The effect of estrogen administration during early pregnancy upon the survival of single implanted pig embryos.

In the present study, we investigated the influence of exogenous estrogen on embryo survival after transfer into prepubertal gilts in which estrus had been induced. In the first experiment, estrus was induced in prepubertal gilts by the administration of 1,000 IU of eCG and 750 IU of hCG every 72 h. Several blastocysts were recovered on d 6 (d 0 is the day of hCG administration), and 1 embryo was transferred to the tip of 1 side of the uterine horn on d 6 (Control). In treated groups, after embryo transfer, 5 mg of estradiol benzoate (EB) was administered on d 11 (EB5mg-1) or d 11, d 13, and d 15 (EB5mg-3) or d 11, 12, 13, 14, and 15 (EB5mg-5) or 20 mg of estradiol dipropionate (EDP) was administered on d 11 (EDP20mg-1) or d 11 and d 14 (EDP20mg-2). Autopsy examinations were performed on d 53 to 60. Although nontreated gilts did not become pregnant, gilts in each of the estradiol-treated groups became pregnant. The greatest pregnancy rate (77.8%, 7/9) was obtained with EDP20mg-2 (EDP20mg-2 > control: P < 0.05). In a second experiment, 1 blastocyst was transferred to prepubertal gilts and treated with EDP20mg-2. Pregnancy in recipient pigs was confirmed by ultrasonography, and pigs were allowed to farrow. Embryo survival rate was high on d 30 of pregnancy (75%, 9/12) but had a tendency (P = 0.0995) to decline from d 30 to delivery (33.3%, 4/12). In a third experiment, prepubertal gilts were administered 5 mg of EDP on d 11 (EDB5mg-1) and d 11 and d 14 (EDP5mg-2). Autopsy examinations were performed on d 53 to 58. Pseudopregnancy rate was high for EDP5mg-2 (63.6%, 7/11) compared with EDP5mg-1 (0%, 0/11; P < 0.05). In a fourth experiment, prepubertal gilts were transferred 1 blastocyst and treated with EDP5mg-2. Pregnancy was confirmed in recipient pigs by ultrasonography, and pigs were subsequently allowed to farrow. Embryo survival rate remained unchanged from d 30 of pregnancy to delivery (66.7%; 8/12). One piglet died from dystocia, and 1 suffered from deformity involving double-breasted hooves and died 6 d after birth. There was no difference (P > 0.05) in survival rate on d 30 of pregnancy and weaning (50%, 6/12). Body weight at birth and at weaning did not differ from that reported in previous studies. In conclusion, this study showed that EDP5mg-2 treatment during early pregnancy leads to full-term development of a single embryo.

Genetic variability in swine leukocyte antigen class II and Toll-like receptors affects immune responses to vaccination for bacterial infections in pigs.

The genes encoding swine leukocyte antigen (SLA) and Toll-like receptor (TLR) are highly polymorphic in pig populations, and likely have influences on infection and the effects of vaccination. We explored the associations of different genotypes of SLA class II and of the genes TLR1, TLR4, TLR5, and TLR6 with antibody responses after vaccination against Erysipelothrix rhusiopathiae (ER) and Actinobacillus pleuropneumoniae (APP) serotypes 1, 2, and 5 in 191 Duroc pigs maintained under specific pathogen-free conditions. We demonstrated close relationships between SLA class II and ER antibody response and between TLR genes other than TLR4 and APP antibody responses. Pigs with specific haplotypes in SLA class II or TLR5 showed decreased antibody response to ER vaccination or increased responses to APP2 and APP5 vaccination, respectively. It might be possible to breed for responsiveness to vaccination and to implement new vaccine development strategies unaffected by genetic backgrounds of pigs.

Co-transfer of parthenogenotes and single porcine embryos leads to full-term development of the embryos.

It has long been known that several embryos are needed to establish and maintain pregnancy during early gestation in pigs. In this study, we assessed whether co-transfer of parthenogenotes with a single embryo was sufficient to maintain development of the embryo. Embryos were recovered at morula and early blastocyst stages from gilts that had been artificially inseminated. Parthenogenotes were produced from oocytes matured in vitro, activated by electrical stimulation, and then cultured for 110h. Those that had developed to morula- or blastocyst-like stages at 110h post-activation were transferred to recipient pigs either with or without morula or blastocyst stage embryos. Parthenogenotes that were transferred to recipients in the absence of embryos were viable up to 30 days post-activation and formed limb-buds; at 40 days, however, all were dead or degenerate. Among pigs that received both parthenogenotes and a single embryo, seven of nine (77.8%) delivered a normal piglet at full-term. This study therefore demonstrates that parthenogenotes can be used in place of embryos to establish pregnancy and promote development of a single co-transferred embryo. This method may be applied to rescue high value porcine embryos that are difficult to produce, such as transgenic cloned embryos, or for embryos frozen as a genetic resource.

Immunoreactive relaxin in seminal plasma of fertile boars and its correlation with sperm motility characteristics determined by computer-assisted digital image analysis.

Ejaculates from 10 mature fertile large white Yorkshire boars were used to examine the correlation between immunoreactive relaxin levels in seminal plasma and sperm motility characteristics. Seminal plasma levels of immunoreactive relaxin were measured by a time-resolved fluoroimmunoassay (TR-FIA). Motility characteristics were assessed using a CellSoft computer-assisted digital image analysis system. The mean +/- SD level of immunoreactive relaxin in seminal plasma was 2.61 +/- 0.62 ng/mL. When the correlation between seminal plasma levels of immunoreactive relaxin and parameters of sperm movement was examined, it was found that relaxin levels were significantly correlated with the percentage of motile spermatozoa (r=0.687, p < 0.05), curvilinear velocity (r=0.745, p < 0.05), straight line velocity (r=0.651, p < 0.05), mean amplitude of lateral head displacement (mean ALH) (r=0.844, p < 0.01) and the maximum amplitude of lateral head displacement (max ALH) (r=0.830, p < 0.01), but not with linearity, beat-cross frequency, or percentage of circular cells. Among these parameters, seminal plasma levels of immunoreactive relaxin showed the strongest correlation with the ALH parameter related to fertilizing ability. These results indicate that immunoreactive relaxin in boar semen may be necessary not only for normal sperm motility but also for normal fertility, suggesting that determination of the profile of immunoreactive relaxin in ejaculates may have value as a potential marker for predicting sperm fertilizing ability of boars.

Sexing of porcine embryo by in situ hybridization using chromosome Y- and 1-specific DNA probes.

This study was carried out to determine if a rapid, simultaneous detection system using chromosome Y- and 1-bearing boar spermatozoa was applicable for sexing embryos. Porcine embryos were recovered from gilts and sows 4 to 6 d after mating, and whole embryos or biopsy cells were mounted on a glass slide with a small amount of fixative (methanol: acetic acid: distilled water = 9:1:4). The samples were then stained by means of a fluorescence in situ hybridization (FISH) procedure developed specifically for the detection of Y-bearing spermatozoa. Hybridization was performed using digoxigenin (dig)-labeled chromosome Y- specific DNA, and biotin-labeled chromosome 1-specific DNA sequences were detected as a signal of FITC and Texas Red on nucleus visualized DAPI-stain. Proportions of whole embryos labeled with chromosome 1-probe were 17 and 97% at the 3 to 16 and > or = 32 cell stage, respectively. Of the 93 biopsied embryos analyzed by FISH, 85 embryos (91%) could be accurately classified as male or female. Of the 65 biopsied embryos, 60 embryos (92%) had a clear blastocoele and a inner cell mass after 48 h of culture in vitro, and these embryos were evaluated as available embryos. One out of 4 recipient gilts which received sexed embryos at transfer farrowed 12 piglets of the expected sex. The results of this study demonstrated that porcine embryos at the > or = 32 cell stage can be sexed within 2 h using the FISH method. Moreover further development of the FISH technique could make it an effective tool for the study of early porcine embryos and for the control of porcine sex.

Precocious estrus and reproductive ability induced by PG 600 in prepuberal gilts.

A total of 29 SPF Large White prepuberal gilts (mean age 152 days at treatment) were examined for estrous and ovulatory responses after PG 600 treatment. After treatment, 85.2% of the gilts showed standing estrus within 6 days. Whereas the treatment-to-estrus interval and duration were 3.7 and 1.9 days respectively. As ovulation occurred on Day 5 to 6, appropriate timing of artificial insemination would be about 4 days after treatment. Fertility of gilts revealed to be excellent, giving rise to a high percentage of normal embryos, 85.3%. Meanwhile, development and growth of fetuses were mostly normal. Other reproductive performances recorded were: mean litter size 6.8; mean birth weight 1.26 kg; weaning-to-return estrus interval 5 to 8 days. In conclusion, PG 600 was found to be useful in inducing fertile estrus in prepuberal gilts, a result which will be of interest for commercial pig farms.

Verification of flow cytometorically-sorted X- and Y-bearing porcine spermatozoa and reanalysis of spermatozoa for DNA content using the fluorescence in situ hybridization (FISH) technique.

Flow cytometric sperm sorting based on X and Y sperm DNA difference has been established as the only effective method for sexing the spermatozoa of mammals. The standard method for verifying the purity of sorted X and Y spermatozoa has been to reanalyze sorted sperm aliquots. We verified the purity of flow-sorted porcine X and Y spermatozoa and accuracy of DNA reanalysis by fluorescence in situ hybridization (FISH) using chromosome Y and 1 DNA probe. Eight ejaculates from 4 boars were sorted according to the Beltsville Sperm Sexing method. Porcine chromosome Y- and chromosome 1-specific DNA probes were used on sorted sperm populations in combination with FISH. Aliquots of the sorted sperm samples were reanalyzed for DNA content by flow cytometry. The purity of the sorted X-bearing spermatozoa was 87.4% for FISH and 87.0% for flow cytometric reanalysis; purity for the sorted Y-bearing spermatozoa was 85.9% for FISH and 84.8% for flow cytometric reanalysis. A total of 4,424 X sperm cells and 4,256 Y sperm cells was examined by FISH across the 8 ejaculates. For flow cytometry, 5,000 sorted X spermatozoa and 5,000 Y spermatozoa were reanalyzed for DNA content for each ejaculate. These results confirm the high purity of flow sorted porcine X and Y sperm cells and the validity of reanalysis of DNA in determining the proportions of X- and Y-sorted spermatozoa from viewing thousands of individual sperm chromosomes directly using FISH.

Rapid and simultaneous detection of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization.

This study was carried out to develop a rapid and simultaneous detection system of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization (FISH). Chromosome Y- and 1-specific DNA probes were produced by polymerase chain reaction with digoxigenin (Dig)- or biotin-dUTP. The hybridization probe mixture of labeled Y-chromosome and chromosome 1-specific DNA was applied to the preparation, immediately denatured at 75 degrees C for 8 min, hybridized for 5 min at 37 degrees C and overall FISH steps were done within a few hours. When double FISH with Dig-labeled chromosome Y-specific and biotin-labeled chromosome 1-specific probes was applied to sperm nuclei pretreated with dithiothreitol, the average of 50.9% of sperm nuclei had the Dig-signal, 99.2% of the sperm nuclei had the biotin-signal and the average of 0.3% of sperm nuclei showed no signal. The putative rate of Y-bearing spermatozoa ranged from 49.8% to 52.8% among 5 boars and the average putative rate of Y-bearing spermatozoa was 51.0%. The results indicated that a rapid and simultaneous FISH with chromosome Y- and 1-specific porcine DNA probes produced by PCR made possible more accurate assessment of Y-bearing porcine spermatozoa.

Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled, porcine male-specific DNA probe produced by polymerase chain reaction.

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5'-AAGTGGTCAGCGTGTCCATA-3' and 5'-TTTCTCCTGTATCCTCCTGC-3') for 236 bp fragment of porcine male-specific DNA sequence and 1.25 x 10(4) template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization.

Evidence for immunoreactive relaxin in boar seminal vesicles using combined light and electron microscope immunocytochemistry.

Light-microscope immunocytochemistry using the peroxidase-antiperoxidase technique and a polyclonal rabbit antiserum raised against purified porcine relaxin showed that cytoplasmic immunostaining for relaxin could be visualized in the epithelial cells of the seminal vesicle. No relaxin immunoreactivity was seen in the testis, epididymis, ductus deferens, prostate or bulbo-urethral gland. A ten times higher concentration of porcine relaxin antiserum was necessary to achieve immunostaining in the seminal vesicle comparable to that in the corpora lutea of pregnant sows. Ultrastructural examination showed that the epithelial cells of the boar seminal vesicle resembled typical protein-secreting cells with prominent rough endoplasmic reticulum and well-developed Golgi apparatus. The most striking feature of these cells was the accumulation of granules with a limiting membrane, which ranged from 200 to 600 nm in diameter and contained flocculent material of moderate electron density. Electron-microscope immunocytochemistry using the protein A-gold technique and relaxin antiserum demonstrated that the granules were the only intracellular organelles that showed immunoreactivity for relaxin. These results indicate that a relaxin-like substance is present in boar seminal vesicles and that the subcellular site of its localization is the granules, suggesting that the seminal vesicle produces and stores a relaxin-like substance, but that it is present at much lower concentrations than in the corpora lutea of pregnant sows.

Response of the lactating and postweaning sow to gonadotropin releasing hormone (GnRH).

Clinical and endocrinological responses to administration of gonadotropin releasing hormone analog (LH-RH-A) during the lactation period and postweaning in the sow were investigated. Plasma LH concentrations in lactating sows rose immediately after administration of LH-RH-A. However, in postweaning sows the increase of LH level was more slowly. Three of 5 postweaning sows came into estrus and ovulated after LH-RH-A treatment. One sow exhibited a distinct LH response, but her ovaries remained quiescent. The remaining one with feeble estrus for a short period became cystic ovaries. Thus, LH response to GnRH in the sow seems to be higher during early lactation than at 2 days postweaning.