Prion protein testis specific (PRNT) gene polymorphisms and transcript level in ovine spermatozoa: Implications in freezability, fertilization and embryo production.

An essential role of prion protein testis specific (PRNT) and prion protein 2 dublet (PRND) genes in the male reproductive function has been highlighted, although a deeper knowledge for the mechanisms involved is still lacking. Our goal was to determine the importance of the PRNT haplotypic variants and mRNA expression levels in ovine spermatozoa freezability and ability for fertilization and embryo developmental processes. Their association with the PRND gene polymorphisms was also analyzed. DNA from rams belonging to three Portuguese sheep breeds (n = 28) was screened by single-strand conformation polymorphism (SSCP) analysis to identify the PRNT and PRND polymorphisms. Semen collected from these rams was cryopreserved and fertility traits evaluated. The SSCP analyses revealed polymorphisms in the codons 6, 38, 43 and 48 of the PRNT coding region - respectively c.17C > T (p.Ser6Phe, which disrupts a consensus arginine-X-X serine/threonine motif); c.112G > C (p.Gly38 > Arg); and synonymous c.129T > C and c.144A > G. The polymorphisms in codons 6, 38 and 48 occur simultaneously while the one in codon 43 occurs independently. Six haplotypes were identified in the PRNT coding region, resulting in three different amino acid polymorphic variants (6S-38G-43C-48V, S6F-G38R-43C-48V and 6F-38R-43C-48V). The PRNT gene mRNA transcript level in spermatozoa was related to the identified haplotypic variants, either considering the codons 6-38-48 (P ≤ 0.0001) or the codon 43 alone (P ≤ 0.0001) or altogether (P ≤ 0.0001). An interaction between PRNT haplotypes and PRND genotypes on PRNT transcript level was also identified (P = 0.0003). Rams carrying the 17C-112G-144A PRNT haplotype had sperm with the highest post-thawed individual motility (P ≤ 0.03). Combined PRNT and PRND polymorphic variation influenced the post-thawed individual motility (P = 0.01). The male PRNT haplotypic, either considering the codons 6-38-48 and 43 altogether or the codon 43 alone, interfered (P ≤ 0.04) in embryo production rates. In conclusion, our data confirm that the PRNT gene is highly polymorphic in sheep and that the PRNT and PRND genotypes are associated. The identified polymorphisms of PRNT coding region seems to interfere on the ram spermatozoa mRNA transcript level and on male fertility, specifically in sperm freezability and ability for embryo development.

Bovine oocyte membrane permeability and cryosurvival: Effects of different cryoprotectants and calcium in the vitrification media.

The cryopreservation process must be improved to enhance oocyte cryosurvival and functionality. Two protocols with different cryoprotectants (CPAs), containing either ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose (EGDMSO) or 1,2-propanediol and sucrose (PrOH) were evaluated. In both protocols, calcium (Ca2+) free or -containing base media were tested. Oocytes were subjected to vitrification or only exposed to CPAs without immersion in liquid nitrogen. Oocyte's viability, cortical granules location and competence for development after fertilization were assessed. Finally, fatty acid composition and membrane permeability of oocytes exposed to CPAs were analyzed. Independently of Ca2+ concentration in the vitrification media, the development rates were higher in oocytes vitrified with EGDMSO protocols (p = 0.0005). After warming, higher cleavage rates were obtained in EGDMSO + Ca2+ compared to the PrOH without Ca2+ protocol (p = 0.02). Oocytes exposed to PrOH without Ca2+ presented lower cleavage rates compared to control (p = 0.04). An enhanced premature zona hardening in vitrified oocytes as well as lower concentrations of the fatty acids c11:18:1 and 20:4n-6 in cumulus oocyte complexes exposed to PrOH protocols were identified. The oocytes minimum volume and permeability were affected by the exposure to PrOH and Ca2+ (p ≤ 0.007). In conclusion, the most effective protocol for bovine oocytes cryopreservation combines EG and DMSO, independently of Ca2+ concentration in the media. A higher toxicity and an incomplete depletion of water during PrOH loading may hamper oocyte viability. The type of CPAs and Ca2+ interfered differentially on oocyte pathways to functionality, and this should be considered when choosing a cryopreservation protocol.

Prion protein 2 (dublet) gene (PRND): role in ovine semen capacitation, cryopreservation and fertility.

The aim of the present study was to examine the role of Doppel protein in the capacitation process and fertilising ability of both fresh and frozen-thawed (FT) spermatozoa from rams carrying different prion protein 2 (dublet) (PRND) gene polymorphisms. The detection efficacy of new anti-Doppel monoclonal antibodies and PRND mRNA quantification were also explored in ovine spermatozoa. Three different genotypes (AA, GA, GG) were identified for codon 26 of ovine PRND-c.78G>A. Using flow cytometry, a higher fluorescence was detected in fresh compared with FT sperm samples incubated with anti-Doppel primary and fluorescein isothiocyanate-conjugated secondary antibodies (P<0.05). Capacitation was affected by semen treatment (fresh and FT) and male PRND genotype (P<0.05). After IVF, the use of fresh semen resulted in a higher cleavage rate than the use of FT spermatozoa (P=0.004). IVF using spermatozoa from individuals classified as carriers of the AA or GA PRND genotypes resulted in higher cleavage rates than seen using spermatozoa from GG carriers (P≤0.0006). Finally, using semen from rams with the AA PRND genotype resulted in the highest Day 6 and Day 8 embryo rates (P≤0.04). In conclusion, the results of the present study confirm that the identification of different PRND genotypes is important for studying the sperm capacitation process and for improving sperm cryoresistance and embryo production. Furthermore, the detection of Doppel in ejaculated ovine spermatozoa, along with its low expression after cryopreservation, strongly suggests an important physiological function of this protein in male fertility.

The prion-related protein (testis-specific) gene (PRNT) is highly polymorphic in Portuguese sheep.

The objective of this study was to search for polymorphisms in the ovine prion-related protein (testis-specific) gene (PRNT). Sampling included 567 sheep from eight Portuguese breeds. The PRNT gene-coding region was analyzed by single-strand conformation polymorphism and sequencing, allowing the identification of the first ovine PRNT polymorphisms, in codons 6, 38, 43 and 48: c.17C>T (p.Ser6Phe, which disrupts a consensus arginine-X-X-serine/threonine motif); c.112G>C (p.Gly38>Arg); c.129T>C and c.144A>G (synonymous) respectively. Polymorphisms in codons 6, 38 and 48 occur simultaneously in 50.6% of the animals, 38.8% presenting as heterozygous. To study the distribution of the polymorphism in codon 43, a restriction fragment length polymorphism analysis was performed. Polymorphic variant c.129C, identified in 89.8% of the animals with 32.8% presented as heterozygous, was considered the wild genotype in Portuguese sheep. Eight different haplotypes which have comparable distribution in all breeds were identified for the PRNT gene. In conclusion, the PRNT coding region is highly polymorphic in sheep, unlike the prion protein 2 dublet gene (PRND), in which we previously found only one synonymous substitution (c.78G>A), in codon 26. The absence or reduced number of PRND heterozygotes (c.78G>A) was significantly associated with three PRNT haplotypes (17C-112G-129T-144A,17CT-112GC-129CT-144AG and 17T-112C-129C-144G), and the only three animals found homozygous at c.78A had the 17C-112G-129C-144A PRNT haplotype. These results constitute evidence of an association between polymorphic variation in PRND and PRNT genes, as has already been observed for PRND and prion protein gene (PRNP).

Cryopreservation of in vitro-produced sheep embryos: Effects of different protocols of lipid reduction.

UNLABELLED: The low survival of sheep in vitro-produced (IVP) embryos after cryopreservation is a key limiting step to the widespread of this technology. In the present work, different approaches for enhancing cryosurvival of these embryos were compared: embryo delipidation by centrifugation in the absence or presence of cytochalasin D, a cytoskeleton stabilizer or by embryo culture in the presence of different doses of the trans-10 cis-12-conjugated linoleic acid isomer (CLA). Three experiments were conducted. In experiment 1, IVP blastocysts before vitrification were randomly distributed into four groups: control; centrifuged (cent), cytochalasin D (cyto-D), centrifuged + cytochalasin D (cent + cyto-D). In experiment 2, different doses of CLA (25, 50, and 100 µM) were supplemented during embryo culture before vitrification of blastocysts. A control group ran simultaneously. A third experiment was performed to compare both approaches from the previous ones but without the groups with the worst results (groups: control, cyto-D, cent + cyto-D, CLA25, CLA50). In all experiments, embryos integrity and reexpansion were assessed after warming and after 3 hours of culture. In experiment 1, the postwarming integrity rate was the lowest (P < 0.05) in embryos from the cent group (cent: 50.6 ± 10.3% vs. CONTROL: 74.6 ± 9.2%, cyto-D: 92.3 ± 9.7%, and cent + cyto-D: 90.5 ± 11.2%), whereas the best (P < 0.05) reexpansion scores were obtained in cent + cyto-D embryos (cent + cyto-D: 2.6 ± 0.28 vs. CONTROL: 1.8 ± 0.08, cent: 1.9 ± 0.2, and cyto-D: 1.8 ± 0.31). In experiments 2 and 3, higher (P < 0.05) cleavage rates were observed in CLA25 (50.9 ± 6.2% and 49.2 ± 5.6%, respectively) and CLA50 (48.9 ± 6.2% and 47.6 ± 5.6%, respectively) than those in the control (41.8 ± 6.1% and 40.4 ± 5.4%, respectively) group. In experiment 2, CLA100 presented the lowest (P < 0.002) Day-6 and -7 embryo production rate and quality. After warming, superior (P < 0.02) expansion scores were achieved in CLA25 (3.1 ± 0.29) and CLA50 (3.8 ± 0.17) than in the control (1.9 ± 0.10) group. Similar results were attained in experiment 3. However, although cent + cyto-D embryos showed higher (P = 0.008) postwarming expansion scores than the control (2.8 ± 0.29 vs. 1.9 ± 0.07) group, this score was lower (P = 0.0009) than that in CLA50 embryos (3.8 ± 0.17). In conclusion, our results showed that different protocols of lipid reduction can be successfully applied to improve the cryotolerance of IVP sheep embryos.

Fatty acid composition of porcine cumulus oocyte complexes (COC) during maturation: effect of the lipid modulators trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA) and forskolin.

The effect of maturation and of two lipid modulators supplementation along in vitro maturation (IVM) on fatty acid (FA) and dimethylacetal (DMA) composition of porcine cumulus oocyte complexes (COC) were studied. Abattoir-derived immature COC were analyzed for FA and DMA or submitted to IVM as follows: control group; t10,c12 CLA group, t10,c12 CLA supplementation for 44 h; Forskolin group, forskolin supplementation during the initial 2 h; t10,c12 CLA + forskolin group, t10,c12 CLA for 44 h and forskolin for just 2h. Each experimental group had five replicates. FA analysis of oocytes, cumulus cells (CC), follicular fluid, and culture media were performed by gas-liquid chromatography. Oocytes and their CC had different FA composition. Oocytes were richer in saturated FA (SFA) preferentially maintaining their FA profile during maturation. Mature CC had the highest polyunsaturated FA (PUFA) content. Five individual and total SFA, and monounsaturated FA (MUFA), notably oleic acid (c9-18:1), percentages were lower (P ≤ 0.023) in mature than in immature CC. t10,c12 CLA was accumulated by COC from t10,c12 CLA and t10,c12 CLA + forskolin groups, mostly in CC where MUFA and an eicosatrienoic isomer decreased (P ≤ 0.043). Nevertheless, PUFA or FA and DMA total content were not affected. Arachidonic acid was reduced in t10,c12 CLA + forskolin CC and hexadecanal-DMA-16:0 in t10,c12 CLA CC. Forskolin alone increased (P ≤ 0.043) c9-18:1 in oocytes. In conclusion, maturation process clearly changed porcine COC FA and DMA profiles, mostly of CC, also more susceptible to modifications induced by t10,c12 CLA. This possibility of manipulating COC lipid composition during IVM could be used to improve oocyte quality/cryopreservation efficiency.

Secondary corpora lutea induced by HCG treatment enhanced demi-embryo survival in lactating high-yielding dairy cows.

Using a novel in vivo model considering a low developmental competence embryo (demi-embryo) and a subnormal fertility recipient (lactating high-yielding dairy cow), this experiment evaluated the effect of human chorionic gonadotrophin (hCG) treatment at embryo transfer (ET) on embryonic size at implantation, embryonic survival and recipient plasma progesterone (P4 ) and bovine pregnancy-specific protein B (PSPB) concentrations until day 63 of pregnancy. Embryos were bisected and each pair of demi-embryos was bilaterally transferred to recipients (n = 61) on day 7 of the oestrous cycle. At ET recipients were randomly assigned to treatment with 1500 IU hCG or to untreated controls. Higher (p < 0.01) pregnancy rates on days 25, 42 and 63, and embryo survival rate on day 63 were observed in hCG-treated cows with secondary CL than in hCG-treated cows without secondary CL and in untreated cows. Pregnancy rates and embryo survival rate were similar in hCG-treated cows without secondary CL and untreated cows. Embryonic size on day 42 was not affected by treatment with hCG, presence of secondary CL and type of pregnancy (single vs twin). Presence of secondary CL increased (p < 0.05) plasma P4 concentrations of pregnant cows on days 14, 19 and 25 but not thereafter and of non-pregnant cows on days 14-21. Treatment with hCG and presence of secondary CL had no effect on plasma PSPB concentrations, which were higher (p < 0.05) in twin than in single pregnancies. In conclusion, secondary CL induced by hCG treatment at ET significantly increased plasma P4 concentrations, the survival rate of demi-embryos and the pregnancy rate of high-yielding lactating dairy cows. Embryos were rescued beyond maternal recognition of pregnancy, but later embryonic survival, growth until implantation and placental PSPB secretion until day 63 of pregnancy were not affected by treatment or presence of secondary CL.

Fat area and lipid droplet morphology of porcine oocytes during in vitro maturation with trans-10, cis-12 conjugated linoleic acid and forskolin.

Lipid droplets (LD) in porcine oocytes form a dark mass reaching almost all cytoplasm. Herein we investigated changes in fat areas, cytoplasmic tone and LD morphology during in vitro maturation (IVM) of porcine oocytes cultured with 100 µM trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA) or 10 µM forskolin at different time periods. Four groups were constituted: control, excipient, t10,c12 CLA and forskolin, with drugs being supplemented during 44 to 48 h and the initial 22 to 24 h in Experiments 1 and 2, respectively. In Experiment 3, forskolin was supplemented for the first 2 h. Matured oocytes were inseminated with frozen-thawed boar semen and cleavage rate recorded. Before and during IVM, samples of oocytes were evaluated for LD, total and fat areas and fat gray value or for meiotic progression. Results showed that forskolin supplementation during 44 to 48 h or 22 to 24 h inhibits oocyte maturation (exp. 1: forskolin = 5.1 ± 8.0%, control = 72.6 ± 5.0%; exp. 2: forskolin = 24.3 ± 7.4%, control = 71.6 ± 5.6%) and cleavage (exp. 1: forskolin = 0.0 ± 0.0%, control = 55.4 ± 4.1%; exp. 2: forskolin = 8.3 ± 3.3%, control = 54.5 ± 3.0%). Forskolin also reduced oocyte and fat areas. In Experiment 3, forskolin negative effect on oocyte maturation and cleavage disappeared, although minor (P ⩽ 0.03) LD and oocyte fat areas were identified at 22 to 24 h of IVM. Oocytes supplemented with t10,c12 CLA during 44 to 48 h presented a lighter (P ⩽ 0.04) colour tone cytoplasm than those of control and forskolin. In conclusion, t10,c12 CLA and forskolin were capable of modifying the distribution and morphology of cytoplasmic LD during porcine oocyte maturation, thus reducing its lipid content in a time-dependent manner.

The prion-like protein Doppel enhances ovine spermatozoa fertilizing ability.

The function of prion-like protein Doppel was suggested to be related to male fertility. In this study, the importance of ovine Doppel polypeptide on spermatozoa capacitation and fertilization was evaluated. After refolding, recombinant Doppel (rDpl) was supplemented with different concentrations (40, 80 or 190 ng/ml) to ovine spermatozoa during the capacitation process. In experiment 1, post-thawed ovine spermatozoa were incubated with different concentrations of rDpl during 1 h for swim-up, and changes in sperm motility, concentration, vigour, viability and capacitation were monitored (10 replicates). In experiment 2, the fertilization ability of post-swim-up spermatozoa incubated as above was tested through heterologous fertilization of bovine in vitro matured oocytes (n = 423, three replicates). Regardless of dosage, rDpl improved (p ≤ 0.03) spermatozoa viability. Sperm individual motility and vigour were the highest (p ≤ 0.04) for the group receiving 190 ng/ml rDpl. Sperm supplemented with the highest doses of rDpl achieved higher (p = 0.02) fertilization rates (56.0 ± 3.0%) than control (39.1 ± 2.2%) and 40 ng/ml rDpl (39.8 ± 2.7%). Preliminary data suggest that Doppel protein may enhance in vitro spermatozoa fertilizing ability.

Effects of embryo size at transfer (whole versus demi) and early pregnancy progesterone supplementation on embryo growth and pregnancy-specific protein bovine concentrations in recipient dairy heifers.

The objectives of this study were to evaluate embryonic size and survival, plasma progesterone (P4) and pregnancy-specific protein bovine (PSPB) concentrations in early pregnancies (n = 99) following the transfer of one whole (n = 66) or one demi (n = 33) embryo to recipient virgin dairy heifers. The experiment was designed to evaluate the fixed effects of embryo size at transfer (whole or demi embryo) on Day 7 of the estrous cycle (Day 0 = estrus) and P4 supplementation between Days 7 to 19 through an intravaginal device (yes or no) on plasma P4 and PSPB concentrations and on embryo measurements. Plasma P4 concentrations were measured by RIA on Days 0, 7, 14, 19, 21, 25, 35, 42, 49, 56 and 63 of pregnancy and, PSPB concentrations were measured by ELISA on Days 7, 21, 25, 35, 42, 49, 56 and 63. The presence of an embryonic vesicle was detected on Day 25, embryonic/fetal movements and heartbeat were evaluated on Days 42 and 63 and embryo measurements [crown-rump length (CRL) and width at mid body] were obtained on Day 42 through ultrasonography. In non-supplemented pregnancies, Day 42 whole embryos had higher (P < 0.05) CRL and width than demi embryos, but the difference averaged only 1 to 2 mm. In P4 supplemented pregnancies, whole and demi embryos attained a similar size on Day 42 of pregnancy. Embryo size at transfer, early exogenous P4 supplementation and their interactions had no effects (P > 0.05) on plasma P4 concentrations. However, the post-hoc LSD evaluation showed that plasma P4 concentrations on Day 25 were higher (P < 0.001) in whole than in demi embryo derived pregnancies and, that exogenous P4 supplementation increased (P < 0.05) plasma P4 concentrations on Day 19 of pregnancy. The plasma PSPB detection rate on Days 7 to 63 of pregnancy was similar in pregnancies resulting from the transfer of whole and demi embryos. From a total of 93 recipients remaining pregnant until Day 63, plasma PSPB was constantly undetectable on Day 7, was detected in 4% of Day 21 samples, 41% of Day 25, 95% of Day 35, 96% of Day 42, 99% of Day 49 and in 100% of samples of Days 56 and 63. Concentrations of PSPB increased (P < 0.05) from Days 21 to 42 and from Days 56 to 63, with a plateau between Days 42 to 56. Demi embryo pregnancies had higher (P < 0.05) plasma PSPB concentrations on Days 35 and 42 than whole embryo pregnancies. Progesterone supplementation had a positive effect (P < 0.01) on PSPB concentrations from Days 35 to 63. Concentrations of PSPB were similar in non-supplemented whole and demi embryo pregnancies from Days 7 to Day 63. In contrast, in supplemented recipients, demi embryo pregnancies had higher (P < 0.05) PSPB concentrations on Days 25 to 42 than whole embryo pregnancies. No significant correlation was found between P4 and PSPB concentrations or between the concentrations of these hormones and embryonic measurements on Day 42. In conclusion, demi embryos experienced a compensatory growth until Day 42 of pregnancy, attaining a similar size to that of whole embryos and originating conceptuses producing similar plasma PSPB concentrations to those of whole embryo derived conceptuses. Embryonic growth and conceptus secretion of PSPB were positively stimulated by early pregnancy exogenous P4 treatment.

Effect of trans-10 cis-12 conjugated linoleic acid on bovine oocyte competence and fatty acid composition.

The reproductive performance of dairy cows may be improved by feeding conjugated linoleic acid (CLA) supplements during early lactation. The mechanism of action of t10,c12 CLA is not clearly known. Our objective was to investigate the effect of t10,c12 CLA on oocyte maturation and lipid composition of cumulus oocyte complexes (COC). The developmental potential of oocytes incubated in in vitro maturation (IVM) medium supplemented with t10,c12 CLA to the blastocyst stage and embryo quality were also assessed. In experiment 1, abattoir-derived oocytes were matured in TCM199 + 10% serum supplemented with 100 µM t10,c12 CLA (t10,c12 CLA n = 672) or without it (control n = 672). Mature oocytes were either stained for chromatin configuration or inseminated and cultured for embryo development assessment. In experiment 2, COC and IVM culture media were subjected to fatty acid (FA) analysis prior and after maturation with t10,c12 CLA or without it (control). Total lipids and FA profiles in oocytes, cumulus cells and culture media were determined by gas chromatography. t10,c12 CLA supplementation to IVM medium improved (p = 0.05) embryo quality evaluated morphologically. This effect was associated with t10,c12 CLA presence (3.1 ± 0.7%, p = 0.04) and lower levels of arachidonic acid in FA profile of t10,c12 CLA mature oocytes (immature oocytes = 4.4 ± 1.9%, t10,c12 CLA mature oocytes = 1.0 ± 0.7%, p = 0.05). Differences in myristic and eicotrienoic acids, saturated and unsaturated FA concentrations between oocytes and cumulus cells were detected (p ≤ 0.05). In conclusion, the presence of t10,c12 CLA during maturation interfered on lipid metabolism improving bovine oocyte competence to develop into higher quality embryos.

Improvement of fertility in artificially inseminated ewes following vaginal treatment with misoprostol plus terbutaline sulphate.

The effect of vaginal administration of misoprostol plus terbutaline sulphate 6 h prior to artificial insemination (AI) upon the site of AI (vaginal or cervical) and fertility was studied using a total of 87 estrous synchronized Serra da Estrela ewes (control n = 42 and treated n = 45). Artificial insemination was performed using refrigerated semen at 54-55 h after sponge removal. Lambing rate (fertility) and prolificacy were compared between control and treated ewes. The effect of the site of semen deposition on fertility was also evaluated. Prolificacy rate was not different between control (1.5) and treated (1.59) ewes. The proportion of cervical AI achieved in control (45.2%) and treated (37.8%) ewes was not significantly different. Overall, fertility was significantly lower in control than in treated ewes (42.9% vs 64.4%; p < 0.04). Fertility following vaginal AI was significantly lower for control for than treated ewes (30.4% vs 60.7%; p < 0.03) but the difference was smaller and not significant for cervical AI (control 57.9% vs 70.6%). It was concluded that vaginal administration of misoprostol plus terbutaline sulphate 6 h prior to artificial insemination did not affect the proportion of cervical inseminations but significantly improved the fertility of treated ewes. Although needing confirmation, it was hypothesized that drugs might have induced local secretory modifications leading to an increase of cervical ability to retain more viable spermatozoa for fertilization.

Prion-like Doppel gene polymorphisms and scrapie susceptibility in Portuguese sheep breeds.

The establishment of an association between prion protein gene (PRNP) polymorphisms and scrapie susceptibility in sheep has enabled the development of breeding programmes to increase scrapie resistance in the European Union. Intense selection for PRNP genotype may lead to correlated selection for genes linked to PRNP. We intended to investigate if any association exists between genetic variation in prion-like protein Doppel gene (PRND) and scrapie susceptibility, determined through PRNP genotyping. Sampling included 460 sheep from eight Portuguese breeds and the PRND gene coding region was analysed by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP), whereas PRNP genotyping was carried out by primer extension. A synonymous substitution (c.78G>A) was detected in codon 26 of the PRND gene, in all breeds except Churra Mondegueira. Linkage disequilibrium was found between the PRND and PRNP loci (P = 0.000). Specifically, PRND was monomorphic in the 45 animals with the more resistant ARR/ARR PRNP genotype (P = 0.003), whereas a higher frequency of PRND heterozygotes (GA) was associated with ARQ/AHQ (P = 0.029). These results constitute preliminary evidence of an association between a polymorphism in the PRND gene and scrapie susceptibility, and indicate that the possibility of undesirable consequences from widespread selection for PRNP genotype on genetic diversity and reproduction traits needs to be further investigated.

In vitro and in vivo fertility of ram semen cryopreserved in different extenders.

Seminal traits of frozen-thawed (FT) ram semen and in vitro and field fertility in native Portuguese breeds were evaluated in 4 experiments. In exp. 1 and 2 the cryopreservation capacity of 2 extenders, E1 (15% egg yolk-EY) and E2 (4.5% EY and trehalose) was compared through morphological evaluation and in vitro fertilizability of FT ram semen. Exp. 3 aimed to determine the usefulness of in vitro homologous/heterologous fertilization tests as tools for predicting ram fertility. Exp. 4 was conducted to verify if the identified differences between the 2 extenders could be confirmed by field fertility. E1 showed a better cryoprotective action expressed by higher in vitro and field fertility results. In conclusion, EY is difficult to be replaced in ram semen extenders. Heterologous fertilization seems to be a useful tool for predicting fertility of FT ram semen.

Doppel gene polymorphisms in Portuguese sheep breeds: insights on ram fertility.

Transgenic knockout of the gene encoding the prion-like protein Doppel leads to male infertility in mice. The precise role of Doppel in male fertility is still unclear, but sperm from Doppel-deficient mice appear to be unable to undergo the normal acrosome reaction necessary to penetrate the zona pellucida of the oocyte. The objective of this study was to characterize Doppel (Prnd) gene polymorphisms in eight Portuguese sheep breeds and to determine a possible relationship between these polymorphisms and ram fertility. Ovine genomic DNA of 364 animals of different breeds (Bordaleira entre Douro e Minho, Churra Badana, Churra Galega Mirandesa, Churra Mondegueira, Merino da Beira Baixa, Merino Branco, Saloia and Serra da Estrela) were analysed by multiple restriction fragment-single-strand conformation polymorphism (MRF-SSCP). This analysis revealed a synonymous substitution G-->A in codon 26 of Prnd gene. Churra Galega Mirandesa and Saloia breeds were more polymorphic (P=0.005 and P=0.04, respectively) than the overall population, while Serra da Estrela and Merino Branco animals were less polymorphic (P=0.007 and P=0.04). No polymorphism was found in Churra Mondegueira breed. Semen from 11 rams of Churra Galega Mirandesa breed (7 homozygous wildtype GG and 4 heterozygous GA) routinely used in the Portuguese Animal Germoplasm Bank was collected and frozen for fertility tests. A classification function was estimated, using data from post-swim-up semen motility and concentration and Day 6 embryo production rate, allowing the identification of the Doppel homozygous GG genotype with 86.7% of accuracy. This preliminary study detected the presence of only one polymorphism in codon 26 of Prnd gene in the Portuguese sheep breeds. In the polymorphic Churra Galega Mirandesa breed, GG genotype could be characterized through a model using three fertility traits, suggesting a relationship with male reproduction. Any future research should investigate not only AA genotype and its influence on ram fertility but also the possible consequences of the European Community selection program to eradicate Scrapie on the Prnd genotypes and indirectly on sheep breed's viability and preservation.

Embryos and culture cells: a model for studying the effect of progesterone.

A positive association between P4 concentration and initial bovine embryo survival has been reported. The objective of this study was to establish two coculture systems as a model to study the influence of progesterone on the initial bovine embryo development. Granulosa cells (GC) or bovine oviduct epithelial cells (BOECs) were used at the base of embryo culture medium microdroplets (TCM199 and 10% of superovulated oestrus cow serum, (SOCS)) supplemented or not with progesterone (P4, 33.4 ng mL(-1)) and/or a progesterone receptor antagonist (onapristone, OP, 2.2x10(-5)M). Presumptive zygotes were transferred to monolayers after in vitro maturation and fertilization of bovine oocytes with thawed swim-up selected sperm. Embryo development was carried out according to the following groups: experiment 1, BOEC (n=378) and BOEC plus OP (n=325); experiment 2, GC (n=514); GC plus OP (n=509); BOEC (n=490); BOEC plus P4 (n=500); BOEC plus P4 and OP (n=502). Embryos were checked for cleavage at day 2 and for stage development between days 8 and 12 of culture. In experiment 1, no differences (P>0.05) were identified between BOEC and BOECOP groups for embryo rates of development, quality or developmental stages. Also in experiment 2, no differences were found in embryo rates of development, quality or developmental stages between embryos cultured under the two coculture systems when no supplementation was added. Embryo development rates were not affected by OP presence in GCOP group. However, P4 negatively affected Day 8 (D8) embryo development rates in BOEC system (BOECP4=16.8+/-2.6% vs. BOEC=23.7+/-1.7%, P=0.02). This negative effect was abolished when P4 antagonist (OP) was added to the culture medium. BOEC supplementation with P4 also induced a delay on embryo development at D8 as confirmed by a lower development score (BOECP4=3.0+/-1.4 vs. GC=3.4+/-0.1, GCOP=3.5+/-0.1, BOEC=3.4+/-0.1 and BOECP4OP=3.5+/-0.1; P<0.05). These results demonstrate that OP supplementation had no harmful effect on embryo development either in granulosa, where P4 is naturally synthesised, or in BOEC coculture systems. Also we can not confirm a direct association between high P4 concentrations and embryo survival during early stages, although P4 may influence early embryo development through different mechanisms mediated by the type of cells present.

Biopsied and vitrified bovine embryos viability is improved by trans10, cis12 conjugated linoleic acid supplementation during in vitro embryo culture.

Bovine embryos cultured in serum-containing media abnormally accumulate lipids in the cytoplasm. This is well known to contribute to their higher susceptibility to cryopreservation and biopsied embryos are even further susceptible. We aimed to improve in vitro produced (IVP) embryos resistance to micromanipulation and cryopreservation by supplementing serum-containing media with trans-10, cis-12 conjugated linoleic acid (t10, c12 CLA). The effect of t10, c12 CLA on lipid deposition and embryonic development was also tested. After in vitro maturation and fertilization (IVF day=D0), zygotes were cultured on granulosa cells+M199+10% serum+100microM GSH supplemented with 100microM of t10, c12 CLA (CLA group, n=1394) or without supplementation (control group, n=1431). Samples of D7/D8 embryos were observed under Nomarsky microscopy for lipid droplets evaluation while others were biopsied and vitrified (group B-Control, n=24; group B-CLA, n=23). Non-biopsied embryos were also frozen (group NB-Control, n=49; group NB-CLA, n=45). Biopsied cells were used for embryo sex determination. Postwarming embryo survival and viability were determined at 0 and 24h of culture, respectively. Supplementation of t10, c12 CLA did not influence cleavage, embryo sex ratio, D7/D8 embryo rate or morphological quality. CLA embryos had higher number of small lipid droplets (P

Cryosurvival of bovine blastocysts is enhanced by culture with trans-10 cis-12 conjugated linoleic acid (10t,12c CLA).

An excessive lipid content in embryo cells is a consequence of embryo culture in the presence of serum which is suggested to be responsible for their high susceptibility to cryopreservation. The objective of the present study was to examine the effects of supplementing serum-containing culture media with trans-10 cis-12 conjugated linoleic acid (10t,12c CLA) on embryo lipid accumulation and its subsequent cryopreservation. Abattoir-derived oocytes were matured and fertilized in vitro (IVF=day 0). On day 1, presumptive zygotes (n=3390) were randomly placed in: (I) (MS), granulosa cell monolayer cultured with M199 and 10% serum; (II) (SCLA), granulosa cell monolayer cultured with M199, 10% serum and 100 microM 10t,12c CLA and (III) (SOF), modified synthetic oviduct fluid, where embryo culture proceeded for 8 days. Cleavage rates or D7/D8 embryo quality did not vary among treatments. D7/D8 embryo production rate was significantly (P<0.001) lower in SOF (17.9+/-1.6%) than in groups MS (29.8+/-2.5%) and SCLA (27.8+/-2.0%). After cytoplasmic lipid droplets observation under Nomarski microscopy, classified embryos were the leanest when cultured in SOF, intermediate in SCLA and the fattest in MS (P<0.02). Post-thawing intact blastocyst rates where significantly higher in the SCLA group (84.7+/-4.1%) than in SOCS (50.3+/-4.8%, P=0.0007) or SOF (65.3+/-6.9%, P=0.03) groups. Post-thawing re-expanding rates were significantly lower when embryos were cultured in MS (34.7+/-3.7%) than in SCLA (63.7+/-5.3%, P=0.0006) or SOF (49.0+/-4.6%, P=0.04). Moreover, re-expanding rates were lower (P=0.05) in SOF than in SCLA cultured embryos. These results clearly show that addition of CLA to serum-containing media reduced lipid accumulation during in vitro culture and significantly improved cryopreservation survival.

Increased cervical electrical activity during oestrus in progestagen treated ewes: Possible role in sperm transport.

The aim of this investigation was to characterize the pattern of cervical myoelectrical activity (EMG) in the sheep, during the periovulatory period, after synchronization of oestrus with progestagen and eCG. EMG was measured with a computerised modular system in five ewes previously fitted with a pair of monopolar Teflon needle electrodes in the muscle layers of the cervix. Each ewe was submitted to oestrus synchronization treatment with intravaginal progestagen sponge during 12 days, and the administration of 500 IU of eCG at the withdrawal of sponge. EMG was recorded in each animal during 19 h, starting 44 h after withdrawal of sponge. The number and duration of events were determined every hour during the experiment. Two distinct event durations were identified: one lasting less than 200 s and another lasting between 300 and 500 s. The two types of events analysed (less than 200 s and lasting between 300 and 500 s) had a similar pattern during the period of observation although they were not in synchrony. For events lasting less than 200 s, activity increased between 48 and 50 h after sponge withdrawal, with the peak of activity being observed between 51 and 53 h. For events of 300-500 s duration, the peak of activity was observed between 48 and 50h after sponge withdrawal and activity was maintained until 51-53 h. The increase in cervical motility observed in progestagen-eCG treated ewes is in keeping with the increase in cervical activity observed by others in natural cycling animals, and suggests that exogenous hormones used in synchronization protocols had no deleterious action on cervical motility during periovulatory period. The enhanced activity of cervical muscle layer found around the time of mating and/or AI suggests it may play an important role as a regulatory mechanism of sperm transport. Taking advantage of the cervical responsiveness to various drugs, experimental modulation of cervical activity could be used to facilitate cervical sperm transport and consequent improve of fertility after cervical AI.