Changes in mononuclear immune cells during bovine pregnancy.

This study aimed to determine the differences in gene expression between mononuclear cells derived from peripheral blood and endometrium during pregnancy in cattle and to determine the proportion of mononuclear cells in the endometrium of pregnant and diestrous cows. Endometrial tissue and peripheral blood were collected from Day 34±2 pregnant cows, and mononuclear cell populations were quantified and sorted (n =5). The relative mRNA levels of inflammatory mediators was assessed by quantitative real time polymerase chain reaction. During pregnancy, the proportion of CD8+ , CD4+ , CD4+ CD25- and CD4+ CD25dim cells among mononuclear cells was greater in blood than endometrium, and cells positive for CD14 and CD68 expressed greater mRNA amounts of interleukin (IL ) 6 , CXCL8 and IL10 in endometrium compared with blood. Cells positive for γ/δ-T cell receptor expressed greater amounts of IL1A transcript in the endometrium than in blood of diestrous cows, CD4+ CD25bright cells expressed more CTLA4 mRNA in the endometrium compared with blood of diestrous cows, and endometrial natural killer cells expressed greater CXCL8 mRNA compared with blood of pregnant and diestrous cows. The percentages of CD21+ , NCR1+ , CD8+ , FoxP3+ , CD3+ and CD68+ cells were greater in the endometrium of Day 35 pregnant cows compared with diestrous cows.

Increased expression of pro-inflammatory cytokines at the fetal-maternal interface in bovine pregnancies produced by cloning.

PROBLEM: A significant rate of spontaneous abortion is observed in cattle pregnancies produced by somatic cell nuclear transfer (SCNT). Major histocompatibility complex class I (MHC-I) proteins are abnormally expressed on the surface of trophoblast cells from SCNT conceptuses. METHOD OF STUDY: MHC-I homozygous compatible (n = 9), homozygous incompatible (n = 8), and heterozygous incompatible (n = 5) pregnancies were established by SCNT. Eight control pregnancies were established by artificial insemination. Uterine and trophoblast samples were collected on day 35 ±1 of pregnancy, the expression of immune-related genes was examined by qPCR, and the expression of trophoblast microRNAs was assessed by sequencing. RESULTS: Compared to the control group, trophoblast from MHC-I heterozygous incompatible pregnancies expressed increased levels of CD28, CTLA4, CXCL8, IFNG, IL1A, IL2, IL10, IL12B, TBX21, and TNF, while GNLY expression was downregulated. The MHC-I homozygous incompatible treatment group expressed increased levels of IFNG, IL1A, and IL2 while the MHC-I homozygous compatible group did not differentially express any genes compared to the control group. In the endometrium, relative to the control group, MHC-I heterozygous incompatible pregnancies expressed increased levels of CD28, CTLA4, CXCL8, IFNG, IL10, IL12B, and TNF, while GATA3 expression was downregulated. The MHC-I homozygous incompatible group expressed decreased amounts of CSF2 transcripts compared with the control group but did not have abnormal expression of any other immune-related genes. MHC-I incompatible pregnancies had 40 deregulated miRNAs compared to control pregnancies and 62 deregulated microRNAs compared to MHC-I compatible pregnancies. CONCLUSIONS: MHC-I compatibility between the dam and fetus prevented an exacerbated maternal immune response from being mounted against fetal antigens.

Lymphocyte soluble factors from pregnant cows modulate mRNA transcript abundances encoding for proteins associated with trophoblast growth and development.

This study was conducted to determine whether T cell populations are responsible for modulating placental development during gestation in cattle. It was hypothesized that CD4+CD25+ and γ/δ+ T cells modulate gene expression, based on mRNA transcript abundances, and promote proliferation and survival of trophoblast cells. Peripheral blood was collected from cows at 160 to 180 days of gestation and non-pregnant cows, T cell populations CD8+, CD4+, CD4+CD25+, CD24+CD25-, and γ/δ+ T cells were isolated, cultured for 48 h, and supernatant was collected. Placental samples were digested, and trophoblast cells were cultured for 24 h. Trophoblast cells were cultured with 50 µL of T cell-conditioned media and 50 µL of fresh culture media for an additional 48 h. Samples in control wells were treated with unconditioned media. Trophoblast cell proliferation, apoptosis, and mRNA transcript assays were conducted. There was no effect of T cell population on trophoblast apoptosis rate, proliferation, and relative mRNA transcript abundances. The T cell supernatant from pregnant and non-pregnant cows induced greater apoptosis rates in trophoblast cells than unconditioned media. Trophoblast cells proliferated less when treated with T cell supernatant from pregnant compared to unconditioned medium and non-pregnant cows. Treatment with the T cell supernatant from pregnant cows resulted in larger abundances of BMP5, IGF1R, PAG10, FGF2, RSPO3 and TMED2 and also a lesser abundance of FGF2 mRNA transcript than non-pregnant group and unconditioned media treatments. Supernatant from T cell derived from pregnant cows modulates trophoblast mRNA transcript abundances differently from T cell supernatant of non-pregnant cows.

Spontaneous Atrial Fibrillation in Transgenic Goats With TGF (Transforming Growth Factor)-ß1 Induced Atrial Myopathy With Endurance Exercise.

BACKGROUND: There is increasing evidence that endurance exercise is associated with increased risk of atrial fibrillation (AF). However, it is unknown if the relationship between endurance exercise and AF is dependent on an atrial myopathy. METHODS: Six cardiac-specific TGF (transforming growth factor)-ß1 transgenic and 6 wild-type (WT) goats were utilized for these studies. Pacemakers were implanted in all animals for continuous arrhythmia monitoring and AF inducibility. AF inducibility was evaluated using 5 separate 10 s bursts of atrial pacing (160-200 ms). Three months of progressive endurance exercise (up to 90 minutes at 4.5 mph) was performed. Quantitative assessment of circulating microRNAs and inflammatory biomarkers was performed. RESULTS: Sustained AF (≥30 s) was induced with 10 s of atrial pacing in 4 out of 6 transgenic goats compared with 0 out of 6 WT controls at baseline (P<0.05). No spontaneous AF was observed at baseline. Interestingly, between 2 and 3 months of exercise 3 out of 6 transgenic animals developed self-terminating spontaneous AF compared with 0 out of 6 WT animals (P<0.05). There was an increase in AF inducibility in both transgenic and WT animals during the first 2 months of exercise with partial normalization at 3 months (transgenic 67%; 100%; 83% versus WT 0%; 67%; 17%). These changes in AF susceptibility were associated with a decrease in circulating microRNA-21 and microRNA-29 during the first 2 months of exercise with partial normalization at 3 months in both transgenic and WT animals. Finally, MMP9 (matrix metallopeptidase 9) was increased during the second and third months of exercise training. CONCLUSIONS: This study demonstrates a novel transgenic goat model of cardiac fibrosis (TGF-ß1 overexpression) to demonstrate that endurance exercise in the setting of an underlying atrial myopathy increases the incidence of spontaneous AF. Furthermore, endurance exercise seems to increase inducible AF secondary to altered expression of key profibrotic biomarkers that is independent of the presence of an atrial myopathy.

Assessment of microchimerism following somatic cell nuclear transfer and natural pregnancies in goats.

Microchimerism is defined as the presence of a small population of cells or DNA in 1 organism originated from a genetically different organism. It is well established that this phenomenon occurs in humans and mice as cells are exchanged between mother and fetus during gestation. Currently, no information is available about the presence of maternal microchimerism in goats, and the only published study is limited to an evaluation of fetal and fetal-fetal microchimerism in blood samples following natural breeding. In order to determine whether bidirectional fetal-maternal cell or DNA trafficking occurs in goats, we assessed: 1) fetal microchimerism in surrogates that gave birth to somatic cell nuclear transfer (SCNT)-derived transgenic offspring (n = 4), 2) maternal microchimerism following natural breeding of SCNT-derived transgenic does with a nontransgenic buck (n = 4), and 3) fetal-fetal microchimerism in nontransgenic twins of transgenic offspring (n = 3). Neomycin-resistance gene (NEO) gene was selected as the marker to detect the presence of the αMHC-TGF-ß1-Neo transgene in kidney, liver, lung, lymph node, and spleen. We found no detectable maternal or fetal-fetal microchimerism in the investigated tissues of nontransgenic offspring. However, fetal microchimerism was detected in lymph node tissue of one of the surrogate dams carrying a SCNT pregnancy. These results indicate occurrence of cell trafficking from fetus to mother during SCNT pregnancies. The findings of this study have direct implications on the use and disposal of nontransgenic surrogates and nontransgenic offspring.

Gene expression and lymphocyte population at the fetal-maternal interface in sheep pregnancies established by somatic cell nuclear transfer.

The hypothesis of this study was that the leukocyte populations and expression levels of genes related to immune response, growth factors and apoptosis would be altered at the fetal-maternal interface in somatic cell nuclear transfer (SCNT)-generated sheep pregnancies. Placental and endometrial samples from sheep pregnancies established by SCNT and natural breeding (control) were collected at 45 days and at term. Expression of genes related to growth factors, apoptosis and immune response was examined using quantitative reverse transcription polymerase chain reaction. Endometrial leukocyte populations and major histocompatibility class I (MHC-I) protein expression were examined by immunohistochemistry. At term we observed altered expression of genes related to apoptosis, growth factors and immune response in placental and endometrial tissue of SCNT pregnancies. In Day-45 pregnancies there was less-pronounced abnormal expression and only genes related to apoptosis and growth factors were abnormal in the placenta. Endometrial gene expression profiles were similar to age-matched controls. Placental MHC-I protein expression was similar in SCNT and controls at 45 days but increased in the SCNT at term. The altered gene expression at the fetal-maternal interface likely contributes to the placental dysfunction and overgrowth observed in sheep SCNT pregnancies.

Genetic and epigenetic regulation of major histocompatibility complex class I gene expression in bovine trophoblast cells.

PROBLEM: The regulatory mechanisms governing differential expression of classical major histocompatibility complex (MHC) class I (MHC-Ia) and non-classical MHC class I (MHC-Ib) genes are poorly understood. METHOD OF STUDY: Quantitative reverse transcription- polymerase chain reaction (PCR) was used to compare the abundance of MHC-I transcripts and related transcription factors in peripheral blood mononuclear cells (PBMC) and placental trophoblast cells (PTC). Methylation of MHC-I CpG islands was detected by bisulfite treatment and next-generation sequencing. Demethylation of PBMC and PTC with 5'-aza-deoxycytidine was used to assess the role of methylation in gene regulation. RESULTS: MHC-I expression was higher in PBMC than PTC and was correlated with expression of IRF1, class II MHC transactivator (CIITA), and STAT1. The MHC-Ia genes and BoLA-NC1 were devoid of CpG methylation in PBMC and PTC. In contrast, CpG sites in the gene body of BoLA-NC2, -NC3, and -NC4 were highly methylated in PBMC but largely unmethylated in normal PTC and moderately methylated in somatic cell nuclear transfer PTC. In PBMC, demethylation resulted in upregulation of MHC-Ib by 2.8- to 6-fold, whereas MHC-Ia transcripts were elevated less than 2-fold. CONCLUSION: DNA methylation regulates bovine MHC-Ib expression and is likely responsible for the different relative levels of MHC-Ib to MHC-Ia transcripts in PBMC and PTC.

Cytokine gene expression at the maternal-fetal interface after somatic cell nuclear transfer pregnancies in small ruminants.

The present retrospective study investigated pregnancy rates, the incidence of pregnancy loss and large offspring syndrome (LOS) and immune-related gene expression of sheep and goat somatic cell nuclear transfer (SCNT) pregnancies. We hypothesised that significantly higher pregnancy losses observed in sheep compared with goat SCNT pregnancies are due to the increased amounts of T-helper 1 cytokines and proinflammatory mediators at the maternal-fetal interface. Sheep and goat SCNT pregnancies were generated using the same procedure. Control pregnancies were established by natural breeding. Although SCNT pregnancy rates at 45 days were similar in both species, pregnancy losses between 45 and 60 days of gestation and the incidence of LOS were significantly greater in sheep than in goats. At term, the expression of proinflammatory genes in sheep SCNT placentas was increased, whereas that in goats was similar to that in control animals. Genes with altered expression in sheep SCNT placentas included cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin 2 receptor alpha (IL2RA), cluster of differentiation 28 (CD28), interferon gamma (IFNG), interleukin 6 (IL6), interleukin 10 (IL10), transforming growth factor beta 1 (TGFB1), tumor necrosis factor alpha (TNF-α), interleukin 1 alpha (IL1A) and chemokine (C-X-C motif) ligand 8 (CXCL8). Major histocompatibility complex-I protein expression was greater in sheep and goat SCNT placentas at term than in control pregnancies. An unfavourable immune environment is present at the maternal-fetal interface in sheep SCNT pregnancies.

Oocytes from small and large follicles exhibit similar development competence following goat cloning despite their differences in meiotic and cytoplasmic maturation.

Reduced developmental competence after IVF has been reported using oocyte derived from small follicles in several species including cattle, sheep, and goats. No information is currently available about the effect of follicle size of the cytoplast donor on in vivo development after somatic cell nuclear transfer (SCNT) in goats. Oocytes collected from large (≥3 mm) and small follicles (<3 mm) were examined for maturation and in vivo developmental competence after SCNT. Significantly greater maturation rate was observed in oocytes derived from large follicles compared with that of small follicles (51.6% and 33.7%, P < 0.05). Greater percent of large follicle oocytes exhibited a low glucose-6-phosphate dehydrogenase activity at germinal vesicle stage compared with small follicle oocytes (54.9% and 38.7%, P < 0.05). Relative mRNA expression analysis of 48 genes associated with embryonic and fetal development revealed that three genes (MATER, IGF2R, and GRB10) had higher level of expression in metaphase II oocytes from large follicles compared with oocytes from small follicles. Nevertheless, no difference was observed in pregnancy rates (33.3% vs. 47.1%) and birth rates (22.2% vs. 16.7%) after SCNT between the large and small follicle groups). These results indicate that metaphase II cytoplasts from small and large follicles have similar developmental competence when used in goat SCNT.

Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane.

Trophoblast Major Histocompatibility Complex Class I Expression Is Associated with Immune-Mediated Rejection of Bovine Fetuses Produced by Cloning.

Trophoblast cells from bovine somatic cell nuclear transfer (SCNT) conceptuses express major histocompatibility complex class I (MHC-I) proteins early in gestation, and this may be one cause of the significant first-trimester embryonic mortality observed in these pregnancies. MHC-I homozygous-compatible (n = 9), homozygous-incompatible (n = 8), and heterozygous-incompatible (n = 5) SCNT pregnancies were established. The control group consisted of eight pregnancies produced by artificial insemination. Uterine and placental samples were collected on Day 35 ± 1 of pregnancy, and expression of MHC-I, leukocyte markers, and cytokines were examined by immunohistochemistry. Trophoblast cells from all SCNT pregnancies expressed MHC-I, while trophoblast cells from age-matched control pregnancies were negative for MHC-I expression. Expression of MHC-I antigens by trophoblast cells from SCNT pregnancies was associated with lymphocytic infiltration in the endometrium. Furthermore, MHC-I-incompatible conceptuses, particularly the heterozygous-incompatible ones, induced a more pronounced lymphocytic infiltration than MHC-I-compatible conceptuses. Cells expressing cluster of differentiation (CD) 3, gamma/deltaTCR, and MHC-II were increased in the endometrium of SCNT pregnancies compared to the control group. CD4(+) lymphocytes were increased in MHC-I-incompatible pregnancies compared to MHC-I-compatible and control pregnancies. CD8(+), FOXP3(+), and natural killer cells were increased in MHC-I heterozygous-incompatible SCNT pregnancies compared to homozygous SCNT and control pregnancies.

Increased Susceptibility to Atrial Fibrillation Secondary to Atrial Fibrosis in Transgenic Goats Expressing Transforming Growth Factor-ß1.

INTRODUCTION: Large animal models of progressive atrial fibrosis would provide an attractive platform to study relationship between structural and electrical remodeling in atrial fibrillation (AF). Here we established a new transgenic goat model of AF with cardiac specific overexpression of TGF-ß1 and investigated the changes in the cardiac structure and function leading to AF. METHODS AND RESULTS: Transgenic goats with cardiac specific overexpression of constitutively active TGF-ß1 were generated by somatic cell nuclear transfer. We examined myocardial tissue, ECGs, echocardiographic data, and AF susceptibility in transgenic and wild-type control goats. Transgenic goats exhibited significant increase in fibrosis and myocyte diameters in the atria compared to controls, but not in the ventricles. P-wave duration was significantly greater in transgenic animals starting at 12 months of age, but no significant chamber enlargement was detected, suggesting conduction slowing in the atria. Furthermore, this transgenic goat model exhibited a significant increase in AF vulnerability. Six of 8 transgenic goats (75%) were susceptible to AF induction and exhibited sustained AF (>2 minutes), whereas none of 6 controls displayed sustained AF (P < 0.01). Length of induced AF episodes was also significantly greater in the transgenic group compared to controls (687 ± 212.02 seconds vs. 2.50 ± 0.88 seconds, P < 0.0001), but no persistent or permanent AF was observed. CONCLUSION: A novel transgenic goat model with a substrate for AF was generated. In this model, cardiac overexpression of TGF-ß1 led to an increase in fibrosis and myocyte size in the atria, and to progressive P-wave prolongation. We suggest that these factors underlie increased AF susceptibility.

Livestock in biomedical research: history, current status and future prospective.

Livestock models have contributed significantly to biomedical and surgical advances. Their contribution is particularly prominent in the areas of physiology and assisted reproductive technologies, including understanding developmental processes and disorders, from ancient to modern times. Over the past 25 years, biomedical research that traditionally embraced a diverse species approach shifted to a small number of model species (e.g. mice and rats). The initial reasons for focusing the main efforts on the mouse were the availability of murine embryonic stem cells (ESCs) and genome sequence data. This powerful combination allowed for precise manipulation of the mouse genome (knockouts, knockins, transcriptional switches etc.) leading to ground-breaking discoveries on gene functions and regulation, and their role in health and disease. Despite the enormous contribution to biomedical research, mouse models have some major limitations. Their substantial differences compared with humans in body and organ size, lifespan and inbreeding result in pronounced metabolic, physiological and behavioural differences. Comparative studies of strategically chosen domestic species can complement mouse research and yield more rigorous findings. Because genome sequence and gene manipulation tools are now available for farm animals (cattle, pigs, sheep and goats), a larger number of livestock genetically engineered (GE) models will be accessible for biomedical research. This paper discusses the use of cattle, goats, sheep and pigs in biomedical research, provides an overview of transgenic technology in farm animals and highlights some of the beneficial characteristics of large animal models of human disease compared with the mouse. In addition, status and origin of current regulation of GE biomedical models is also reviewed.

Effect of time and dose of recombinant follicle stimulating hormone agonist on the superovulatory response of sheep.

The objective of this study was to determine the superovulatory potential of a single-chain analog of human FSH (Fcα) when administered to ewes either 3 days before, or coincident with, simulated luteolysis (pessary removal [PR]). A total of 40 animals were randomly assigned to receive Fcα at doses of 0.62, 1.25, or 2.5 IU/kg of body weight (bwt) 3 days before PR or 0.31, 0.62, 1.25, or 2.5 IU/kg of bwt at PR. Control ewes received protein without FSH activity. Blood samples were collected during the periovulatory period and ovarian tissue was collected 11 days after PR. Ovulation rate did not differ from the control group in ewes receiving the smallest doses of Fcα (0.31 and 0.62 IU/kg). However, a significant superovulatory response was noted in sheep receiving Fcα at doses of 1.25 and 2.5 IU/kg and this response was comparable in animals receiving the largest dose levels of Fcα at, or 3 days before, PR. The interval between PR and the LH surge was significantly extended and the LH surges were less synchronous in animals receiving Fcα at PR when compared with animals receiving the potent FSH agonist 3 days before PR. Taken together, these data indicate that the human single-chain gonadotropin with FSH activity promotes superovulation in ewe lambs in the breeding season. A single injection of the recombinant gonadotropin 3 days before luteolysis synchronizes the LH surge. The use of the single-chain analog of FSH in assisted reproduction for domestic animals is likely to be of practical significance as an alternative to conventional gonadotropins in superovulation protocols in livestock species.

Effect of single-chain ovine gonadotropins with dual activity on ovarian function in sheep.

We examined the half-life and biological activity of two single-chain proteins that combined portions of ovine FSH and LH. We proposed the hypothesis that these chimeric proteins would display LH and FSH activities and would promote follicle maturation in ewes. Estrus activity was synchronized using progestogen-impregnated vaginal pessaries. To negate the impact of endogenous LH and FSH, animals received serum-containing antibodies against GNRH 1 day before pessary removal (PR). At PR sheep (five animals per group) received a single injection (10 IU/kg, i.v.) of either the ovine-based (oFcLcα) gonadotropin analog, an ovine-based analog containing oLHß truncated at the carboxyl terminus (oFcL(ΔT)cα), or a human-based gonadotropin analog (hFcLcα). Control animals received a comparable amount of gonadotropin-free protein. Ovulation was induced 3 days after PR using human chorionic gonadotropin (1000 IU, i.v.). Ovaries were collected 11 days after PR. Neither estradiol (E2) or progesterone (P4) production, development of preovulatory follicles or corpora lutea (CL) were noted in control animals receiving gonadotropin-free protein. Significant increase in the synthesis of E2 and P4 was noted in sheep receiving the dually active gonadotropin analogs. The number of CLs present 11 days after PR was significantly increased in sheep receiving the chimeric glycoproteins compared with control animals. The magnitude of the secretory and ovarian responses did not differ between hFcLcα and oFcLcα or between oFcLcα and oFcL(ΔT)cα. Immunoactivity of LH and FSH was low in control animals, but was significantly elevated in sheep receiving the gonadotropin analogs. In conclusion, ovine-based gonadotropin analogs are functionally active in sheep and a single injection is adequate to induce the development of multiple ovulatory follicles.

Period of dominance of the ovulatory follicle influences embryo quality in lactating dairy cows.

Length of dominance of the ovulatory follicle and exposure to oestradiol (OE(2)) during proestrus can affect fertility. Lactating cows had their oestrous cycle pre-synchronized and were subjected to one of the four synchronization treatments. Cows in the oestrus detection (OD) treatment received GnRH on day 6 of the oestrous cycle, PGF(2alpha) 7 days later, and were inseminated at detected oestrus. The remaining cows were subjected to the Ovsynch (OVS) protocol (day 0 GnRH, day 7 PGF(2alpha), day 9 GnRH, and timed artificial insemination (AI) 12 h later) starting on day 3 (OVS3) or day 6 (OVS6 and OVS6E) of the oestrous cycle. Cows in the OVS6E treatment received an injection of 0.5 mg oestradiol cypionate 36 h before AI. Ovaries were examined by ultrasonography and blood was sampled for progesterone and OE(2) concentrations. Uteri were flushed 6 days after AI and recovered embryos-oocytes evaluated. Diameter of the ovulatory follicle at AI differed (P<0.01) among treatments, and it was the largest for OVS3 cows, which also had extended (P<0.01) length of follicular dominance. During proestrus, OD and OVS6E cows had increased (P<0.01) OE(2) concentrations. Fertilization was not altered by treatments, and maximum fertilization was achieved when the number of accessory spermatozoa was >7. Proportions of viable embryos in relation to embryos and embryos-oocytes recovered were smaller for OVS3 cows (P<0.01) than the other treatments, and embryos from OVS3 cows also had fewer (P<0.01) blastomeres and tended (P=0.09) to have a lower proportion of live blastomeres. Extending the period of follicle dominance did not alter fertilization but reduced (P<0.001) embryo quality. Embryo quality was compromised even when the dominance of the ovulatory follicle was extended by only 1.5 days.

Efficacy of an injection of dinoprost tromethamine when given subcutaneously on luteal regression in lactating Holstein cows.

The objectives of these studies were to evaluate the efficacy of a PGF(2alpha) (PGF) analog given through different routes on causing luteal regression in lactating dairy cows. In Experiment 1, lactating Holstein cows (n=118) at random stages of lactation were blocked by parity and days in milk (DIM) and, within each block, randomly assigned to receive PGF as an intra-muscular (IM) injection in the semimembranous/semitendinous muscle (CON), subcutaneous (SC) injection in the cervical area (SCN), or SC injection in the ischio-rectal fossa (IRF). Blood was sampled at 0, 12, 24, 36, and 48 h after treatment for assessment of progesterone concentration. In Experiment 2, a total of 379 lactating Holstein cows, 46+/-7 DIM, were blocked by DIM and, within each block, randomly assigned to receive treatment similar to CON or IRF groups from Experiment 1. Blood was sampled 0 and 48 h after treatment for assessment of progesterone concentration. Cows were classified as experiencing luteal regression when progesterone concentration was <1.0 ng/mL or <40% of initial concentration (0 h=100%). In Experiment 1, there was no effect of route of PGF treatment on decline in progesterone concentration and on the proportion of cows experiencing luteal regression by 12, 24, 36, and 48 h after treatment. Similarly, in Experiment 2, route of treatment did not affect either the decline in progesterone concentration or the proportion of cows that had luteal regression by 48 h after treatment. Treatment of lactating dairy cows with 25mg of PGF given SC in the ischio-rectal fossa did not affect either the decline in progesterone concentration or the proportion of cows that experienced luteal regression by 12, 24, 36, and 48 h after PGF treatment.