Uterine serpin (SERPINA 14) correlates negatively with cytokine production at the foetal-maternal interface but not in the corpus luteum in pregnant dairy heifers experimentally infected with Neospora caninum.

This study examines gene expression patterns in dairy heifers experimentally infected with N. caninum during on Day 110 of pregnancy with live foetuses at euthanasia, 42 days later. The study population was constituted of four non-infected controls and three infected dams. Gene expression was determined on gamma interferon (IFNγ), (Th1 pro-inflammatory cytokine), interleukin-4 (IL4) (Th2 pro-gestation cytokine) or interleukin-10 (IL10) (T regulatory cytokine) and the serine peptidase inhibitor SERPINA14 in intercaruncular, placental, uterine lymph node (UTLN) and luteal tissue samples. Intercaruncular SERPINA14 expression was negatively correlated with IFNγ expression in cotyledon samples and with IL4 expression in UTLN. No relationships were detected between cytokine gene expression at the foetal-maternal interface and SERPINA14 expression in the luteal samples. Our findings suggest that gene expression of the uterine serpin SERPINA14 correlates negatively with the expression of Th1 and Th2 cytokines at the foetal-maternal interface but not in the corpus luteum.

Quality of human spermatozoa: relationship between high-magnification sperm morphology and DNA integrity.

The aim of this work is to establish the relationship between the morphology of Intracytoplasmic Morphologically Selected Sperm Injection (IMSI)-selected spermatozoa and their DNA integrity. The 45 ejaculates were randomly distributed into three treatment groups: normozoospermic, oligoasthenozoospermic and oligoasthenotheratozoospermic samples. The evaluation of DNA integrity was performed using the sperm chromatin dispersion test. It was established that DNA integrity of spermatozoa is strongly dependent on ejaculate quality (P < 0.05). The count of spermatozoa with nonfragmented DNA in normozoospermic samples was high and independent from IMSI-morphological classes (Class 1 versus Class 3, respectively) (P > 0.1). With decreased ejaculate quality, the percentage of spermatozoa with nonfragmented DNA decreased significantly (P < 0.05) independent from morphological class. Nevertheless, the rate of IMSI-selected spermatozoa with fragmented DNA within of Class 1 in normozoospermic (Group 1), in oligoasthenozoospermic (Group 2) and in oligoasthenotheratozoospermic (Group 3) samples was 21.1%, 31.8% and 54.1%, respectively. In conclusion, there is a direct relationship between morphological parameters of spermatozoa and their DNA integrity. However, the IMSI technique alone is not enough for the selection of spermatozoa with intact nuclei.

Influence of the FecX(R) allele in heterozygous ewes on follicular population and outcomes of IVP and ET using LOPU-derived oocytes.

Ewes heterozygous for the FecX(R) allele (R+) in the bone morphogenetic protein 15 (BMP15) gene display increased ovulation rate and prolificacy. Besides this phenotypic advantage, the influence of the FecX(R) allele on follicle number and size, oocyte competence and in vitro production (IVP) remains undefined. With these aims, 8 R+ and 8 wild-type (++) ewes were subjected to 2 laparoscopic ovum pick-up (LOPU) trials (four sessions per trial; two with and two without FSH) and subsequent IVP and fresh embryo transfer. All follicles >3 mm were punctured (n = 1673). Genotype did not significantly affect the number of punctured follicles per ewe and session (10.4 and 10.2 in R+ and ++ untreated ewes, 17.4 and 14.3 in R+ and ++ FSH-treated ewes, respectively), but follicular diameter of R+ ewes was significantly reduced compared with ++ ewes (-0.2 mm in untreated and -0.8 mm in FSH-treated ewes; p < 0.01). R+ ewes showed higher recovery rate and increased numbers of total and suitable cumulus-oocyte complexes for in vitro maturation (IVM). Similar rates of day 8 blastocysts were observed in R+ (36.1%, 147/407) and ++ (32.6%, 100/307) ewes, but the final output of day 8 blastocysts per ewe and session was higher in R+ ewes (+0.75; p < 0.005), without differences in survival rate at birth of the transferred embryos (40.4%, 21/52 vs 36.4%, 16/44, respectively). In conclusion, a higher number of oocytes proven to be competent for in vitro development and embryo survival after transfer are recovered from R+ ewes, despite the lower mean size of their follicles at puncture.

A suitable duplex PCR for ovine embryo sex and genotype of PrnP gene determination for MOET-based selection programmes.

The objective of this study was to test the suitability of a duplex PCR assay for sex and scrapie resistance genotype determination in fresh embryos. Duplex PCR amplified a repetitive and specific fragment of Y chromosome, used for sex diagnosis, and a PrnP fragment. PrnP codons 134 and 156, and codon 171 were genotyped by restriction fragment length polymorphisms and allele-specific PCR, respectively, after re-amplification of PrnP fragment. The specificity of the method was first assessed by testing 359 blood samples from Rasa Aragonesa sheep breed (161 males and 198 females). No amplification failures and total agreement between genotypic and phenotypic sex were found. In the same way, PrnP genotype determination by duplex PCR assay was in agreement with the PrnP animal's genotype established by sequencing. Finally, 73 samples of 1-10 cells from compact morulae were aspirated through the zona pellucida and genotyped for sex and PrnP. The efficiency was 96% when three or more cells were sampled. These results confirm that the duplex PCR assay reported in this work can be used for rapid sex determination in ovine embryos, with a high efficiency and accuracy (96%) when three or more cells are sampled, allowing sexed fresh embryos of known PrnP genotype to be transferred in multiple ovulation and embryo transfer programmes.

The efficiency of in vitro ovine embryo production using an undefined or a defined maturation medium is determined by the source of the oocyte.

In vitro oocyte maturation can be influenced by oocyte source and maturation media composition. The aim of the present study was to compare the efficiency of a defined in vitro maturation medium (TCM199 supplemented with cysteamine and epidermal growth factor; Cys + EGF) with an undefined medium (TCM199 supplemented with follicle-stimulating hormone and follicular fluid; FSH + FF) for in vitro production (IVP) of ovine embryos, using oocytes obtained by laparoscopic ovum pick-up from FSH-stimulated [n=11; 158 cumulus-oocyte complexes (COCs)] and non-stimulated (n=16; 120 COCs) live ewes, as well as abattoir-derived oocytes (170 COCs). The produced blastocysts were vitrified and some of them were transferred to synchronized recipients. The best and the worst final yields of embryo IVP observed in this study were obtained using oocytes from FSH-stimulated ewes matured in FSH + FF (41.3%; 33/80) and in Cys + EGF (19.2%; 15/78) medium, respectively (p<0.01). No significant differences between both media were attained in the blastocyst development rate or in the final yield of embryo IVP using oocytes from non-stimulated ewes or abattoir-derived oocytes. The overall in vivo survival rate of the transferred vitrified blastocysts was 13.1% (8/61), without significant differences between oocyte sources or maturation media. In conclusion, under the experimental conditions of the present study, TCM199 supplemented with cysteamine and EGF is a convenient defined maturation medium for IVP of embryos from oocytes of live non-stimulated ewes or from oocytes of abattoir-derived ovaries. However, the best final yield of embryo IVP observed in this study was attained when oocytes came from FSH-stimulated donors and TCM199 was supplemented with FSH and follicular fluid.

First birth of an animal from an extinct subspecies (Capra pyrenaica pyrenaica) by cloning.

Two experiments have been performed to clone the bucardo, an extinct wild goat. The karyoplasts were thawed fibroblasts derived from skin biopsies, obtained and cryopreserved in 1999 from the last living specimen, a female, which died in 2000. Cytoplasts were mature oocytes collected from the oviducts of superovulated domestic goats. Oocytes were enucleated and coupled to bucardo's fibroblasts by electrofusion. Reconstructed embryos were cultured for 36h or 7d and transferred to either Spanish ibex or hybrid (Spanish ibex malex domestic goat) synchronized recipients. Embryos were placed, according to their developmental stage, into the oviduct or into the uterine horn ipsilateral to an ovulated ovary. Pregnancy was monitored through their plasmatic PAG levels. In Experiment 1, 285 embryos were reconstructed and 30 of them were transferred at the 3- to 6-cells stage to 5 recipients. The remaining embryos were further cultured to day 7, and 24 of them transferred at compact morula/blastocyst stage to 8 recipients. In Experiment 2, 154 reconstructed embryos were transferred to 44 recipients at the 3- to 6-cells stage. Pregnancies were attained in 0/8 and 7/49 of the uterine and oviduct-transferred recipients, respectively. One recipient maintained pregnancy to term, displaying very high PAG levels. One morphologically normal bucardo female was obtained by caesarean section. The newborn died some minutes after birth due to physical defects in lungs. Nuclear DNA confirmed that the clone was genetically identical to the bucardo's donor cells. To our knowledge, this is the first animal born from an extinct subspecies.

Short-term dietary protein supplementation improves reproductive performance of estrous-synchronized ewes when there are long intervals of prostaglandin or progesterone-based treatments for timed AI.

To evaluate the reproductive effects of a short-term dietary protein supplementation (Days -10 to -3) before timed AI (TAI = Day 0), 471 Merino ewes grazing native pastures were estrous-synchronized when there were either long intervals between prostaglandin administrations (two prostaglandin injections 15 or 16 d apart; PG15 and PG16, respectively) or with a progesterone-eCG (P4-eCG) protocol, resulting in a 3 × 2 experimental design. Ovulation rate on Day 8 (OR), non-estrous-return to Day 21 (NRR21), and fertility, prolificacy and fecundity on Day 70 were evaluated. The interaction between estrous synchronization protocol and supplementation was not significant for any of these variables (P > 0.05). Supplementation increased OR, prolificacy and fecundity (+0.14, +0.15 and +0.14, respectively, P < 0.01), but did not affect NRR21 or fertility of ewes (+6.2% and +6.7% respectively, P > 0.05). Ewes treated using the PG15 and PG16 protocols had a lesser OR (-0.27), prolificacy (-0.22) and fecundity (-0.20) than ewes treated using P4-eCG protocol (P < 0.01 for each), and similar NRR21 and fertility (-5.4% and -7.9% respectively, P > 0.05 for both variables), without significant differences between the PG15 and PG16 groups. In conclusion, a short-term dietary protein supplementation before TAI improved OR, prolificacy and fecundity of ewes which were estrous-synchronized by imposing long interval PG (15 or 16 d apart) or P4-eCG-based protocols. There was a greater OR, prolificacy and fecundity when there was use of the P4-eCG compared to long interval PG-based protocols. Estrous-non-return rate after AI and fertility as a result TAI were not affected by either the supplementation or the estrous synchronization protocols used.

A deletion in the bone morphogenetic protein 15 gene causes sterility and increased prolificacy in Rasa Aragonesa sheep.

Bone morphogenetic protein 15 (BMP15) is a member of the transforming growth factor beta superfamily, is specifically expressed in oocytes and is essential for sheep prolificacy. Reported mutations in this gene cause increased ovulation rate and infertility in a dosage-sensitive manner. In this work, a new naturally occurring mutation in the BMP15 gene from the ovine Rasa Aragonesa breed is described. This mutation is a deletion of 17 bp that leads to an altered amino acid sequence and introduces a premature stop codon in the protein. Highly significant associations (P < 0.0001) were found between the estimated breeding value for prolificacy and the genotype of BMP15 in Rasa Aragonesa animals with high and low breeding values for this trait. As for other mutations in BMP15, this new mutation is associated with increased prolificacy and sterility in heterozygous and homozygous ewes respectively.

Reliability of sex determination in ovine embryos using amelogenin gene (AMEL).

Sex specific sequence variability of the amelogenin gene has been used for sex determination in the family of Bovidae. In our study, suitability and reliability of the amelogenin gene for ovine sex determination in embryos was studied. The specificity of the method was previously demonstrated by testing 579 blood samples from several Spanish sheep breeds (161 males and 198 females). No amplification failures and very high agreement between genotypic and phenotypic sex was found (578/579), in contrast to humans, where errors in sex determination has been reported because of mutations in AMELX or AMELY genes. However, one female animal showed a male genotypic sex, being the most plausible explanation that a recombination event has happened during the meiosis. In our study only 0.17% (1/579) of the samples tested has been misdiagnosed using the amelogenin gene. Finally, 1-10 cells from each of 67 compact morulae were aspirated through the zona pellucida, and genotyped for sex determination. The efficiency in sex determination was 95 and 98% when more than two and more than three cells were sampled, respectively. The total time required for the genetic test, was less than 4h. These results confirm that the amelogenin gene can be used for rapid sex determination in ovine embryos, with a high efficiency and accuracy (100%) when three or more cells are sampled, allowing transferring sexed fresh embryos in MOET programmes. To our knowledge, this was the first time that sex determination using the amelogenin gene was performed in ovine embryos.

SNP rs403212791 in exon 2 of the MTNR1A gene is associated with reproductive seasonality in the Rasa aragonesa sheep breed.

The aim of this study was to characterize and identify causative SNPs in the MTNR1A gene responsible for the reproductive seasonality traits in the Rasa aragonesa sheep breed. A total of 290 ewes (155, 84 and 51 mature, young and ewe lambs, respectively) from one flock were controlled from January to August. The following three reproductive seasonality traits were considered: the total days of anoestrus (TDA) and the progesterone cycling months (P4CM); both ovarian function seasonality traits based on blood progesterone levels; and the oestrus cycling months (OCM) based on oestrous detection, which indicate behavioural signs of oestrous. We have sequenced the total coding region plus 733 and 251 bp from the promoter and 3'-UTR regions, respectively, from the gene in 268 ewes. We found 9 and 4 SNPs associated with seasonality traits in the promoter (for TDA and P4CM) and exon 2 (for the three traits), respectively. The SNPs located in the gene promoter modify the putative binding sites for various trans-acting factors. In exon 2, two synonymous SNPs affect RFLP sites, rs406779174/RsaI (for the three traits) and rs430181568/MnlI (for OCM), and they have been related with seasonal reproductive activity in previous association studies with other breeds. SNP rs400830807, which is located in the 3'-UTR, was associated with the three traits, but this did not modify the putative target sites for ovine miRNAs according to in silico predictions. Finally, the SNP rs403212791 (NW_014639035.1: g.15099004G > A), which is also associated with the three seasonality phenotypes, was the most significant SNP detected in this study and was a non-synonymous polymorphism, leading a change from an Arginine to a Cysteine (R336C). Haplotype analyses confirmed the association results and showed that the effects found for the seasonality traits were caused by the SNPs located in exon 2. We have demonstrated that the T allele in the SNP rs403212791 in the MNTR1A gene is associated with a lower TDA and higher P4CM and OCM values in the Rasa Aragonesa breed.

Effects of solid storage of sheep spermatozoa at 15 degrees C on their survival and penetrating capacity.

This study was designed to evaluate the possible benefits of adding gelatin to a standard milk extender, for solid storage of sheep semen at 15 degrees C. Solid storage was assessed in terms of effects on sperm motility and membrane integrity up to 2 days (Study 1), and on in vitro penetration capacity after storage for 24h (Study 2). In both studies, semen was diluted in CONTROL (standard milk extender) and GEL (1.5 g gelatin/100ml extender) diluents to a final concentration of 400 x 10(6)sperm/ml. In Study 1, semen samples were stored at 15 degrees C, and sperm quality variables analyzed after 2, 24 and 48 h of storage. Motility and viability values were significantly lowered using the liquid compared to the gel extender for all storage periods, except for motility after 2h of storage, whose values were similar. After 2h of incubation at 37 degrees C, motile cell percentages and membrane integrity were significantly lower in the CONTROL group than in the GEL group for all storage periods. In Study 2, in vitro matured lamb oocytes were randomly divided into three groups and fertilized with CONTROL diluted semen stored for 2h or 24h, or with GEL diluted semen stored for 24h. After co-incubation, oocytes were evaluated for signs of penetration. Storage of semen in the GEL diluent for 24h gave rise to increased in vitro fertilization rates in comparison with the CONTROL diluent. Our findings indicate that the solid storage at 15 degrees C of ram spermatozoa by adding gelatin to the extender leads to improved survival and in vitro penetrating ability over the use of the normal liquid extender. A solid diluent could thus be a useful option for the preservation of fresh ovine semen for extended periods.

Effect of in vitro maedi-visna virus infection on adherence and phagocytosis of staphylococci by ovine cells.

This work was aimed at studying the effect of maedi-visna virus (MVV) infection in vitro on the ability of sheep cells to adhere to staphylococci (Staphylococcus aureus and Staphylococcus epidermidis), and phagocytose these bacteria. Adherence was studied in sheep choroid plexus cells (SCPC) using an ELISA test and phagocytosis was studied in pulmonary alveolar macrophages (PAM) by chemiluminescence. A 5- and 7-day of in vitro MVV infection resulted in syncytium formation and a significant increased adherence (P < 0.01) of SCPC to bacteria. SCPC endogenous fibronectin was significantly higher (P < 0.01) on days 5 and 7 than on day 0 of MVV infection. A significantly decreased phagocytosis (P < 0.05) was also observed on days 5 and 7 of MVV infection in PAM when compared to MVV-free controls. Comparatively, phagocytosis was highest for S. aureus non-slime producing strains, followed by S. epidermidis, and S. aureus slime producing strains, in that order. Finally, increased expression of both, class I and class II major histocompatibility antigens was also observed in MVV-infected PAM on days 5 and 7, whereas SCPC only demonstrated upregulation of MHC class I. These results, indicative of an alteration of some cell functions in MVV-infected cells, may help to understand interactions between MVV-infected cells and bacteria in simultaneous infections and may provide clues to the possible in vivo interactions of both pathogens.

Embryo losses in Rasa Aragonesa ewes actively immunized against androstenedione or passively immunized against testosterone.

Two-day-old embryos from untreated ewes were transferred to the oviducts of ewes actively immunized against androstenedione (n=26, Group A), passively immunized against testosterone (n=19, Group B) or left untreated (n=25, Group C). Donor ewes superovulated after treatment with follicle-stimulating hormone and fluorogestone acetate (FGA). Recipient ewes were treated with FGA and pregnant mare serum gonadotropin (PMSG, 300 I.U.). Group A received two injections of Fecundin at a 4-wk interval. FGA sponges were inserted when the second injection was given. Group B was treated with antitestosterone antiserum (35 ml) at sponge withdrawal. Each recipient received two morphologically viable embryos 52 to 62 h after the onset of estrus. Antibody titre at embryo transfer and progesterone concentration on Days 2, 4, 6, and 12 after estrus were determined. Fertility was lower in Group A when compared to Group C (42.3 vs 84.1%; P<0.01) while that of Group B (63.2%) did not differ from those of Groups A and C. In immunized groups, most of the embryo losses occurring were complete (both embryos were lost), resulting in a decreased fertility, while in the untreated group embryo losses were mainly partial (only one embryo was lost), hence lowering prolificacy. Fertility in immunized groups changed according to the antibody titre reached. Ewes from Groups A and B with higher antibody titres displayed lower fertility than control ewes. On Days 4 and 12 of the cycle, Group A plasma progesterone concentrations positively correlated with antibody titres and were higher with respect to those of Group C (P<0.05). Progesterone levels in Group B were similar to those of Group C. These results indicate that ewes reaching higher antibody levels had more embryo losses, attributable to the adverse influences of the oviductal and/or uterine environment on embryo development.

Effect of immunization on reproducvive performance, embryo quality and progesterone in Rasa Aragonesa ewes actively immunized against androstenedione or passively immunized against testosterone.

A total of 217 Rasa Aragonesa ewes were used to test two immunization treatments: 1.Active immunization against androstenedione: ewes immunized in previous matings (androstenedione, reimmunized; AR groups, n=58) or not (first immunization; AF groups n=64) were boosted either 2 or 4 wk before mating. 2.Passive immunization against testosterone: antisera were injected either at sponge withdrawal (zero time; T0 group, n=21) or 1 wk previously (Tl group, n=22). We used 52 ewes as controls (C group). Half of each group was used either to record reproductive performance or to embryo viability assessment. Prolificacy was significantly increased in ewes which reached a moderate antibody level, independently of the treatment. Fertility was lower in AR ewes that attained a high antibody titre (P<0.01). The percentage of viable embryos recovered was lower in AF ewes (P<0.01), and in ewes whose testosterone antibody titre was high (P<0.05), compared to C group. It was proven that similar or lower antibody levels were more harmful for ewes from AF and Tl than for ewes from AR or T0 groups. The proportion of nonfertilized recovered ova was not significant. Progesterone levels were notably increased in AR ewes (P<0.001) independently of ovulation rate and were positively correlated to antibody titre at mating (P<0.01) but these events were not observed in T ewes. These findings indicate that after androgen immunoneutralization, only those ewes having antibody titres within a limited range at mating had improved reproductive performance. Further research is needed in order to understand the role that progesterone plays in immunized ewes.

Interspecies pregnancy of Spanish ibex (Capra pyrenaica) fetus in domestic goat (Capra hircus) recipients induces abnormally high plasmatic levels of pregnancy-associated glycoprotein.

Interspecies embryo transfer could be a valuable tool in preservation programs of endangered species. In this work the results of both interspecific-monospecific (ibex-in-goat) and interspecific-bispecific (mixed-species; ibex+goat-in-goat) embryo transfers in the capra genus are reported. The aim of this work was to compare the PAG plasmatic profiles occurring in these interspecific gestations to those encountered in normal (i.e. intraspecies) pregnancies of Spanish ibex and domestic goat. Spanish Ibex females were superovulated with 9 mg NIADDK-oFSH-17 and embryos were surgically collected 5.5 d after estrus. Two embryos were transferred per recipient. Domestic goat recipients were previously mated either to vasectomized domestic bucks (n=17 females; interspecific-monospecific gestations) or to fertile ones (n=9 females; interspecific-bispecific gestations). Intraspecific pregnancies were obtained by natural mating between males and females of the same species (Spanish ibex: n=6; domestic goat: n=1). Pregnancy rate diagnosed by progesterone was low in both interspecific-monospecific (7/17) and interspecific-bispecific (3/9) transfers. None of the monospecific (0/7) and 2 (2/3) of the bispecific established pregnancies developed to term. Ibex-in-ibex PAG profile showed 2 similar peaks of 60 to 70 ng/mL on Days 34 and 153 of pregnancy, while goat-in-goat had the maximum value (60 to 70 ng/mL) at Day 50, decreasing slightly afterwards until parturition. Mixed-species gestations (ibex+goat in goat) showed a first peak of 500 to 1000 ng/mL on Day 70 and a second one (200 to 500 ng/mL) on Day 140 of pregnancy. Four ibex-in-goat gestations that terminated with the expulsion of dead fetuses at Days 110 to 170 had their maximum PAG values (100 to 700 ng/mL) on Days 60 to 90. We conclude that it is possible to achieve pregnancies after transfer of ibex embryos into domestic goats, but this requires a great change of the PAG profiles, which increase significantly. Live ibex kids can be produced when embryos from both species share the uterus. This is the first report of successful interspecific pregnancies in the capra genus.

Exogenous growth hormone improves the number of transferable embryos in superovulated ewes.

The application of pGH (porcine Growth Hormone) to superovulated ewes was studied with the aim of improving the embryo yield. Thirty-seven ewes were superovulated with pFSH for 3 d and 18 of them were cotreated the third day with 0.50 mg of pGH. Embryos were surgically recovered on Day 7 after sponge withdrawal. Then, 102 morphologically healthy embryos were immediately transferred in pairs to 51 synchronized recipient ewes. The GH treatment did not significantly affect the percentage of ewes in estrus, the time of estrus onset or the ovulation rate. However, it improved synchronization by grouping estrus in a narrower range (12 h) in comparison to the control group (24 h); (16 to 28 h after sponge withdrawal vs 12 to 36 h; P < 0.05). The total amount of LH released during the preovulatory surge was lower in the GH than in the control group (P < 0.05). No differences were found between groups for other LH-related parameters such as basal levels, peak values or peak time from sponge removal. The proportions of unfertilized oocytes and degenerate embryos recovered were lower in the GH cotreated group (P < 0.05 and P < 0.01, respectively). This resulted in higher rates of transferable embryos and lambs born per donor ewe in the GH than in the untreated group (3.9 vs 1.7 and 2.28 vs 0.84, respectively; both, P < 0.05). These beneficial effects of GH would likely be due either to a direct action on oocyte maturation or to an indirect action on the oviductal environment.

Screening of some variables influencing the results of embryo transfer in the ewe. I. Five-day-old embryos.

Rasa Aragonesa ewes (n = 89) received 2 embryos on Day 6 of the estrous cycle (Day 0 = estrus) from 46 donors of the same breed that had been superovulated with FSH-p. The influence of several variables on fertility and prolificacy after transfer was studied by discriminant analysis. Our results showed that the main variables that contributed to a high fertility rate were the degree of synchrony (better outcome if donors come into estrus later than the recipients); Fluorogestone acetate (FGA) to estrus interval and interval from previous lambing in the recipients, ovulation rate of the donors and recipients (better if superior to the mean); prolificacy of recipients in the previous lambing; and difference in developmental stage of the pair of transferred embryos (better if inferior to the mean). The main variables affecting prolificacy were the ovulation rate and weight of the donors and progesterone concentrations of the recipients (better if lower than the mean); age of the donors and difference in progesterone concentrations between donors minus those of the recipients (better if higher than the mean). The percentage of ewes correctly classified into lambing or not lambing status was 73% (P < 0.001) and that of the ewes correctly classified as lambing 1 or 2 lambs was 81.8% (P < 0.001). Whether or not the criteria we have established for optimum transfer results are applicable to conditions other than our own still needs to be confirmed.

Screening of some variables influencing the results of embryo transfer in the ewe. Part II: two-day-old embryos.

Each of sixty Rasa aragonesa ewes received two embryos on Days 2-3 of the estrous cycle (Day 0=estrus) from 27 donors of the same breed that were superovulated with pFSH. The influence of several variables on fertility and prolificacy after transfer was studied by discriminant analysis. Our results showed that the main variables contributing to higher fertility were: in the donor-recipient couple, degree of estrus synchrony between them (better if donors were in estrus before recipients); in recipients, interval from FGA withdrawal to estrus onset, prolificacy in the previous lambing, age (all, better if inferior to the mean) and interval from the previous lambing (better if superior to the mean); in donors, ovulation rate (better if lower than the mean); and in embryos, developmental stage (better if superior to the mean). Likewise, the main variables contributing to higher prolificacy were: in donors, body condition score (better if higher than the mean) and weight (better if inferior to the mean); and in recipients, plasma progesterone concentration at transfer (better if inferior to the mean). The percentage of ewes correctly classified as lambing or not was 71.7% (P<0.01), and 72.5% of the ewes were correctly classified as having one or two lambs (P<0.05). Whether the criteria we have found for optimum results after transfer are applicable or not to conditions other than ours will need to be confirmed.

Influence of follicle size on the penetrability of immature pig oocytes for homologous in vitro penetration assay.

In order to improve the performance of homologous in vitro penetration (hIVP) assays using immature oocytes to assess the penetrating ability of boar sperm, the present study was designed to evaluate the influence of oocyte and follicle size on the penetrability of immature pig oocytes obtained from slaughterhouse ovaries. Nonatretic antral follicles were isolated, measured with a computerized image analysis system and grouped according to their diameter: Group 1 (0.40-0.99 mm), Group 2 (1.00-2.19 mm), Group 3 (2.20-2.79 mm), and Group 4 (2.80-6.50 mm). After sperm coincubation and before penetrability evaluation, the immature oocytes were classified into four size categories according to their diameter excluding zona pellucida: <105, 105-109, 110-114, and > or =115 microm. As regards follicle size, the highest viability and penetrability were obtained with oocytes from follicles >2.20 mm (P>0.05). Regarding oocyte size, significant differences (P<0.05) were observed for all parameters evaluated between oocytes with a diameter above or below 110 microm. However, our results revealed that such differences were due to follicle size rather than oocyte diameter, since oocytes with the same diameter but from different follicle size groups showed different penetration rates. With increasing follicle size, the percentage of penetrated oocytes increased (P<0.05). Finally, our results showed that the greater penetrability of immature oocytes from larger follicles is not due to variations in the thickness of the zona pellucida. There were no significant differences in zona pellucida thickness between oocytes from the four follicular size groups. In summary, these results indicate that follicle size directly affects the penetrability of immature pig oocytes used in hIVP.

Relationship between antral follicle size, oocyte diameters and nuclear maturation of immature oocytes in pigs.

We designed the present study to examine the possible relationship between oocyte, antral follicle size and the nuclear heterogeneity of immature pig oocytes, in order to study the heterogeneity of oocyte populations in ovaries obtained from slaughterhouses. Previously, we carried out an initial experiment to determine, by histological analysis, the effectiveness of the macroscopic criteria (MC) used to screen atretic and nonatretic antral follicles. We recovered 239 follicles by mechanical dissection, measured them with a computerized image analysis system, and classified them into five size categories according to their diameter (FD): Group 1 (0.40-0.99 mm), Group 2 (1.00-2.19 mm), Group 3 (2.20-2.79 mm), Group 4 (2.80-3.59 mm) and Group 5 (3.60-6.50 mm). In relation to histological analysis, the results showed that MC is an effective method to select atretic and nonatretic antral follicles from 0.40 to 6.50 mm in diameter (overall accuracy was 80.75%, with sensitivity and specificity rates of 79.33 and 82.20%, respectively). In a second experiment, we recovered 454 nonatretic follicles, then measured and classified them as mentioned above. We removed oocytes individually from follicles and measured their size (oocyte diameter without and with zona pellucida, OD and TOD, respectively). Finally, we evaluated the relationship between OD, FD and nuclear maturation of immature oocytes (germinal vesicles (GV) Stages 0, I, II, III and IV; diakinesis, prophase I, and metaphase I). Overall OD was 101.77 +/- 0.65, 109.19 +/- 0.45, 113.55 +/- 0.50, 116.92 +/- 0.46 and 117.13 +/- 0.47 microm (Groups 1, 2, 3, 4, and 5, respectively). Differences in OD between groups were significant (P < 0.01), although from 2.80 to 6.50 mm follicles, the oocytes were not different in size. There was a certain heterogeneity in OD within each follicular group. Although we observed a certain degree of nuclear variability, regardless of FD or OD, the present study showed a clear progression in GV when FD increased from 0.40 to 6.50 mm. A positive correlation (r2 = 0.4248; P > 0.05) was established mainly between the nuclear stage and oocyte diameter.