bluuebunny/crossref_metadata_2025 huggingface dataset:
113 PRELIMINARY CHARACTERIZATION OF OXYTOCINASE IN EQUINE SERUM
Authors: Diel de Amorim M. , Nielsen K. , Card C.
Published: December 2014

Oxytocinase/insulin regulated aminopeptidase (IRAP) or leucyl-cystinyl aminopeptidase (LNPEP) is an enzyme that is involved in the regulation of hormones such as oxytocin, vasopressin, and angiotensin in both humans and sheep. Historically, very low levels of this aminopeptidase were reported in monthly samples obtained from cycling and pregnant mares using an enzymatic colourimetric method. The regulation of oxytocin in horses is of interest because of its central role in uterine clearance, luteal maintenance, parturition, passage of fetal membranes, maternal foal bonding, and milk let-down. A preliminary study was performed with the objective of re-examining the level of serum oxytocinase in nonpregnant control (n = 3 mares sampled every other day; EOD of the oestrous cycle), and n = 5 mares sampled Day 12 to 15; oxytocin-treated (n = 2 mares sampled Day 12 to 15), and early pregnant mares (n = 6 mares sampled EOD), using more sensitive ELISA methodology. Mares were examined daily in oestrus until ovulation (Day 0) and from Days 10 to 21, using transrectal ultrasonography of the reproductive tract. Palpable changes in uterine and cervical tone, ultrasound measurement of dominant follicles, oedema scores (0 to 4 with 4 being maximal oedema), and changes in luteal echotexture and size were recorded. Pregnant mares were bred using AI (>200 million motile and normal sperm) from a proven stallion while in oestrus until ovulation, beginning when the dominant follicle was >35 mm. Oxytocin-treated mares were administered 60 IU IM oxytocin SID from Day 7 to 14. Blood was collected to obtain serum. Changes in serum oxytocinase levels were measured using a commercially available ELISA kit for Horse LNPEP according to the manufacturer's instructions (MyBioSource, San Diego, CA, USA) and validated for use in our laboratory using serial dilutions of pooled serum with an intra-assay and inter-assay CV <15%. The lowest standard of the assay was 31.2 ng mL–1. Preliminary results of this pilot study demonstrated in control cycles median serum oxytocinase levels below the lowest standard (2.0 to 30.1 ng mL–1). Oxytocin-treated mares had median serum oxytocinase levels from 39 to 61 ng mL–1 on Days 12 to 15. Early pregnant mares had detectable levels from Day 8 to 21 (medians ranging from 40 to 89 ng mL–1). We concluded that serum oxytocinase levels were below the lowest standard in diestrus, and were low but detectable in oxytocin-treated mares. The highest oxytocinase levels were measured during early pregnancy. Further studies of serum oxytocinase in a larger population of mares, along with studies of tissue mRNA levels of oxytocinase, are required to better understand the regulation of oxytocin in horses.
Research was supported by the Natural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada) and the Equine Health Research Fund (University of Saskatchewan, SK, Canada).
116 EFFECT OF EXOGENOUS PROGESTERONE AND GONADOTROPIN-RELEASING HORMONE APPLICATION ON MAINTENANCE OF PREGNANCY IN EARLY PREGNANT EWES AFTER PROSTAGLANDIN F2 ALPHA INJECTION
Authors: Kose M. , Kaya M. S. , Atli M. O.
Published: December 2014

In this experimental study, we tested the hypothesis that exogenous progesterone and GnRH applications would prevent the detrimental effect of prostaglandin F2α (PGF2α; a) on early pregnancy in ewes by sustaining high plasma progesterone level. For this purpose, 9 pregnant ewes (mating day = 0) were divided into 2 groups on Day 18 as follows: (1) PGF2α group (125 μg of d-cloprostenol injection on Day 18, n = 5); (2) PGF2α + progesterone sponge group (125 μg of d-cloprostenol injection on Day 18 + 20 mg flugestone acetate for 7 days, n = 4). Moreover, gonadotropin-releasing hormone (GnRH; 10 μg of buserelin acetate, n = 4) was injected intramuscularly to PGF2α + progesterone sponge group ewes on Day 22 after mating to induce luteinization in dominant follicles. The progesterone sponge was withdrawn on Day 25. Plasma progesterone (P4) concentration was measured on Days 18 and 19 by ELISA. Pregnancies were examined by using transrectal ultrasonography on Days 18, 22, 25, and 35 after mating. Statistical difference between groups was analysed by Chi-squared test. P4 concentration declined to below 1 ng mL–1 on Day 19 in both groups. While all pregnancies were terminated (5/5) in the PGF2α group by Day 25, progesterone sponge application prevented pregnancy loss (4/4) in the PGF2α + progesterone sponge group (P < 0.02). When the progesterone sponge was withdrawn on Day 25, half of the pregnancies (2/4) continued after GnRH application in the PGF2α + progesterone sponge group until birth. In conclusion, results suggest that exogenous progesterone application is sufficient for maintaining early pregnancy in ewes, even if the corpus luteum is regressed. Furthermore, GnRH application on Day 22 after mating might luteinize dominant follicles and could be sufficient to maintain pregnancy after exogenous progesterone is removed.
12 EFFECT OF HUMAN CHORIONIC GONADOTROPIN (hCG) ADMINISTRATION ON DAY 2 OR DAY 5 AFTER OESTRUS ON PREGNANCY RATE IN HIGH-YIELDING DAIRY COWS
Authors: Sanchez J. M. , Maillo V. , Molina L. , Perez-Marin C. C. , Lonergan P. , Rizos D.
Published: December 2014

In cattle, ~40% of embryonic loss occurs in the period from Day 8 to Day 16 of pregnancy. A significant proportion of embryo loss may be due to inadequate circulating progesterone (P4) concentrations. Low P4 concentrations have also been implicated as a causative factor in the low pregnancy rates (PR) observed in high-yielding dairy cows. Administration of hCG during the early luteal phase stimulates hypertrophy of the original corpus luteum (CL) and, depending on the day of administration, induces ovulation of the first-wave dominant follicle and formation of a functional accessory CL, which increases circulating P4 concentrations. The aim of this study was to examine whether administration of hCG on Day 2 or Day 5 after oestrus after timed AI (TAI) would lead to an increase in pregnancy rates in dairy cattle. Lactating Holstein cows (n = 194) from 12 commercial dairy herds in Southern Spain (37.8833° N, 4.7667° W) with an average milk production at 37.8 L/cow per day and typically with a PR to first AI of ~30% were randomly assigned based on their body condition score (2.65 ± 0.05; mean ± SEM), parity (2.60 ± 0.09), and days in milk (75.06 ± 0.63) to 1 of 3 treatments and administered a single intramuscular injection of 3000 IU of hCG (4 mL of Veterin Corion) either (1) on Day 2 = 36 h after TAI (n = 65; hCG2 group), (2) Day 5 = 108 h after TAI (n = 64; hCG5 group), or (3) 4 mL of saline on Day 2 = 36 h after TAI (n = 65; control group). Cows were synchronized using a 7-day Ovsynch TAI protocol that included a P4-releasing intravaginal device (PRID DELTA 1.55 g). First, gonadotropin-releasing hormone (Cystorelin 100 mg) treatment was administered at PRID insertion (Day 0) followed by 25 mg Dinoprost (prostaglandin F2α: Enzaprost T) on Day 7 at PRID withdrawal. Then, 56 h later, the second gonadotropin-releasing hormone (100 mg) treatment was administered and all cows were inseminated 16 h later. Pregnancy was diagnosed by ultrasonography 28 to 32 days after TAI. Logistic regression model and chi-squared test were used to analyse data. Pregnancy rate to AI was significantly higher in the hCG2 and hCG5 groups than in the control group (43.1 and 45.3%, v. 27.7%; P < 0.05). A treatment-by-parity interaction was observed; while pregnancy rate for primiparous cows was not affected by treatment, multiparous cows from the hCG2 group had greater pregnancies per AI than those in the control group (47.2% v. 21.1%, respectively; P < 0.05). In conclusion, these preliminary results suggest that hCG administration on Day 2 and 5 after oestrus increases PR at first postpartum AI in Holstein cows. In addition, hCG on Day 2 increases the fertility in multiparous cows.This study was funded by the Spanish Ministry of Science and Innovation (AGL2012–37510) and partially supported by Ceva Salud Animal S.A., Spain – synchronization protocol and DFV, Spain – hCG treatments.
120 MATERNAL EXPOSURE TO DIESEL ENGINE EXHAUST DURING PREGNANCY AFFECTS EARLY EMBRYO DEVELOPMENT IN A RABBIT MODEL
Authors: Valentino S. , Dahirel M. , Mourier E. , Archilla C. , Richard C. , Daniel N. , Maulny L. , Peynot N. , Canon E. , Slama R. , Cassee F. , Tarrade A. , Duranthon V. , Chavatte-Palmer P.
Published: December 2014

Airborne pollution has been associated with various adverse effects on human reproductive health, especially intrauterine growth retardation and early pregnancy loss. However, few studies have analysed its effect on early development. Diesel exhaust particles (DEP) have been shown to alter blastocyst formation when diluted in embryo culture medium (2010 Toxicol Sci. 117, 200–208), but no data are available concerning the effect of maternal inhalation of diesel exhaust on early embryo development. Our study has been designed to answer this question using rabbit as a model and DEP doses mimicking daily exposure to traffic in large European cities. New Zealand female rabbits were superovulated by means of 5 subcutaneous administration of pFSH for 3 days before mating, followed 10 to 12 h later by an intravenous administration of 30 IU of hCG at the time of mating (natural mating). Dams were exposed to a representative air pollution mixture; that is, diluted diesel engine exhaust (1 mg m–3; N = 14) or clean air (N = 12), for 1 h every morning and afternoon, from Day 3 to Day 6 post-coitum (dpc). At 6 dpc, in vivo-developed embryos were collected from uteri perfused with PBS and counted; their diameter was measured on pictures using ImageJ software (NIH, Bethesda, MD, USA). Another group of female rabbits was exposed to the same inhalation conditions from 3 to 27 dpc without superovulation treatment. Measures by ultrasound were performed on these dams at 7 dpc. Data were analysed by Mann-Whitney test and ANOVA, including dams as cofactor. At 6 dpc, number of embryos per dams was higher in exposed group compared with control (P < 0.05). In contrast, embryo diameter was significantly lower in the DEP exposed group compared with the clean air exposed group (P < 0.01). Gene expression analysis is being performed in these embryos. At 7 dpc, ultrasound measurements evidenced a decrease in embryo diameter, perimeter, and volume in the exposed group compared with control (P < 0.01, P < 0.01, and P < 0.01, respectively). These data indicate that repeated exposure to airborne pollution even for daily short periods affects early development. Consequences of maternal DEP exposure on feto-placental development are under investigation.
121 EXPRESSION OF HYALURONAN SYSTEM COMPONENTS IN THE EARLY PREGNANT OVINE ENDOMETRIUM
Authors: Kiyma Z. , Hitit M. , Ozel C. , Sen G. , Kose M. , Guzeloglu A. , Kaya M. , Atli M. O. , Kurar E. , Kayis S. A. , Kaya M. S.
Published: December 2014

Hyaluronan (HA; hyaluronic acid), a member of glycosaminoglycans (GAG) family, is the main polysaccharide and is expressed in almost all tissues including those of the reproductive tract. Three different hyaluronic acid-synthase (HAS) enzymes, HAS1, HAS2, and HAS3, synthesise HA. The action of HA depends on its molecular size and its cell surface receptor (CD44). Hyaluronidases (HYAL) are a group of enzymes, HYAL-1 and HYAL-2, that degrade HA. Roles of the HA system in reproductive events include oocyte maturation, fertilization, and implantation. It is also known that ovarian steroids, specifically progesterone, regulate the HA system in the endometrium. Moreover, HA-mediated cell signalling participates in embryonic development. The aim of this study was to evaluate expression of the HA system at the mRNA level in the early pregnant ovine endometrium at pre-implantation stage. Therefore, endometrial tissue samples were collected on Day 13 of the oestrous cycle (n = 10) and pregnancy (n = 14). Relative mRNA expression levels of HA system genes were quantified using quantitative RT-qPCR. Data were analysed using one-way ANOVA and l.s.d. test. All of the studied components of the HA system (HAS1, HAS2, HAS3, HYAL1, HYAL2, and CD44) were expressed in the ovine endometrium. Steady-state mRNA levels of HAS1, HYAL1, HYAL2, and CD44 were not significantly different between cyclic and early pregnant ovine endometrium on Day 13. However, expression of HAS2 and HAS3 appeared to be down-regulated in early pregnancy. Considering that both cyclic and pregnant endometrium on Day 13 is under the influence of progesterone, detected differential regulation of some components of HA system in the ovine endometrium may be directly related to the effects of the presence of an embryo. The role of the HA system in ovine endometrium at later stages of pregnancy warrants further investigation.
Partially funded by TUBITAK-112R022 to ZK and SUBAP to AG.
124 CRYOPRESERVED BOVINE OVIDUCTAL EPITHELIAL CELLS SURVIVAL, GROWTH, AND ABILITY TO SUPPORT EARLY EMBRYO DEVELOPMENT
Authors: Corbin E. , Cordova A. , Grosbois J. , Mermillod P.
Published: December 2014

Previous experiments demonstrated that co-culture of bovine embryos with bovine oviducal epithelial cells (BOEC) improved blastocyst rate and quality (Cordova et al. 2014). However, the use of primary cell support for improving embryo development in vitro may introduce a higher variability of the results between different BOEC batches used, as well as sanitary risks. The use of well-controlled large batches of frozen BOEC may help to solve these problems. Therefore, the aim of the present study was to characterise the survival and functionality of frozen-thawed BOEC. Bovine oviducts attached to ovaries showing recent ovulation were collected at a local slaughterhouse during 4 replicates (3 oviducts per replicate). Epithelial cells were expelled by gentle squeezing and washed 3 times. Half of the cell pellet was diluted 100-fold in culture medium (TCM199 + 10% FCS) for culture of fresh cells. The other half was diluted 10-fold in cell freezing medium (TCM199 + 20% FCS + 10% dimethyl sulfoxide), allowed to equilibrate in this medium for 10 min, and frozen at –80°C in a container filled with isopropyl alcohol. After 4 h, the tubes were transferred into LN for at least 1 h. The tubes were then thawed (5 min in 37°C water bath), diluted 1 : 1 in cell culture medium, and centrifuged for 10 min at 100 × g. The pellet was then diluted 100× in cell culture medium. Fresh or frozen-thawed cells were seeded in 4-well NUNC plates for 7 days at 38.8°C in a humidified atmosphere with 5% CO2 in air. The medium was renewed every 48 h, and the viability of cells was assessed by calcein-AM and ethidium homodimer labelling. After 7 days of culture, the medium was replaced by SOF medium + 5% FCS, and bovine in vitro-produced zygotes were added the day after and co-cultured for 8 days at 38.8°C in a humidified atmosphere with 5% CO2 in air to evaluate embryo development. Half of the medium was renewed every 48 h. Frozen-thawed cells showed the same viability than fresh ones at Days 0 and 7 of culture and reached confluence at the same time (Day 7). Development results are shown in Table 1. Frozen and fresh cells support early embryo development at the same rate. In conclusion, the present study showed that BOEC frozen on the day of collection are equivalent to fresh BOEC in regards to their survival and proliferation and their ability to support early embryo development. At collection, the cells may face stresses that are just as considerable as freezing/thawing (temperature shock, scrapping, change of environment). This may explain why they are not affected by freezing than at collection. The differentiation status of these cells is now under analysis by immunocytochemistry.

Table 1.Cleavage rate and blastocyst rate in 3 different types of culture systems
127 VERO CELLS CONDITIONED MEDIUM IMPROVES EMBRYONIC DEVELOPMENT DURING IN VITRO CULTURE OF BOVINE EMBRYOS
Authors: Barcelo-Fimbres M. , Gouze J. N. , Mtango N. R. , Koppang R. , Verstegen J. P.
Published: December 2014

Beneficial effects on embryonic development of co-culture with Vero cells during in vitro culture have been previously reported and related to Vero cells growth factors/cytokines secretion into the medium (1998 Human Reprod. 1600–1605). However, co-culture increases the probability of microorganism contamination during culture. In this study, we aimed to overcome this problem by culturing bovine embryos in conditioned medium derived from Vero cells culture (CM). Vero cells (GenBiotech, Antibes, France) were cultured for 24 h, and then the medium was removed, filtered, and frozen until its use. A total of 701 slaughterhouse bovine cumulus oocytes complexes were matured and fertilized in vitro under standard conditions. After fertilization, zygotes were allocated to 4 different treatments [0 (control), 5, 10, and 20% of CM v/v] for in vitro culture in BBH7 medium (BoviPro®, MOFA Global, Verona, WI, USA) at 38.5°C in 5% O2, 5% CO2, 90% N2 atmosphere for 7 days. A total of 8 replicates were done. Cleavage rates were assessed at 2.5 days, blastocyst rates and embryonic stage at 7 days, and blastocyst total cell number at 7.5 days after fertilization. Data were analysed by ANOVA using GLM; the percentages were transformed using arcsin square root. If the ANOVA was significant (P < 0.05), means were separated by Tukey's procedure using Statistix 10 software (Tallahassee, FL, USA). No differences in cleavage rates were found between treatments (P > 0.1; Table 1). However, the addition of conditioned medium at all levels increased the blastocyst production at Day 7 of culture (between 11 to 19 percentage points) compared with the control group (P < 0.01; Table 1); likewise, CM addition was superior in total embryo production (Day 7 morulae plus all blastocysts; P < 0.01; Table 1), between 12.5 to 15 percentage points higher than the control group. No differences were found between treatments for blastocyst total cell number at 7.5 days of culture (P > 0.1; Table 1) and overall embryonic developmental stage (P > 0.1; Table 1). In conclusion, we found a superior embryonic development when Vero cells conditioned medium was added at Day 0 of in vitro culture; the positive effect on total embryo production was observed even at 5% of conditioned medium.

Table 1.Means (± s.e.m.) of embryonic development of bovine embryos
14 IS THE LOW NUMBER OF OVARIAN ANTRAL FOLLICLES ≥3mm IN DIAMETER ASSOCIATED WITH LOW FERTILITY IN LACTATING NELORE COWS?
Authors: Pinheiro V. G. , Ereno R. L. , Razza E. M. , Barros C. M. , Nogueira M. F. G.
Published: December 2014

The follicular growth in cattle occurs in a wave pattern of 2 to 3 waves per oestrous cycle and is characterised by synchronous growth of a cohort of antral follicles, where usually only one of these will become dominant. The amount of recruited follicles per wave is variable between animals and breeds but is highly repeatable among individuals. Some studies report that in Bos indicus the amount of follicles recruited by wave is higher when compared with Bos taurus. The variation in the size of the ovarian follicular population can affect fertility by influencing oocyte competence (Ireland et al. 2007). We aimed to identify Nelore (Bos indicus) cows with high and low numbers of antral follicles recruited by follicular wave and to compare their pregnancy rates. We used 268 multiparous Nelore cows between 40 and 70 days postpartum and body condition score between 3.5 to 4.5 (5-point scale). The cows were sorted in ascending order according to the average number of follicles at all examinations (≥3 mm in diameter). Hence, 33% of animals with the greater follicular population were enrolled in the high population group (HG, n = 89, ≥38 follicles), whereas the intermediate animals (33%) were placed in the intermediate group (IG, n = 88, between 28 and 38 follicles), and animals (33%) with lower follicular population were included in the low population group (LG, n = 91, ≤28 follicles). The animals underwent 3 ultrasound examinations (days D–10, D0, and D28). In D0, at random day of the oestrous cycle, all cows received an intravaginal device containing progesterone (1.0 g, DIB®) and oestradiol benzoate (EB, 2.0 mg, IM, Estrogin®). Eight days later (D8) we administered 75 μg of d-cloprostenol (Croniben®), and the intravaginal device was removed. Twenty-four hours after DIB removal, the cows were treated with EB (1.0 mg, IM), and after 30 to 36 h animals were AI at a fixed time. Data were analysed using PROC GENMOD SAS System 9.1 for Windows (2002–2003). The mean (±SD) of antral follicles in both ovaries was 32.7 ± 17.8. There was no difference (P = 0.144) in pregnancy rates between the HG, LG, and IG animals (32.6, 46.6, and 42.9%, respectively). Thus, we conclude that there was no difference in pregnancy rates between Nelore cows either with high or low population of ovarian antral follicles after AI at a fixed time.Research was supported by grants #2013/02201-3 (VGP), #2011/50259-5 (RLE), #2012/23409-9 (EMR), #2012/50533-2 (MFGN), São Paulo Research Foundation (FAPESP).
145 THE INFLUENCE OF PROPYLENE GLYCOL DRENCHING ON THE SUPEROVULATORY RESPONSE AND EMBRYO QUALITY IN HIGH-YIELDING DAIRY COWS
Authors: Othman T. , Ismael S. , Ayoub M.
Published: December 2014

Genetic improvement of dairy cows increased markedly over the last decades; this has marked increased milk yield, which has been associated with reduced fertility parameters. The objective of this study was to determine the effects of feeding propylene glycol (PG) on superovulatory responses of 50 dairy cows and on their embryo quality and quantity. Starting at 1 week before the application of superovulatory regimen, each cow received once daily an oral dose of 150 g of PG (PG group) or water (control group). All cows were superovulated with a total dose of 400 mg of FSH administrated twice daily in decreasing doses over 4 consecutive days. Embryos were recovered nonsurgically 7.5 days after the onset of oestrus using a 2-way catheter. Evaluation of embryo quality was done according to the IETS manual based on 1 to 4 grades. Grade 1 embryos were transferred after freezing; grade 2 and 3 embryos were freshly transferred. The results showed that the number of total ova/embryos recovered, grade 1 embryos, and the number of transferable embryos were significantly higher (P = 0.048, 0.015, and 0.014, respectively) in the PG group (10.33 ± 1.9, 7.08 ± 1.7, and 7.92 ± 1.6, respectively) when compared with control group (6.09 ± 0.9, 2.11 ± 0.45, and 3.92 ± 1.25, respectively). Insignificant increase in grade 2 and grade 3 embryos was indicated in the control group (1.37 ± 0.3 and 0.51 ± 0.18, respectively) when compared with PG group (0.75 ± 0.28 and 0.25 ± 0.13, respectively). On the other hand, the results indicated that there was an increase in the number of degenerated embryos and unfertilized ovum in the PG group (2.33 ± 0.85) compared with control group (2.09 ± 0.6). In conclusion, these results suggested that administration of PG has the ability to positively improve the superovulatory response and embryo quality in high-yielding dairy cows.
154 EVALUATION OF EMBRYO RECOVERY RATE AND EMBRYO TRANSFER IN ANGORA GOATS
Authors: Karakas K. , Alkan H. , Onur G. , Ozen D. , Kaymaz M. , Izgur H.
Published: December 2014

The aims of this study were to compare the embryo recovery rate in Angora goats based on application timing; at the beginning (September – October; Group 1) and end (December; Group 2) of the breeding season and to evaluate the viability and survivability of fresh or vitrified-thawed embryos when transferred. For this purpose, nine Angora goats were used as donors and thirthy Angora goats were used as recipients. Donor goats were synchronized and superovulated with traditional protocol and were mated with fertile bucks. At the 156th hour of the mating, embryos were collected surgically and evaluated under a stereo microscope. In group 1, 103 embryos and in group 2, 63 embryos were collected from nine goats. Fresh or vitrified-thawed embryos were transferred surgically to synchronized recipients. In Group 1 fresh/thawed embryos were transferred to 15/15 goats and in group 2, fresh/thawed embryos were transferred to 8/8 goats, respectively. Each recipient received 1 or 2 embryos ipsilateral to the ovary containing one or more corpora lutea. On day 30 of the transfer, goats were examined by transrectal ultrasonography, pregnancy rates of fresh/thawed embryos were 66.6%/26.6% for group 1 and 62.5%/62.5% for group 2. On day 100 of the transfer, goats were examined again by ultrasonography, and pregnancy rates were 46.6%/0% for group 1 and 37.5%/0% for group 2, respectively. After about 50 days, goats were kidded. In group 1, 3 twins and 4 single kids were born; in group 2, 2 twins were born. The total number of collected embryos and pregnancy rates among the groups were analysed using SPSS® (version 14.01, Chicago, IL, USA) and for all comparisons, differences were considered with a minimum of 5% significance level. After statistical analyses, the numbers of collected embryos at the beginning and at the end of the breeding season were compared. There was no difference in freezable/transferable embryo quality. As a result, embryos could be collected after superovulation protocols in Angora goats both at beginning and end of the breeding season, however there might be a decrease in numbers of collected embryos and the reasons for this might not be only the seasonal factors but also the environment, care, nutrition and previous superovulation protocols. The pregnancy rate following transfer of fresh embryos was satisfactory but not all does confirmed pregnant kidded; hence, reducing the number of recipients kidding. The pregnancy rate following transfer of vitrified-thawed embryos was generally low and unsatisfactory. Further research is warrented in improving the cryopreservation techniques and thus the embryo survival rate of Angora goat embryos.
This study was financed with the University of Ankara Grant.
155 EFFECT OF VITRIFICATION OF MORULAE ON PREGNANCY RATE IN GOAT
Authors: Toishibekov M. M. , Valieva G. A. , Askarov S. M.
Published: December 2014

This work evaluated alternative methods for goat morulae cryopreservation by using the High Security Vitrification Kit (Cryobiosystem): vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). Vitrification was applied according to the method described by Vajta et al. (1998). Both treatments used a vitrification solution (VS) containing 20% ethylene glycol (EG), 20% dimethylsulfoxide (Me2SO), 0.5 mol L–1 sucrose in DPBS with 10% BSA. In our experiment we used the Vit-Master™ (MTG, Germany). Super-cooled liquid nitrogen (LN) facilitates heat transmission between LN and the cryosolution interface suggested to be beneficial for bovine semen and blastocyst cryoconservation. By surgical flushing of 30 super-stimulated goats, 137 transferable morulae were harvested; 41 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 47) or SCURV (n = 49), respectively thawed, and transferred to recipients. Embryos were vitrified using the HSV Kit. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS followed by vitrification (group V). Accordingly, morula of SCURV group were exposed to 50% VS for 2 min and to 100% VS for 30 s at 37°C. Thereafter, embryos were transferred into the VIT-Master for freezing with liquid nitrogen using negative pressure. Thawing of vitrified embryos was accomplished by placing the vitrified embryos in solutions of 0.25 M sucrose for 2 min and 0.125 M sucrose for 3 min, respectively. After thawing only survived embryos were transferred. Statistical analyses were performed with Student's t-test. After transfer of fresh or frozen-thawed embryos of V and SCURV groups, 25, 17, and 19 kids were born. No statistical difference was found for the percentage of viability of thawed embryos after vitrification (36.2 ± 4.4%), and SCURV methods (38.7 ± 6.5%). The survival of fresh embryos, however, was significantly higher (60.9 ± 5.3%). Differences were statistically significant (P < 0.05). Importantly, our data suggest that the SCURV method can be used for cryopresevation of goat morulae. Nevertheless, further work regarding the developmental competence of embryos cryopreserved with the SCURV method is needed.
160 STRATEGIC VACCINATION AGAINST BOVINE VIRAL DIARRHEA (BVD), INFECTIOUS BOVINE RHINOTRACHEITIS (IBR) AND LEPTOSPIROSIS IMPROVES PREGNANCY RATE IN FTAI PROTOCOLS IN NELORE BEEF COWS
Authors: Souto L. A. S. , Maturana Filho M. , Lemes K. M. , Torres F. D. , Madureira E. H.
Published: December 2014

The negative effect of some diseases, such as bovine viral diarrhea (BVD), infectious bovine rhinotracheitis (IBR), and Leptospirosis, on bovine reproduction rates are well known. The uses of vaccines are considered to be an important tool available in order to control reproductive losses but their efficiency is still controversial. The aim of this study was to evaluate the effects of vaccination against BVD, IBR, and Leptospirosis to improve pregnancy rate in beef cattle submitted to fixed-timed AI (FTAI). Nelore cows (n = 1172) from 4 beef cattle farms in Brazil were randomly distributed in two experimental groups: Group 1 (treated, n = 584) received the first dose of the inactivated vaccines (Bioleptogen and Bioabortogen H, Biogénesis Bagó, Garín, Argentina) at the beginning of the FTAI protocol and the second dose on the pregnancy diagnostic 40 days later; Group 2 (control group, n = 588) received 0.9% saline solution. Serum samples from 3–5% of animals in each farm herd were collected to determinate IBR, BVD, and Leptospirosis challenges, by using ELISA protocol for BVD and IBR (Synbiotics BVD p80 ab monoblocking test and Synbiotics BoHV-1 gB monoblocking test, respectively; Synbiotics Corp., Kansas City, MO, USA); and microaglutination test for Leptospirosis. All animals were submitted to the FTAI protocol: D0 = intravaginal P4 device (Cronipress, Biogenesis Bagó) and application of 2 mg oestradiol benzoate (Bioestrogen, Biogénesis Bagó); Day 8 = intravaginal device removal + 0.5 mg oestradiol cipionate (E.C.P, Zoetis Inc., Florham Park, NJ, USA) and 25 mg D-cloprostenol sodium (Croniben, Biogénesis Bagó) and FTAI after 48 h. Body condition scores (BCS) were measured on Day 0 and pregnancy diagnostic were performed on Day 40. Data were analysed by logistic regression using PROC LOGISTIC procedure of the SAS software (SAS 9.3, SAS Institute, Inc., Cary, NC, USA) as well as the significant differences between the factors was analysed to nonparametric statistical frequency (chi-square test; PROC FREQ). Pregnancy rates on day 40 were greater (P < 0.0001) in Group 1 (58.21%; 340/584) compared to Group 2 (44.73%; 263/588). Effect in BCS by pregnancy rates was observed (P = 0.0165) among animals with higher compared to lower BCS (61.40% v. 47.98%, respectively). Results from BVD, IBR and Leptospirosis prevalence were respectively (78.26%; 95.65%, and 10.20%), demonstrating that all herds were challenger for the 3 agents and a positive correlation with pregnancy rates (P < 0.001) in vaccinated group. In conclusion, vaccination with Bioabortogen H and Bioleptogen contributed to increase pregnancy rates in beef cattle submitted to FTAI; and the positive correlation between high prevalence of BVD, IBR, and leptospirosis, and an increased pregnancy rate by 13.48% can be due to fewer losses caused by the vaccination protection up to Day 40.
162 CAN CHLAMYDIA ABORTUS BE TRANSMITTED BY EMBRYO TRANSFER IN GOATS?
Authors: Pellerin J. L. , Ashraf A. , Oseikria M. , Laroucau K. , Vorimore F. , Roux C. , Larrat M. , Michaud S. , Fieni F.
Published: December 2014

Chlamydia abortus is a gram-negative obligate intracellular bacterium. Its lifecycle includes a resistant infectious form and a metabolically active non-infectious form. Chlamydia abortus infection results in abortion in goats; in nonpregnant animals the infection is usually subclinical. Chlamydia abortus presents a major zoonotic risk for pregnant women. The aim of this study was to investigate whether the embryonic zona pellucida (ZP) protects early embryo cells from infection and to test the efficacy of the washing protocol recommended by the IETS for bovine embryos. The study was performed in triple replicate: 14 donor goats, certified negative by ELISA and PCR to C. abortus, were synchronized, superovulated, and subsequently inseminated by males controlled negative for C. abortus. Fifty-two ZP-intact embryos (8–16 cells) were collected 4 days later, by laparotomy. The embryos were randomly divided into 12 batches. Nine batches of 5 embryos were incubated in a medium containing 4 × 107 Chlamydia mL–1, AB7 strain. After incubation for 18 h at 37°C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of PBS and 5% FCS solution in accordance with IETS guidelines for bovine embryos. In parallel, 3 batches of ZP-intact embryos (2, 2, and 3 embryos in the first, second, and third batches, respectively) were used as controls by being subjected to similar procedures, but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 h at 13 000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at –20°C before examination for evidence of C. abortus using RT-PCR. Chlamydia abortus DNA was found in all batches of infected ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the tenth wash fluid for 4 batches (4/9) of infected embryos. As expected, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goat to healthy recipients and/or their offspring. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of infected caprine embryos can eliminate C. abortus from the ZP.
169 INVASION OF MURINE ANNEXIN A1 IN BOVINE OVARIAN CORTEX TISSUE DURING SHORT-TIME XENOTRANSPLANTATION IN CONVENTIONAL AND IMMUNE DEFICIENT MICE
Authors: Bartholomeus E. , Langbeen A. , Leblon D. , Fransen E. , Ponsaerts P. , Leroy J. L. M. R. , Bols P. E. J.
Published: December 2014

In 2012, 6.7 million women worldwide were diagnosed with cancer, including young women and children. Almost 85% of the latter survive the disease. However, the necessary cytotoxic treatment reduces their reproductive potential by damaging the stock of, basically preantral, ovarian follicles. Current fertility preservation techniques try to overcome this problem. Still, the success rate is low due to a lack of optimization and standardization. Nowadays, culturing preantral follicles in vitro is not yet routinely possible. Therefore, we used an in vivo model based on xenotransplantation of bovine ovarian cortex tissue in recipient mice, as an alternative to studying human reproduction. To further characterise this model, we assessed the influence from the host on the graft at the protein level. Visualising murine annexin A1, an anti-inflammatory protein, in the transplanted bovine cortex tissue should help to elucidate the process of immunological rejection. Hereto its distribution around the present bovine follicles is measured. In total, 12 mice (6 conventional fluorescent C57BL/6-eGFP and 6 immune deficient Balb/c-Nu) were used as graft recipients. All mice were anesthetized with an intraperitoneal injection and subsequently sterilized. Small pieces (max. 9 mm3) of adult bovine ovarian cortex, retrieved from slaughterhouse ovaries, were grafted retroperitoneal. After a transplantation period of 14 (3 mice/mice strain) or 28 (3 mice/mice strain) days, the mice were killed and all grafts were successfully recovered. These were fixed (4% formaldehyde) and processed into histological paraffin slides. Murine annexin A1 (Sigma-Aldrich HPA011271) was localised using immunofluorescence through a rhodamine label (RITC filter). We measured the shortest distances between 10 annexin fluorescent signals and the basal membrane of each follicle, found in the graft. We calculated the average of these measurements, resulting in one data point per follicle. The dataset was fitted in a linear mixed model with annexin as dependent variable, and transplantation period and mice strain as fixed effects. Fluorescent signals of murine annexin A1 were found in all grafts form both mice strains, surrounding bovine follicles. Data show no interaction between mice strain and the duration of the transplantation period. The influence of the mice strain showed a trend towards significance (P = 0.08), possibly due to the immunological state of the host. To our knowledge, this is the first time an attempt has been made to characterise the host/donor interaction in xenografting procedures. We can conclude that annexin A1 from the murine host (whether immunodeficient or immunocompetent) invades the bovine graft tissue in a short period of time.
172 RELATIONSHIPS AMONG GRANULOSA CELL FGF9 mRNA, FOLLICLE SIZE, AND APOPTOSIS IN CATTLE
Authors: Schutz L. F. , Robinson C. , Zhang L. , Totty M. , Albonico M. , Spicer L. J.
Published: December 2014

Fibroblast growth factor 9 (FGF9) has been suggested to act as a dedifferentiation factor during bovine folliculogenesis, reducing steroidogenesis and increasing cell proliferation in granulosa (GC) and theca (TC) cells, but whether endogenous GC production of FGF9 change during bovine folliculogenesis and atresia/apoptosis is unknown. The objective of these studies was to investigate the relationship between FGF9 mRNA, follicle size, and health status of follicles. Ovaries (n = 10 cows) from a local abattoir classified visually as in midcycle phase (i.e. presence of corpus luteum and large follicles) were collected and categorized as small (1–5 mm), medium (5.1–8 mm) or large (8.1–22 mm) in size (Experiment 1). Follicular fluid (FFL) was aspirated for measurement of oestradiol (E2) and progesterone (P4) via radioimmunoassay and GC collected for RNA extraction. Abundance of mRNA for FGF9 and Caspase-3 (CASP3), an effector of apoptosis, were measured by real-time PCR (qPCR). Data were analysed via factorial ANOVA with main factors: follicle size, follicle estrogenic status, and their interaction. The abundance of GC FGF9 mRNA was greater (P < 0.05) in large E2-inactive (E2P4 concentrations) follicles (10.5 ± 22). The abundance of GC CASP3 mRNA was greater (P < 0.01) in small E2-inactive follicles than in large and medium E2-active and E2-inactive follicles. FGF9 mRNA abundance was not correlated with E2/P4 ratio in FFL, but it was positively correlated with CASP3 mRNA abundance (r = 0.35; P < 0.05). GC CASP3 mRNA abundance was negatively correlated with E2/P4 ratio (r = –0.48; P < 0.01). To investigate the relationship between FGF9 and CASP3 mRNA abundance during experimentally-induced apoptosis, GC from large and small follicles were collected (Experiment 2) and GC were plated in medium containing 10% FCS. GC (n = 3 independent pools for small and large follicles) were then treated with or without 10% FCS for an additional 24 h or 48 h followed by RNA extraction and qPCR for measurement of abundance of FGF9 and CASP-3 mRNA. Statistical analyses with ANOVA included main factors: treatment, duration of treatment, and their interaction. In small-follicle GC, FGF9 and CASP3 mRNA abundance were not correlated and were not affected by treatments. In large follicles, FGF9 mRNA abundance was greater in GC treated without FCS (27.5 ± 2.7) than in GC treated with 10% FCS (6.6 ± 2.7) and tended to differ (P < 0.08) between 24 h (22.5 ± 2.7) and 48 h (11.6 ± 2.7). CASP3 mRNA abundance was greater in GC treated without FCS (310 ± 36) than in GC treated with 10% FCS (140 ± 36) but did not differ (P > 0.10) between 24 h and 48 h. In Experiment 2, there was no significant correlation between FGF9 and CASP3 mRNA (r = 0.28; P = 0.2). These results indicate that FGF9 mRNA abundance is greater in GC from large E2-inactive than from E2-active follicles and its production may be increased in large follicles undergoing apoptosis.
182 IMMUNOHISTOCHEMISTRY LOCALIZATION OF GROWTH DIFFERENTIATION FACTOR 9 (GDF-9) AND BONE MORPHOGENETIC PROTEIN 15 (BMP-15) IN CANINE FOLLICLES THROUGHOUT ESTRUS CYCLE
Authors: Maupeu D. , Palomino J. , De los Reyes M.
Published: December 2014

Among different developmental proteins, growth differentiation factor-9 (GDF-9) and bone morphogenic protein 15 (BMP-15), members of the transforming growth factor β (TGFb) superfamily, have been described in many species as key mediators for folliculogenesis. The aim of this study was to analyse in canines the presence and localization of GDF-9 and BMP-15 proteins in follicle cells and oocytes during follicle development. To determine the immunolocalization of GDF-9 and BMP-15, sections of 4% paraformaldehyde in PBS ovaries obtained from 15 bitches at different stage of oestrous cycle (proestrus-oestrus n = 5; diestrus n = 5; anestrus n = 5) were dehydrated (xylene–ethanol solutions) and embedded into paraffin blocks. Individual paraffin sections (5 µm) were inactivated with methanol/H2O2. Sections were blocked at 37°C in the peroxidase reagent and incubated with rabbit anti-human GDF-9 (1 : 200) or goat anti-human BMP-15 (1 : 150) antibodies overnight at 4°C and with secondary antibodies goat anti-rabbit IgG and donkey anti-goat IgG, both HRP conjugated. Previously negative control sections received rabbit serum. Sections were stained with 1 mg mL–1 3,3-diaminobenzidine (DAB) and counterstained with haematoxylin. All sections were examined at magnification × 200 using an IX71 inverted microscope (Olympus, Center Valley, PA, USA), with an IX2-RFA lamp and a ProgRes CapturePro camera (Jenoptik AG, Jena, Germany). The same exposure time was applied to all samples and digital photos were taken and subsequently analysed. The intensity of immunostaining in both oocyte and follicular cells of each follicle was evaluated with Image J version 1.45s software (National Institutes of Health, Bethesda, MD, USA). The images were transformed to grey scale and the corrected total cell mark was calculated by subtracting the mean staining of background reading to the integrated density of each sample. The results showed that samples of all slices contained primordial, primary, secondary and pre-antral follicles as well as different sizes of antral follicles showed positive GDF-9 and BMP-15 immunoreactions in the oocyte cytoplasm and in the follicles cells. No such labelling was found in the negative controls. Follicle sizes differed in GDF-9 and BMP-15 immunoreactions according to oestrous cycle. The pattern of GDF-9 immunostaining in proestrus, oestrus, and diestrus was more intense within preantral rather than antral follicles/oocytes, whereas BMP-15 immunoreaction was prominent mainly in proestrus stage increasing to antral stage. These preliminary results indicated temporal pattern of GDF-9 and BMP-15 in canine ovarian follicles which might play important roles in follicular/oocyte development and ovulation during oestrous cycle.
Research was funded by FONDECYT Grant 1140658.
195 SUPPRESSION OF SETDB1 DURING BOVINE EMBRYONIC DEVELOPMENT RESULTS IN PRE-IMPLANTATION MORTALITY AND DECREASED TRANSCRIPTION OF TRANSPOSABLE ELEMENTS
Authors: Snyder M. D. , Pryor J. H. , Veazey K. J. , Peoples M. D. , Williamson G. L. , Golding M. C. , Westhusin M. E. , Long C. R.
Published: December 2014

Organization of chromatin structure by the combinatorial patterns of DNA methylation and post-translational histone modification is essential for the establishment and maintenance of proper transcriptional programs that result in the coordination of embryonic development. We previously observed that suppression of transcripts encoding SET domain, bifurcated 1 (SETDB1) using small interfering RNAs (siRNA) is embryonic lethal, with SETDB1-suppressed embryos (n = 361) arresting immediately before the blastocyst stage (blastocyst rate: Control 44.9 ± 4.9% and NULL injected 25.7 ± 6.0%). Studies in rodents indicate SETDB1 is a crucial regulator of transposable elements and that the precise epigenetic regulation of these elements is a key aspect of transcriptional programs controlling pluripotency and placentation. To better characterise the molecular basis of the observed mortality, we analysed expression of the bovine Long Interspersed Nuclear Element 1 family (LINE1) of transposable elements via quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). Mature bovine oocytes were obtained from a commercial supplier (De Soto Biosciences, Seymour, TN, USA) and IVF performed by standard laboratory protocol. Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes divided into 3 different treatment groups: non-injected control (CNTL), non-targeting siRNA injected control (siNULL), and zygotes injected with siRNAs targeting SETDB1 (siSETDB1). Each embryo was injected with ~100 pL of siRNAs (10 µM) in fluorescent dextran solution. All zygotes were verified as injected by fluorescent microscopy and then cultured in Bovine Evolve (Zenith Biotech, Guilford, CT, USA) medium supplemented with 4 mg mL of BSA (Probumin, EMD Millipore, Darmstadt, Germany). Groups of embryos (15–20) from each treatment were lysed at the 4-cell, 8-cell, and morula stages, RNA extracted, and analysed by RT-qPCR using GAPDH and YWHAZ as reference genes. A two-way ANOVA and a Student's t-test were used to analyse the results from the RT-qPCR. As expected, siSETDB1-injected morulae displayed dramatic reduction in the level of Setdb1 transcripts as compared to siNULL control (96%; P < 0.05). Preliminary analysis of LINE1 transcripts at the morula stage indicated siSETDB1-injected embryos displayed a 75% reduction compared to the siNULL. Whether alteration in LINE1 regulation contributes to the developmental arrest and embryonic mortality of siSETDB1-injected embryos is under investigation.