Development of a surgical technique for long-term catheterisation of bovine fetuses.

The aim of this study was to further develop and extensively describe a surgical technique in order to realise long-term fetal blood sampling in the bovine species. Eleven Holstein-Friesian 6- to 8-month pregnant cows (4-10 years old) were used for this study. Gestational age on the day of surgery varied from approximately 180 days (n = 1) to 240 days (minimum: 232 days, maximum 252 days; n = 10). The fetal medial tarsal artery was catheterised in pregnant cows with a polyvinyl catheter in dorsal recumbency under general anaesthesia. Although 5 out of 11 operations (45.5%) performed between 232 and 252 days of gestation were lost due to different causes mainly associated with peritonitis and septicaemia, the mean interval between operations and calvings was 42.5 days (between 27 and 95 days). It is important to emphasise that a well-trained surgical team is needed for bovine fetal cannulation in order to be able to decrease the risk factors during the operations. Due to the fact that after 5 unsuccessful cases none of the pregnancies were lost, this skill can be reached, and our technique can enable bovine fetal blood sampling for long-term endocrinological and physiological investigations before and during parturition.

Onset of puberty and regularity of oestral cycles in ewe lambs of four breeds under high-altitude conditions in a non-seasonal country.

Twenty-four Colombian Creole, Romney Marsh, Hampshire and Corriedale ewes raised under high-altitude conditions in a non-seasonal country were used to determine and to characterize the onset of puberty and of regularity ovarian function since the age of 3months. They underwent blood collection for determination of progesterone and monthly weight assessment. Oestrus was assessed daily by a teaser ram. In all breeds, age at onset of puberty ranged from 5.4 to 6.9 months and age at onset of regular ovarian function ranged from 7.4 to 8.6 months. Colombian Creole showed a higher body development at onset of puberty: 73.5 ± 8.3% versus 56.2 ± 7.4 in Romney Marsh, 58.8 ± 10.4 in Hampshire, and 57.3 ± 8.0 in Corriedale (p < 0.05), as well as a higher relative daily weight gain (+17%, p < 0.05). A negative correlation between daily weight gain and age at onset of puberty was established. Progesterone at onset of puberty and of regularity did not differ between breeds, but characterization of the luteal phase by the progesterone area under the curve (P4-AUC) revealed at both time points significantly lower values for Creole lambs (p < 0.05). Decreased P4-AUC paralleled a higher proportion of short oestral cycles observed prior to onset of regularity in Creoles, whereas an increased proportion of extra-long cycles were recorded in Romney Marsh (p < 0.05). These results establish first reference data for economically important breeds raised under tropical conditions. In comparison with other breeds, Colombian Creole requires a higher body development to achieve puberty and that an important proportion of short characterizes its prepubertal period cycles.

Evaluation of different hormonal treatments on oestral and ovarian responses in Moroccan Beni Arouss goats during anoestrus and breeding season.

The efficacy of eight combinations of fluorogestone acetate (FGA, 20 or 40 mg as intravaginal device during 11 days), equine chorionic gonadotropin (eCG, 300 or 500 UI injected 48 hr before FGA removal) and prostaglandin F2α (cloprostenol, 0 or 50 µg injected 48 hr before FGA removal) aiming at induction and synchronization of oestrus and ovulation was evaluated during the anoestrus season in spring and during the breeding season in autumn in adult Beni Arouss goats. Oestrous behaviour was recorded between 12 and 60 hr after FGA removal. Blood samplings allowing to assess onset of the pre-ovulatory LH surge and increase of progesterone as sign of an active corpus luteum were performed, respectively, between 20 and 60 hr and 3, 5, 8 and 15 days after FGA removal. No season-related differences (spring vs. autumn) were observed for oestrous response (95% vs. 93%), pre-ovulatory LH surge (94% vs. 84%) and luteal response after 3-8 and 11-15 days post-treatment (respectively 92% vs. 66% and 92% vs. 98%). The onset of oestrus (21 [13-53] vs. 32 [12-54] hr) and LH surge (26 [20-60] vs. 38 [22-60] hr) occurred significantly later in autumn. FGA (40 vs. 20 mg) in autumn significantly delayed the onset of oestrus (36 [16-54] vs. 23 [12-47] hr) and LH surge (44 [26-58] vs. 33 [22-60] hr). Significant treatment-related differences were recorded for onset of LH surge (earliest for 20 mg FGA, 300 IU eCG, 50 µg PGF2α ) and onset of luteal phase (latest for 40 mg FGA, 300 IU eCG, 50 µg PGF2α ). In conclusion, the hormone combinations tested appeared equally effective in terms of oestrous and ovulation rates. Season has influenced significantly the onset of oestrus and LH surge, and the high dose regimen of FGA delayed the ovarian response in autumn.

Correlation of two radioimmunoassay systems for measuring plasma pregnancy-associated glycoproteins concentrations during early pregnancy and postpartum periods in water buffalo.

This is the first time that PAG determination using two different antisera raised against PAG molecules purified from both caprine (RIA-706) and bubaline placentas (RIA-860) is reported in water buffalo. Ninety-eight buffalo cows, belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) programme, were enrolled in this study. Blood samples were taken on days 0 (AI), 23, 25, 28, 30 and 45. Pregnancy was confirmed by ultrasonography on days 28 and 45. The blood of 20 buffaloes that had calved was tested every five days from the day of calving until day 50 postcalving. Differences in PAG concentrations were observed between pregnant and nonpregnant buffaloes starting from day 23 post AI using both RIA-706 and RIA-860 (p < 0.001). However, estimated mean concentrations of PAG measured by RIA-706 were higher than RIA-860 (p < 0.001) and Bland-Altman analysis showed biases ranged from 0.0 ng/ml at day 23 to 0.79 ng/ml at day 28 post AI. Moreover, RIA-706 showed greater sensitivity and accuracy both at 23 and 25 days of pregnancy. RIA-706 and RIA-860 decreased below 1 ng/ml from 40 and 30 days postpartum, respectively, suggesting that PAG are better recognized by the antisera raised against the caprine PAG in the postpartum period also. This is essential when using PAG as an appropriate marker of early pregnancy after postpartum for detecting new pregnancies. The results of this study show that the ability of RIA systems to recognize early PAG could be improved using antisera raised against PAG molecules isolated from caprine placenta.

Relationship of progesterone, bovine pregnancy-associated glycoprotein-1 and nitric oxide with late embryonic and early fetal mortalities in dairy cows.

The aim of the present study was to determine the relationship of progesterone (P4), bovine pregnancy-associated glycoprotein-1 (bPAG-1) and nitric oxide (NO) levels with late embryonic (LEM; day 28 to day 42) and early fetal mortalities (EFM; > day 42 to day 56) in dairy cows. Transrectal ultrasonography (6-8 MHz) was performed in 100 Holstein-Friesian cows at days 28, 42 and 56 after artificial insemination (AI; day 0) to diagnose pregnancy and to monitor the fate of the embryo. After ultrasound scanning of each cow, a milk sample was collected for assessment of P4 by an ELISA test and a blood sample was collected for assessment of bPAG-1, by using a double-antibody radioimmunoassay, and serum NO metabolites (nitrate + nitrite). Based on ultrasonographic examinations and bPAG-1-RIA, 41 of 100 inseminated cows were confirmed pregnant at day 28 after AI. Nine cows suffered of LEM, and 6 cows suffered of EFM and the overall pregnancy loss rate was 36.6% (15/41) between days 28 and 56 of pregnancy. By logistic regression analysis, there were no significant relationships between the level of P4 and bPAG-1 at day 28 after AI and the occurrence of LEM and EFM. Also, there were no significant relationships between the levels of P4 and bPAG-1 at day 42 and the occurrence of EFM. On the other hand, a significant relationship (P<0.05) was found between NO level at day 28 and the occurrence of LEM. In conclusion, measurement of the serum NO concentration at day 28 of pregnancy might help to predict the outcome of pregnancy by day 42 in dairy cows but further studies are needed to confirm this.

Purification of pregnancy-associated glycoproteins from late-pregnancy Bubalus bubalis placentas and development of a radioimmunoassay for pregnancy diagnosis in water buffalo females.

BACKGROUND: Pregnancy-associated glycoproteins (PAGs) were first described as placental antigens present in the blood serum of the mother soon after implantation. Here, we describe the purification of several pregnancy-associated glycoproteins from water buffalo placenta (wbPAGs). A specific radioimmunoassay (RIA) was developed for early pregnancy diagnosis in buffalo species. RESULTS: Amino-terminal microsequencing of immunoreactive placental proteins allowed the identification of eleven wbPAGs sequences [Swiss-Prot accession numbers: P86369 to P86379]. Three polyclonal antisera (AS#858, AS#859 and AS#860) were raised in rabbits against distinct wbPAG fractions. A new RIA (RIA-860) was developed and used to distinguish between pregnant (n=33) and non-pregnant (n=26) water buffalo females. CONCLUSIONS: Our results confirmed the multiplicity of PAG expression in buffalo placenta. In addition, the RIA-860 system was shown to be sensitive, linear, reproducible, accurate and specific in measuring PAG concentrations in buffalo plasma samples from Day 37 of gestation onwards.

Ultrasound measurements of uterine height, horns diameter and presence of intraluminal fluid to investigate uterine involution in lactating sows housed in farrowing crates.

The uterine involution of sows housed in farrowing crates was investigated during lactation using B-mode trans-abdominal ultrasonography. The objectives were to describe uterine involution, detect any delay or uterine disorders and assess possible associations between involution and subsequent reproductive performance. Three parameters were measured: uterine height (H), horns diameter (D) and the percentage of sows with intraluminal fluid (F). During lactation (3-4 weeks), H decreased from 11.0±1.6 the first week to 5.9±1.5 cm the last week (p<0.001), and D from 2.6±0.7 to 1.4±0.2 cm (p<0.001). Between days 1-7, H and D decreased significantly faster, i.e. respectively 0.38 cm (p<0.0001) and 0.20 cm (p<0.0001) per day than between days 22-28, i.e. respectively 0.02 cm (p=0.49) and 0.00 cm (p=0.75) per day. F decreased significantly (p<0.0001) from 78% at the beginning to 16% at the end of lactation. Between days 1-7, F decreased significantly (p<0.001) faster than during the last week of lactation (p=0.41). Between days 22-28, H of sows from parity ≥3 were significantly higher than those of sows from parity 1 and 2 (p=0.007). During that period, F was significantly higher in sows of higher parity. This effect of parity on F was significantly higher during the entire lactation period in sows of parity ≥6. Some sows were monitored after weaning. There was no significant relationship between the 3 parameters measured at the end of lactation and the subsequent performance. A small number of sows was suspected of endometritis (2%) and one case of fœtoplacental retention was detected. In conclusion, B-mode ultrasonography is a suitable tool to monitor uterine involution in lactating sows. When examination is conducted during the last week of lactation, it may help the farmer to verify whether uterine involution is complete, and to decide whether a sow should be either culled or maintained on farm.

In vivo oocyte developmental competence is reduced in lean but not in obese superovulated dairy cows after intraovarian administration of IGF1.

The present study investigated the role of IGF1 in lactating lean and non-lactating obese dairy cows by injecting 1 µg IGF1 into the ovaries prior to superovulation. This amount of IGF1 has been linked with pregnancy loss in women with the polycystic ovary syndrome (PCOS) and was associated with impaired bovine oocyte competence in vitro. Transcript abundance and protein expression of selected genes involved in apoptosis, glucose metabolism, and the IGF system were analyzed. Plasma concentrations of IGF1 and leptin, and IGF1 in uterine luminal fluid (ULF), were also measured. IGF1 treatment decreased embryo viability in lean cows to the levels observed in obese cows. Obese cows were not affected by IGF1 treatment and showed elevated levels of IGF1 (in both plasma and ULF) and leptin. Blastocysts from lean cows treated with IGF1 showed a higher abundance of SLC2A1 and IGFBP3 transcripts. IGF1 treatment reduced protein expression of tumor protein 53 in blastocysts of lean cows, whereas the opposite was observed in obese cows. IGF1 in plasma and ULF was correlated only in the control groups. Blastocyst transcript abundance of IGF1 receptor and IGFBP3 correlated positively with IGF1 concentrations in both plasma and ULF in lean cows. The detrimental microenvironment created by IGF1 injection in lean cows and the lack of effect in obese cows resemble to a certain extent the situation observed in PCOS patients, where IGF1 bioavailability is increased in normal-weight women but reduced in obese women, suggesting that this bovine model could be useful for studying IGF1 involvement in PCOS.

Approaches to Identify Pregnancy Failure in Buffalo Cows.

The aim of this work was to find the best strategy to diagnose pregnancy failures in buffalo. A total of 109 animals belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) program were enrolled in this study. Blood samples were collected at days 0, 14, 25, 28 and 40 after AI for the determination of progesterone (P4) and pregnancy-associated glycoproteins (PAGs) by the radioimmunoassay (RIA) method. Transrectal ultrasonography was performed on day 25, 28 and 40 after AI to monitor pregnancy. The animals included in the data analysis were assigned ex post in pregnant (n = 50) and mortality (n = 12) groups. By ultrasonography, the predictive sign of mortality was the heartbeat. At day 25, the PAGs concentration was significant in predicting embryonic mortality with respect to ultrasonography and P4, at the cut-off of 1.1 ng/mL. At day 28, either PAGs, at a cut-off of 2.2 ng/mL, or ultrasonography, with no detection of heartbeat, were highly predictive of embryonic mortality. PAGs were the best marker (p < 0.05) for predicting embryonic mortality between 25 and 40 days of gestation in buffalo. Its utilization as a diagnostic tool can influence management decisions in order to improve farm reproductive management.

Transmission of Bluetongue Virus Serotype 8 by Artificial Insemination with Frozen-Thawed Semen from Naturally Infected Bulls.

Transmission of bluetongue (BT) virus serotype 8 (BTV-8) via artificial insemination of contaminated frozen semen from naturally infected bulls was investigated in two independent experiments. Healthy, BT negative heifers were hormonally synchronized and artificially inseminated at oestrus. In total, six groups of three heifers received semen from four batches derived from three bulls naturally infected with BTV-8. Each experiment included one control heifer that was not inseminated and that remained BT negative throughout. BTV viraemia and seroconversion were determined in 8 out of 18 inseminated heifers, and BTV was isolated from five of these animals. These eight heifers only displayed mild clinical signs of BT, if any at all, but six of them experienced pregnancy loss between weeks four and eight of gestation, and five of them became BT PCR and antibody positive. The other two infected heifers gave birth at term to two healthy and BT negative calves. The BT viral load varied among the semen batches used and this had a significant impact on the infection rate, the time of onset of viraemia post artificial insemination, and the gestational stage at which pregnancy loss occurred. These results, which confirm unusual features of BTV-8 infection, should not be extrapolated to infection with other BTV strains without thorough evaluation. This study also adds weight to the hypothesis that the re-emergence of BTV-8 in France in 2015 may be attributable to the use of contaminated bovine semen.

Determination of sex and scrapie resistance genotype in preimplantation ovine embryos.

The aim of this study was to test the accuracy of genotype diagnosis after pre-amplification of DNA extracted from biopsies obtained by microblade cutting of ovine embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer to recipients. Sex and PrP genotypes were determined. Sex diagnosis was done by PCR amplification of ZFX/ZFY and SRY sequences after PEP-PCR while PrP genotype determination was performed after specific pre-amplification of specific target including codons 136, 154 and 171. Embryos were collected at Day 7 after oestrus. Blastocysts and expanded blastocysts were biopsied immediately after collection whereas compacted morulae were biopsied after 24 hr of in vitro culture. Eighty-nine biopsied embryos were frozen by vitrification. Fresh and vitrified whole embryos were kept as control. DNA of biopsies was extracted and pre-amplified. Sex diagnosis was efficient for 96.6% of biopsies and PrP genotyping was determined in 95.8% of codons. After embryo transfer, no significant difference was observed in lambing rate between biopsied, vitrified control and fresh embryos (54.5%, 60% and 66.6%, respectively). Embryo survival rate was not different between biopsied and whole vitrified embryos (P = 0.38). At birth, 96.7% of diagnosed sex and 95.4% of predetermined codons were correct. Lamb PrP profiles were in agreement with parental genotype. PEP-PCR coupled with sex diagnosis and nested PCR coupled with PrP genotype predetermination are very accurate techniques to genotype ovine embryo before transfer. These original results allow planning of selection of resistant genotype to scrapie and sex of offspring before transfer of cryopreserved embryo.

Characterization of CD79αcy+ cells in placentas from ruminants.

Previous work carried out to characterise different immune cells in ruminant placentas found strong CD79αcy nuclear labelling in cells histologically resembling trophoblast cells. In the attempt to characterize this cell population, placentomes collected from cattle, sheep and water buffaloes were examined by immunohistochemistry with single and double labelling using monoclonal antibodies (mAb) against B lymphocytes and trophoblast cells. Most CD79αcy + cells co-expressed placental lactogen or cytokeratin and were CD21 and MHC class II negative strongly suggesting they do not have a B cell origin. However, a potential immunological role of these cells cannot be ruled out and it is currently unknown if the findings described may have an impact on physiological knowledge, health, and or diseases pathogenesis in ruminants.

Investigation of PAG2 mRNA Expression in Water Buffalo Peripheral Blood Mononuclear Cells and Polymorphonuclear Leukocytes from Maternal Blood at the Peri-Implantation Period.

: The main objective of this study was to assess PAG2 mRNA expression in maternal blood cells at the peri-implantation period in water buffalo; moreover, we wanted to evaluate the earliest time in which PAG-2 could be detected in maternal blood. Thirty-two lactating buffaloes artificially inseminated (AI) were utilized. Blood was collected at Days 0, 14, 18, 28, 40 after AI (AI = day 0). Pregnancy was diagnosed by ultrasound at Days 28 and 40 post AI. Out of 32 buffaloes, 14 were pregnant (P group) and 18 were not pregnant (NP group). The plasma PAG-2 threshold of 1.0 ng/mL in the P group was reached at day 40 post AI. PAG2 mRNA expression differed between the P and NP groups, and was either evaluated in Peripheral Blood Mononuclear Cells (PBMC) or Polymorphonuclear Leukocytes (PMN), starting from day 14. However, both the estimated marginal means and multiple comparisons showed that PAG2 mRNA expression was higher in PMN than PBMC. In the present study, PAG-2 appeared in the blood (40 Days post AI), and an early expression of PAG2 mRNA at Day 14 post AI was also observed. Although further research is undoubtedly required, PAG2 mRNA in peripheral blood leukocytes could be using to better understand the role that PAGs play during pregnancy in buffalo.

Native and recombinant bovine placental lactogens.

The bovine placenta produces a wide variety of proteins that are structurally and functionally similar to the pituitary proteins from the GH/PRL gene family. Bovine placental lactogen (bPL) is a 200-amino acid long glycoprotein hormone that exhibits both lactogenic and somatogenic properties. The apparent molecular masses of purified native (n) bPL molecules (31-33 kDa) exceed 23 041 Da, which is the theoretical molecular mass of the protein core. At least six isoelectric variants (pI: 4.85-6.3) of bPL were described in cotyledonary extracts and three different bPL isoforms (pI: 4.85-5.25) were found in fetal sera. The bPL molecules that are detected in higher concentrations in peripheral circulation exhibit a more acidic pI than those present in placental homogenates. This may reflect an important glycosylation process occurring just prior to the bPL secretion. The bPL mRNA is transcribed in trophectoderm binucleate cells starting from Day 30 of pregnancy until the end of gestation. In mothers, bPL is involved in the regulation of ovarian function, mammogenesis, lactogenesis, and pregnancy stage-dependent adaptation of nutrient supplies to the fetus. Due to the higher fetal, compared to maternal concentrations of circulating hormone, it has been suggested that bPL primarily targets fetal tissues.

Development and validation of a radioimmunoassay for thyrotropin in cattle.

In mammals, thyrotropin, or thyroid-stimulating hormone (TSH), assay is used for the diagnosis of primary hypothyroidism. Hypothyroidism is the most common type of thyroid disorder in cattle. The aim of this study was to develop and validate, under physiologic and pathologic conditions, a radioimmunoassay (RIA) for bovine TSH (bTSH). Double RIA was performed with purified bTSH and specific bovine antiserum. Laboratory validation included research of minimal detection limit, accuracy, and reproducibility. The physiologic validation included a thyrotropin-releasing hormone (TRH) challenge performed on euthyroid cows and a follow-up of bTSH concentration over a 24-hour period. Furthermore, bTSH concentration was assayed in a large population of healthy dairy and beef cows to define reference interval. The pathologic validation was made by assaying bTSH and thyroid hormones on healthy and goitrous newborn calves. The minimum detection limit (MDL) for bTSH assay was 1.3 microU/ml. The recovery was 101% to 106%. The intra- and interassay coefficients of variation (CVs) ranged from 5% to 11% and 11% to 15%, respectively. The RIA covered the whole range of physiologic bTSH values, as shown by bTSH values induced by TRH-challenge. A pulsatile secretion of bTSH was observed, accompanied by a diurnal variation with lower night values than day values. Reference intervals of bTSH ranged from 1.3 to 13.0 microU/ml for beef and dairy breeds. Finally, bTSH easily discriminated goitrous newborn calves from healthy ones, leading to the definition of a cutoff value of 35 microU/ml. The bTSH assay positively reacted to physiologic and pathologic conditions. The accuracy and precision of the RIA were satisfying.

Effect of the luteinizing hormone on embryo production in superovulated rabbit does.

For most domestic animals, the responses to superovulation treatments are not controlled as a consequence of the lack of knowledge on exogenous gonadotrophins effects on the ovarian function. The role of luteinizing hormone (LH) on the number and quality of embryos produced was evaluated on rabbit does superovulated with porcine FSH (pFSH). Parameters of embryos recovery, in vitro and in vivo embryo development rates after freezing/thawing were compared. We used three experimental groups: (1) control group without superovulation treatment, (2) "pFSH+pLH" and (3) "pFSH" groups where females were treated with pFSH, respectively, with (20%) or without (0%) porcine LH supplementation. The number of corpora lutea and the number of embryos produced were significantly higher (p<0.001) in superovulated does than in control group (27.1, 26.7 versus 11.9 corpora lutea and 20.3, 21.2 versus 9.6 embryos produced for pFSH+pLH, pFSH and control group, respectively). However, both gonadotrophins administrations (groups 2 and 3) led to defaults of ovulation when compared with untreated does. No significant difference was observed between the number and quality of the embryos produced by does treated with pFSH+pLH or with pFSH alone. Moreover, we observed no significant difference between results of in vivo and in vitro viability assays after thawing. We concluded that pFSH alone seems to be sufficient to stimulate the follicles growth and that exogenous pLH administrated has no effect on the quantity and quality of embryos. Further studies are needed to evaluate the hormonal patterns before and after the gonadotrophins injections in the rabbit species.

Evaluation of GnRH treatment 12 days after AI in the reproductive performance of dairy cows.

The objective of this study was to evaluate the effects of GnRH administered at Day 12 post-AI on the reproductive performance of dairy cows. Holstein-Friesian dairy cows (n=103) on a large Hungarian dairy farm were allocated randomly to treated (n=54) or control (n=49) groups. Twelve days after AI, treated cows received a GnRH agonist i.m., while the control group received a placebo (physiological saline). Progesterone radioimmunoassay was used to determine the correct timing of artificial insemination (Day 0) and the incidence of luteal insufficiency on Day 12. Ultrasonography and radioimmunoassay for pregnancy-associated glycoprotein were used to detect pregnancy and late embryonic/fetal mortality between Days 32 and 55 after AI. Three cows from each group were inseminated when progesterone concentrations were >1.0 ng/mL, and six cows (four from the treated and two from the control group) had luteal insufficiency (progesterone<1.0 ng/mL) on Day 12. Late embryonic/fetal mortality occurred in three treated cows and in two control cows. When these cows were removed from the model, calving rates after first service were 59.6% (28/47) and 59.1% (26/44) for treated and control cows, respectively (P>0.05). There was no significant difference between treated and control cows when they were inseminated before or after Day 100 from calving. In summary, administration of a GnRH agonist on Day 12 after AI did not improve reproductive performance in dairy cows. However, our approach may be used for the field evaluation of different treatment protocols.

Intrinsic quality of goat oocytes already found denuded at collection for in vitro embryo production.

Although cumulus cells are essential for efficient oocyte maturation, the establishment of protocols that support IVD of embryos obtained from denuded oocytes (DOCs) is important for optimizing the use of reproductive biotechnologies. Thus, this study aimed to establish a protocol for IVD of goat DOC using different strategies of IVM and methods of oocyte activation. Four experiments were performed. Similar developmental competence of slaughterhouse DOC was obtained, regardless of maturation media (complex, semidefined or simplified). However, the ability to reach the blastocyst stage was affected by the activation method. Denuded oocytes subjected to parthenogenetic activation had greater (P < 0.05) development capacity, compared with those undergoing IVF with average cleavage rate of 83% and 75%, blastocyst rate of 49% and 28%, and blastocysts in relation to the cleaved embryos of 59% and 38, respectively. In addition, the quality of embryos evaluated after vitrification/warming was similar between parthenogenetic activation and IVF. Finally, we demonstrated that the coculture of cumulus-oocyte complex (COC) with DOC increased the competence of DOC at a ratio of 1:1 and 1:9 (DOC:COC). We believe that presence of cumulus cells (CCs) is not essential to the meiotic maturation, if at the time of removal of the oocyte from follicular environment, they already acquired competence to development. However, when the oocytes still need to acquire competence, the presence of CC may significantly contribute in their developmental capacity acquisition during IVM. Thus, regardless of the source, these oocytes will require longer time in IVM, contrary to what happens in the absence of CC. In conclusion, although DOC had a lower developmental potential, especially after IVF, they were able to produce blastocysts and the coculture of DOC with COC increased this developmental capacity.

Multiple forms of Pregnancy-Associated Glycoproteins released in vitro by porcine chorion or placentomal and interplacentomal explants of wild and domestic ruminants.

Characterization of the Pregnancy-Associated Glycoproteins (PAG) is important for studies of reproduction of various eutherian domestic, wild and endangered mammals. Distinct chorionic PAG genes are expressed in embryo-origin cells: pre-placental trophoblast (TR) and in placental trophectoderm (TRD) of various entherians. This study demonstrates in vitro production of the PAG proteins during long-term cultures of various chorionic explants: porcine TR or TRD, cotyledonary (CT) of European bison (Eb), and CT or intercotyledonary (intCT)-TRD of the cattle. Chorionic proteins isolated from media were analyzed by homologous or heterologous Western immunoblotting with anti-PAG sera, raised against cellular bovine or secretory porcine antigens. Used anti-PAG sera identified diverse molecular forms of released PAG proteins: 43-69 kDa for EbPAG proteins, 40-85 kDa for bovine PAG (bPAG), and 43-73 kDa for porcine PAG (pPAG). Immunoblotting revealed also that both CT and intCT-TRD explants secreted equivalent amounts of bPAG proteins. This useful system of in vitro protein production can provide native chorionic PAG proteins with placental unique carbohydrate chains. The PAG proteins are required as standard markers for diagnostic tests of pregnancy in domestic and wild mammals, in which seasonal reproductive processes are relatively difficult to control.

Use of a homologous radioimmunoassay (RIA) to evaluate the effect of maternal and foetal parameters on pregnancy-associated glycoprotein (PAG) concentrations in sheep.

Early pregnancy detection and prediction of the number of lambs would be profitable for sheep breeders because it enables them to adjust nourishment of pregnant ewes according to the individual needs in order to prevent health problems around parturition. The concentration of ovPAG has previously been reported to be related with maternal parameters (farm, breed and age) as well as foetal parameters (number of lambs, their sex and birth weight), but contradictory results were obtained in different small-scale studies. This large-scale study evaluates the effect of these parameters on the ovPAG concentration, determined by a homologous radioimmunoassay (RIA), and it further investigates the possibility to predict the number of lambs by means of homologous ovPAG determination. Eighty-three and ninety-five ewes of the Suffolk and Texel breed, respectively, housed on four different farms (experiment 1) and 68 ewes of the Suffolk breed, housed on two different farms (experiment 2) were included in this study, and their estrous cycles were synchronised using a progesterone analogue. On the day of synchronisation (D-14) and at 25 (D25), 35 (D35) and 45 (D45) days after insemination, blood samples were taken and a homologous radioimmunoassay (RIA) was used to determine the ovPAG concentrations. At parturition, age of the ewe, number and sex of the lambs (experiment 1) and birth weight (experiment 2) were registered. OvPAG concentrations were not affected by age of the ewe and sex of the lambs. Farm and breed of the ewes, number and birth weight of the lambs had a significant effect on ovPAG concentrations at all time points (P<0.05). The odds of multiple lambs increased significantly with increasing ovPAG concentration, although prediction of litter size based on ovPAG concentration at the individual ewe level was not useful due to small sensitivity and/or specificity whatever the cutoff value used. In conclusion, the ovPAG concentration is affected by farm and breed of the ewes, and number and birth weight of the lambs.