 or 10μg/mL of gossypol acetic acid. Plasma and uterine gossypol concentrations increased with increasing gossypol intake. The number of low-quality embryos-ova was greater for the high than for the moderate and control diets. Embryos collected from the high diet had the least number of cells because of fewer live cells, and were smaller in diameter. Greater dietary gossypol reduced blastocyst development and extended the time to reach the blastocyst stage. Similarly, gossypol concentration at 10μg/mL compromised in vitro development and increased the proportion of degenerated embryos at 96h in culture. These findings provide in vivo and in vitro evidence that intake of 36.8mg of FG/kg of BW per d and gossypol concentrations >7μg/mL in plasma, in uterine flush, or in vitro compromise early embryo development, which might explain some of the negative effects of gossypol on the fertility of dairy cows.

Reproductive research in non-human primates at Institute of Primate Research in Nairobi, Kenya (WHO Collaborating Center): a platform for the development of clinical infertility services?

The Institute of Primate Research (IPR; www.ipr.or.ke) is a WHO collaborating center for research in reproductive biology, infectious diseases and ecology/conservation. It includes a fully equipped surgical complex, >5000 square feet of laboratory space, a quarantaine facility, library, conference room, administrative offices, etc. More than 500 primates can be housed at IPR, mainly baboons. Reproductive research at IPR is applied to endometriosis, assisted reproduction, prevention of heterosexual transmission of HIV and includes the investigation of immunocontraceptives and placental retroviruses. Reproductive research capacities of IPR include: videolaparoscopic surgical equipment, surgical experience, endometrial biopsies and uterine flushes, ovarian stimulation, laparoscopic oocyte aspiration, hormonal analyses in baboon blood and urine, sperm assessment, in vitro culture and reproductive immunological investigations. During the last years, simultaneously with the development of baboon IVF, there have been contacts with several Kenyan gynecologists at the level of KEMRI (Kenya Medical Research Institute), KOGS (Kenyan Obstetrical and Gynecological Society), Kenyatta National Hospital and Aga Khan Hospital in Nairobi to develop clinical infertility services including low-budget high-quality IVF in Nairobi. The logic behind this initiative is that the Kenyans trained in non-human primate embryology, and IVF would be natural partners to develop human IVF in Kenya.

Conceptus development during blastocyst elongation in lines of pigs selected for increased uterine capacity or ovulation rate1,2

Lines of pigs selected for increased uterine capacity have improved conceptus survival, whereas pigs selected for increased ovulation rate have decreased conceptus survival relative to an unselected control line. The objective of this study was to evaluate conceptus development during blastocyst elongation as a potential contributing factor to differences in conceptus survival rate among these pig lines. Conceptuses were recovered from pregnant control, uterine capacity, and ovulation rate line gilts at d 10 and 12 of gestation. At d 10 of gestation, conceptus morphologic diversity was assessed by comparing within-litter average conceptus diameter and the standard deviation of conceptus diameters. At d 12 of gestation, conceptus morphologic diversity was assessed by comparing blastocyst populations obtained from individual gilts. Real-time PCR analyses for transcripts involved in steroidogenesis, cellular differentiation, and immune responsiveness were performed on spherical, ovoid, and filamentous conceptuses recovered from these selection lines. Uterine flushings were also assayed for total protein and estradiol-17beta at d 10 and 12 of gestation. Morphological data were analyzed using ANOVA with the fixed effects of line, farrowing season, and their interactions. Conceptus mortality, uterine flushing, and real-time PCR data were analyzed using ANOVA with the fixed effects of line, day or blastocyst morphology, farrowing season, and their interactions. Conceptus mortality, measured as the ratio of conceptus recovery to ovulation rate, was not different between the lines on d 10 and 12 of gestation. There were no significant line effects for conceptus morphologic diversity at d 10 and 12 of gestation. Expression of transcripts associated with steroidogenesis (steroidogenic acute regulatory protein, cytochrome P450 side chain cleavage, and aromatase), cellular differentiation (cytokeratin-18 and vimentin), and immune responsiveness (interleukin-1beta) in spherical, ovoid, and filamentous conceptuses was not different between the lines. Furthermore, protein and estradiol-17beta in uterine flushings at d 10 and 12 of gestation were not different between the selection lines. These findings indicate limited, if any, deviations between these lines of pigs in conceptus development during blastocyst elongation and suggest that mechanisms involved in generating line differences in survival rate likely are manifested later in gestation.

Excessive ovarian response is associated with increased expression of interleukin-2 in the periimplantation endometrium

To evaluate and compare in vivo expression of T helper type 1 (Th1) cytokines in the peri-implantation endometrium of infertile patients between natural and stimulated cycles. An observational study. A tertiary assisted reproduction center. Infertile patients. Uterine flushings and endometrial biopsies were collected 7 days after LH surge in natural cycles or after hCG injection in stimulated cycles. T helper type 1 cytokines were determined by immunolocalization and by ELISA. Natural cycles were classified as group A (n = 17), whereas stimulated cycles with peak serum E(2) of <or=20,000 pmol/L (moderate responders) and with peak serum E(2) of >20,000 pmol/L (excessive responders) were classified as group B (n = 32) and group C (n = 32), respectively. Higher serum E(2) concentration was associated with increased expressions of interleukin (IL)-2 in the endometrium and uterine flushing. Group C demonstrated a statistically significantly higher IL-2 expression in endometrial biopsies by glandular and stromal immunostaining and by ELISA, compared with group A and group B. There was no difference in IL-2 concentration between group A and group B. Interferon-gamma protein concentration was comparable for the three groups. Increased expression of IL-2 in the peri-implantation endometrium may account for the lower implantation rate in excessive responders.

Embryo recovery from exercised mares

The effect of exercise on mare reproductive efficiency was evaluated by comparing rates of embryo recovery from mares assigned to either an exercise regimen or a non-exercise (control) regimen. Exercised mares were worked daily for 30 min under average ambient conditions of >30 °C and >50% humidity. Mares were inseminated during estrus and subjected to uterine flush for embryo recovery on d 7 after ovulation for two consecutive cycles. After this, mares were allocated to the opposite group and allowed an estrous cycle without reproductive manipulation; then insemination and uterine flushing were conducted on two more consecutive cycles. Prostaglandin F 2α was administered on the day of uterine flush. Mare rectal temperature increased during exercise from a mean of 38 °C to a mean of 39.9 °C. Mares had ovulations from smaller follicles when exercised than they did under control conditions (39.8 ± 0.5 compared with 41.5 ± 0.5 mm diameter; P < 0.05), and had an increased time from PGF 2α administration to subsequent ovulation (8.47 ± 0.337 compared with 9.27 ± 0.294 d; P < 0.05). Embryo recovery from control mares was 22 of 35 (63%). Fewer embryos were recovered from exercised mares (11 of 32, 34%; P < 0.05). The proportion of embryos classified as Grade 1 tended to be less in exercised than in non-exercised mares (4 of 11, 36% compared with 16 of 22, 73%; P = 0.051). These data indicate that exercising mares in a hot and humid environment are associated with changes in ovarian follicle development and ovulation, and a reduction in embryo recovery.

Effects of flaxseed supplementation on endometrial expression of ISG17 and intrauterine prostaglandin concentrations in primiparous dairy cows submitted to GnRH-based synchronized ovulation

The objective of the experiment was to determine the effects of feeding whole flaxseed on ovarian function and uterine concentration of prostaglandins on the 17th day post-ovulation. Fortyeight primiparous Holstein cows averaging 550 kg of body weight (BW) were allotted at 2 wk postpartum to two groups of 24 animals: a control diet with no added fat (CON) or a diet with 9% whole flaxseed (FLA). At 6 wk postpartum cows were subjected to a Presynch-Ovsynch protocol with and without artificial insemination on day 0. Real-time transrectal ultrasonography and plasma progesterone concentrations were used to evaluate ovarian function. On day 17, uterine flushings and biopsies of uterine endometrium were collected. Synchronized ovulation rates based on low and high plasma progesterone concentrations on days 0 and 7, respectively, were the same for cows fed CON and those fed FLA (87.5 vs. 87.5%). The 13,14-dihydro-15-keto-PGF2α (PGFM) to prostaglandin E metabolite (PGEM) ratio in uterine flush was significantly higher for non-inseminated cows fed CON (32.0) than for non-inseminated cows fed FLA (17.5) and there was no difference between pregnant cows fed CON (12.2) and those fed FLA (12.0). Feeding flaxseed before breeding may contribute to increased fertility by improving uterine conditions for embryo survival. Key words: Flaxseed, dairy cattle, ovarian function, prostaglandins

Use of a low-volume uterine flush for diagnosing endometritis in chronically infertile mares

Low-volume uterine flush ( n = 401) was performed in 308 infertile mares to diagnose endometritis. Mares evaluated were either barren after three or more breedings or had two or more unsuccessful embryo recovery attempts during consecutive cycles. Culture results were compared with cytological and histological findings, efflux clarity and pH to substantiate that the micro-organisms recovered were truly pathogens. Cytological specimens were evaluated for presence of epithelial and inflammatory cells, bacteria, yeast and debris. Endometrial biopsies ( n = 110) were examined for the presence of neutrophils in the stratum compactum. Micro-organisms were recovered in 282/401 (70%) of low-volume flushes; E. coli was most frequently isolated (42.2%), followed by β hemolytic Streptococcus (37.6%). Efflux clarity of 318 flushes was clear ( n = 109), cloudy ( n = 149), or mucoid ( n = 60). Isolation of micro-organisms was highly associated with cloudy and mucoid effluxes ( P < 0.001), debris on cytological specimens ( P < 0.001), increased efflux pH ( P < 0.003), and neutrophils on endometrial biopsy ( P < 0.01). E. coli was associated with debris on cytological smear ( P < 0.002), whereas β hemolytic Streptococcus was associated with increased efflux pH ( P < 0.002). Using the presence of neutrophils in a tissue specimen as the “best standard” for diagnosing endometritis, the sensitivity of flush culture was 0.71 and for flush cytology was 0.8, whereas the specificity was 0.86 and 0.67, respectively. Neutrophils in uterine flushes under-reported inflammation; only 86/282 positive cultures were positive on cytology. The clinical estimate of a contaminated (false positive) flush culture was 11%, if a false positive was defined as positive culture with clear efflux and no debris or neutrophils on cytology (26/228). In conclusion, a low-volume uterine flush was a rapid, accurate method for identifying mares with chronic endometritis. When micro-organisms were recovered, endometritis was confirmed by efflux clarity, pH and cytological findings of debris, bacteria, or neutrophils. E. coli was most commonly isolated and it appeared to differ in pathogenicity from β hemolytic Streptococcus.