Vaginal microbiota differences associated with pelvic organ prolapse risk during late gestation in commercial sows†.

During the last decade, sow mortality due to pelvic organ prolapse (POP) has increased. To better understand the biology associated with POP, sows were phenotypically assessed and assigned a perineal score (PS) based on presumed POP risk and categorized as PS1 (low), PS2 (moderate), or PS3 (high). The study objective was to identify changes in sow vaginal microbiota that may be associated with POP. The hypothesis is that vaginal microbiota differs between sows with variable risk for POP, and changes in microbiota during late gestation exist between sows with differing risk. Of the 2864 sows scored during gestation week 15, 1.0, 2.7, and 23.4% of PS1, PS2, and PS3 sows, respectively, subsequently experienced POP. Vaginal swabs subjected to 16S rRNA gene sequencing revealed differences in community composition (Bray-Curtis; P < 0.05) and individual operational taxonomic unit (OTU) comparisons between vaginal microbiota of PS1 and PS3 sows at gestation week 15. Further, differences (P < 0.05) in community composition and OTUs (Q < 0.05) were observed in PS3 sows that either did or did not subsequently experience POP. Differences in community structure (alpha diversity measurements; P < 0.05), composition (P < 0.05), and OTUs (Q < 0.05) were observed in gestation week 12 sows scored PS1 compared to week 15 sows scored PS1 or PS3, suggesting that sow vaginal microbiota shifts during late gestation differently as POP risk changes. Collectively, these data demonstrate that sows with greater POP risk have unique vaginal microflora, for which a better understanding could aid in the development of mitigation strategies.

Evaluation of the molecular response of corpora lutea to manganese-amino acid complex supplementation in gilts.

Porcine pregnancy establishment and maintenance are dependent on the formation of functional corpora lutea (CL). Manganese (Mn) is critical for CL function as it is a cofactor for Mn superoxide dismutase and enzymes involved in cholesterol synthesis. Previously, we have shown that luteal Mn content increased and luteal progesterone (P4) concentration decreased in the CL of gilts fed diets supplemented with an Mn-amino acid complex (Availa-Mn; Zinpro Corporation) compared with controls fed Mn sulfate. Importantly, serum P4 increased from 0 (estrus onset) to 12 d post estrus (dpe), as expected, but P4 abundance in circulation was not affected by dietary Mn source (P = 0.15). We hypothesized that a more bioavailable Mn source (which results in increased luteal Mn content) would alter the luteal proteome and abundance of mRNA associated with steroid biogenesis during the mid-luteal phase of the estrous cycle. Postpubertal gilts (n = 32) were assigned to one of the four gestation diets. The control diet (CON) contained 20 ppm of supplemental Mn in the form of Mn sulfate. Three additional diets included 20 (TRT1), 40 (TRT2), or 60 (TRT3) ppm of supplemental Mn in the form of a Mn-amino acid complex instead of Mn sulfate. Dietary treatment began at estrus synchronization (approximately 20 d before estrus) and continued through 12 dpe when gilts were euthanized and tissues were collected. Protein and total RNA extracts from the CL were used for proteomic analysis via label-free liquid chromatography with tandem mass spectrometry to assess global protein abundance and quantitative real-time polymerase chain reaction (qRT-PCR) to assess specific mRNA abundance, respectively. Compared with CON, 188, 382, and 401 proteins were differentially abundant (P < 0.10) in TRT1, TRT2, and TRT3, respectively. Gene Ontology enrichment software revealed that proteins involved in P4 signaling and cholesterol synthesis were downregulated in CL of gilts fed Mn-amino acid complex compared with controls. Quantitative RT-PCR showed that relative transcript abundance of genes encoding steroidogenic enzymes (CYP11A1 and StAR) in CL tissue was decreased in gilts from TRT2 compared with CON (P = 0.02), but TRT1 and TRT3 were not affected (P ≥ 0.30). Collectively, these data support our hypothesis that a more bioavailable dietary Mn source may influence luteal function by altering the abundance of protein and mRNA involved in steroidogenesis.

Circulating biomarkers associated with pelvic organ prolapse risk in late gestation sows.

Sow mortality, as the result of pelvic organ prolapse (POP), has been increasing in the last decade in the U.S. swine industry. The objective of this study was to identify potential biological markers associated with risk of POP in sows. We hypothesized that sows differing in perineal score (PS) from PS1-PS3 (PS1-a presumed low POP risk; PS2-a presumed moderate POP risk; and PS3-a presumed high POP risk) would differ in circulatory biomarkers of inflammation and hormonal profiles. On gestation week 15, 2,864 individual sows were assigned a PS, and subsequently, 1.0%, 2.7%, and 23.4% of PS1, PS2, or PS3 sows, respectively, experienced POP. During PS assignment at days 107-116 of gestation, blood samples were collected from sows on two farms of similar genetics, feed sources, and health status. Whole blood was subjected to complete blood count (CBC) analysis (n = 212) and steroid hormones were measured in serum from a subset (n = 110) of animals assigned PS3 parity matched to PS1. Lipopolysaccharide-binding protein (LBP), tumor necrosis factor-alpha (TNF-α), haptoglobin, C-reactive protein (CRP), and creatine kinase (CK) levels were also evaluated. Complete blood count analysis revealed decreased (P ≤ 0.05) mean platelet volume (3.9%), lymphocytes (6.5%), and monocytes (7.5%) in PS3 compared to PS1 sows. Increased (P ≤ 0.02) abundance of androstenedione (13.4%), androsterone (18.2%), estrone (24.8%), and 17ß-estradiol (26.2%) was observed in PS3 compared to PS1 sows. Additionally, a 25.8% increase (P = 0.04) in LBP in PS3 compared to PS1 sows was observed. Many dynamic physiological changes occur in sows during late gestation as they approach farrowing. The data presented herein demonstrate that distinct differences in concentrations of circulating biomarkers exist between late gestation sows at high or low risk for POP and may serve as a useful tool for understanding the etiology of POP and evaluation of mitigation strategies.

Impact of manganese amino acid complex on tissue-specific trace mineral distribution and corpus luteum function in gilts.

Functional corpora lutea (CL) are required for pregnancy establishment and gestational maintenance in swine, and CL function is susceptible to environmental influences. Manganese (Mn) could be critical in regulating CL function since it is a component of the antioxidant enzyme Mn superoxide dismutase (MnSOD) as well as enzymes involved in cholesterol and steroid hormone synthesis. We hypothesized that a more bioavailable dietary Mn source would increase Mn content in the CL thereby influencing luteal function during the mid-luteal phase of the estrous cycle. Postpubertal gilts (n = 32) were assigned to one of four gestation diets. The control diet (CON) met or exceeded National Research Council (2012) requirements and was formulated to contain 20 parts per million (ppm) of added Mn in the form of Mn sulfate. Three additional diets included 20 (treatment [TRT]1), 40 (TRT2), or 60 (TRT3) ppm of added Mn from a Mn-amino acid complex (Availa-Mn; Zinpro Corporation) instead of Mn sulfate. Dietary treatment began at estrus synchronization onset and continued through 12 days post estrus (dpe) of the ensuing estrous cycle. Blood samples were collected at estrus onset, which was assigned as 0 dpe, as well as 4, 8, and 12 dpe. Gilts were euthanized and tissues were collected at 12 dpe. Serum progesterone (P4) increased (P < 0.01) from 0 to 12 dpe but was unaffected by dietary treatment (P = 0.15) and there was no effect of the interaction between day and treatment (P = 0.85). Luteal Mn content increased (P ≤ 0.05) by 19%, 21%, and 24% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Luteal P4 concentrations decreased (P = 0.03) 25%, 26%, and 32% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Relative to CON gilts, CL calcium content decreased (P = 0.02) by 36%, 24%, and 34% for TRT1, TRT2, and TRT3 gilts, respectively. Collectively, these data support the hypothesis that feeding a more bioavailable Mn source increases Mn accumulation in CL tissue. If and how this influences CL function may be related to altered luteal P4 concentrations.

Vaginal microbiome and serum metabolite differences in late gestation commercial sows at risk for pelvic organ prolapse.

Sow mortality attributable to pelvic organ prolapse (POP) has increased in the U.S. swine industry and continues to worsen. Two main objectives of this study were, (1) to develop a perineal scoring system that can be correlated with POP risk, and (2) identify POP risk-associated biological factors. To assess POP risk during late gestation, sows (n = 213) were scored using a newly developed perineal scoring (PS) system. Sows scored as PS1 (low), PS2 (moderate), or PS3 (high) based on POP risk. Subsequently, 1.5, 0.8, and 23.1% of sows scored PS1, PS2, or PS3, respectively, experienced POP. To identify biomarkers, serum and vaginal swabs were collected from late gestation sows differing in PS. Using GC-MS, 82 serum metabolite differences between PS1 and PS3 animals (P < 0.05) were identified. Vaginal swabs were utilized for 16S rRNA gene sequencing and differences in vaginal microbiomes between PS1 and PS3 animals were detected on a community level (P < 0.01) along with differences in abundances of 89 operational taxonomic units (P < 0.05). Collectively, these data demonstrate that sows with greater POP risk have differential serum metabolites and vaginal microflora. Additionally, an initial and novel characterization of the sow vaginal microbiome was determined.

Novel Engraftment and T Cell Differentiation of Human Hematopoietic Cells in ART-/-IL2RG-/Y SCID Pigs.

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T- B- NK- SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (ARTEMIS)-/- genetic background. We confirmed ART-/-IL2RG-/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one ART-/-IL2RG-/Y male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal ART-/-IL2RG-/Y SCID pigs. At birth, human CD45+ CD3ε+ cells were detected in cord and peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.

Differentiating between the effects of heat stress and lipopolysaccharide on the porcine ovarian heat shock protein response1.

Heat stress (HS) negatively affects both human and farm-animal health and undermines efficiency in a variety of economically important agricultural variables, including reproduction. HS impairs the intestinal barrier, allowing for translocation of the resident microflora and endotoxins, such as lipopolysaccharide (LPS), from the gastrointestinal lumen into systemic circulation. While much is known about the cellular function of heat shock proteins (HSPs) in most tissues, the in vivo ovarian HSP response to stressful stimuli remains ill-defined. The purpose of this study was to compare the effects of HS or LPS on ovarian HSP expression in pigs. We hypothesized that ovarian HSPs are responsive to both HS and LPS. Altrenogest (15 mg/d) was administered per os for estrus synchronization (14 d) prior to treatment and three animal paradigms were used: (i) gilts were exposed to cyclical HS (31 ± 1.4 °C) or thermoneutral (TN; 20 ± 0.5 °C) conditions immediately following altrenogest withdrawal for 5 d during follicular development; (ii) gilts were subjected to repeated (4×/d) saline (CON) or LPS (0.1 µg/kg BW) i.v. infusion immediately following altrenogest withdrawal for 5 d; and (iii) gilts were subjected to TN (20 ± 1 °C) or cyclical HS (31 to 35 °C) conditions 2 d post estrus (dpe) until 12 dpe during the luteal phase. While no differences were detected for transcript abundances of the assessed ovarian HSP, the protein abundance of specific HSP was influenced by stressors during the follicular and luteal phases. HS during the follicular phase tended (P < 0.1) to increase ovarian protein abundance of HSP90AA1 and HSPA1A, and increased (P ≤ 0.05) HSF1, HSPD1, and HSPB1 compared with TN controls, while HS decreased HSP90AB1 (P = 0.01). Exposure to LPS increased (P < 0.05) HSP90AA1 and HSPA1A and tended (P < 0.1) to increase HSF1 and HSPB1 compared with CON gilts, while HSP90AB1 and HSPD1 were not affected by LPS. HS during the luteal phase increased (P < 0.05) abundance of HSPB1 in corpora lutea (CL), decreased (P < 0.05) CL HSP90AB1, but did not impact HSF1, HSPD1, HSP90AA1, or HSPA1A abundance. Thus, these data support that HS and LPS similarly regulate expression of specific ovarian HSP, which suggest that HS effects on the ovary are in part mediated by LPS.

The impact of dietary supplementation of arginine during gestation in a commercial swine herd: II. Offspring performance.

Arginine (Arg) is an important amino acid of pig fetal development; however, whether Arg improves postnatal performance is ill-defined. Therefore, the influence of Arg supplementation at different gestational stages on offspring performance was evaluated in a commercial swine herd. Sows (n = 548) were allocated into 4, diet by stage of gestation treatments: Control (n = 143; 0% suppl. Arg), or dietary treatments supplemented with 1% L-Arg (free-base; Ajinomoto Animal Nutrition North America, Inc., Chicago, IL): from 15 to 45 d of gestation (n = 138; Early-Arg); 15 d of gestation to farrowing (n = 139; Full-Arg); and from day 85 of gestation to farrowing (n = 128; Late-Arg). All offspring were individually identified and weighed at birth; at weaning, a subset was selected for evaluation of carcass performance at market. All data were analyzed using birth weight (BiWt) and age as covariates. Wean weights (WW) and prewean (PW) ADG tended to increase (P = 0.06) in progeny from sows supplemented with Arg, as compared to progeny from Control sows. Preplanned contrast comparisons revealed an increased (P = 0.03) BiWt for pigs from sows receiving 1% L-Arg prior to day 45 of gestation (Early-Arg and Full-Arg; 1.38 kg/pig), as compared to pigs from sows not supplemented prior to day 45 of gestation (Control and Late-Arg; 1.34 kg/pig). No difference in BiWt was observed (1.36 kg/pig; P = 0.68) for Arg supplementation after day 85 of gestation (Full-Arg and Late-Arg), as compared to those not receiving Arg supplementation after day 85 (Control and Early-Arg); although WW and PW ADG were greater (P = 0.02), respectively. A 3.6% decrease (P = 0.05) in peak lean accretion ADG occurred when dams received 1% L-Arg prior to day 45 of gestation (Early-Arg and Full-Arg), however, no other significant differences were detected in finishing growth parameters or carcass characteristics (P ≥ 0.1). Pig mortality rates tended (P = 0.07) to decrease in progeny of dams supplemented Arg after day 85 (3.6%) compared to dams not provided additional Arg during late gestation (4.9%). Collectively, these data suggest that Arg provided during late gestation may improve WW and PW ADG, however, finishing performance was not affected. While Arg supplementation provided some moderate production benefits, further investigation is warranted to comprehensively understand the gestational timing and biological role of Arg supplementation during fetal and postnatal development in commercial production systems.

The impact of dietary supplementation of arginine during gestation in a commercial swine herd: I. Gilt reproductive performance.

Supplemental arginine (Arg) during gestation purportedly benefits fetal development. However, the benefits of a gestational Arg dietary strategy in commercial production are unclear. Therefore, the objectives of this study examined Arg supplementation during different gestational stages and the effects on gilt reproductive performance. Pubertal gilts (n = 548) were allocated into 4 treatment groups: Control (n = 143; 0% supplemental Arg) or 1 of 3 supplemental Arg (1% as fed) treatments: from 15 to 45 d of gestation (n = 138; Early-Arg); from 15 d of gestation until farrowing (n = 139; Full-Arg); or from 85 d of gestation until farrowing (n = 128; Late-Arg). At farrowing, the number of total born (TB), born alive (BA), stillborn piglets (SB), mummified fetuses (MM), and individual piglet birth weights (BiWt) were recorded. The wean-to-estrus interval (WEI) and subsequent sow reproductive performance (to third parity) were also monitored. No significant effect of supplemental Arg during any part of P0 gestation was observed for TB, BA, SB, or MM (P ≥ 0.29). Offspring BiWt and variation among individual piglet birth weights did not differ (P = 0.42 and 0.89, respectively) among treatment groups. Following weaning, the WEI was similar among treatments (average of 8.0 ± 0.8 d; P = 0.88). Litter performance over 3 parities revealed a decrease (P = 0.02) in BA for Early-Arg fed gilts compared with all other treatments, whereas TB and WEI were similar among treatments over 3 parities (P > 0.05). There was an increased proportion of sows with average size litters (12 to 16 TB) from the Full-Arg treatment sows (76.8% ± 3.7%) when compared with Control (58.7% ± 4.2%; P = 0.01); however, the proportion of sows with high (>16 TB) and low (<12 TB) litters was not different among treatments (P = 0.20). These results suggest that gestational Arg supplementation had a minimal impact on reproductive performance in first parity sows. These data underscore the complexity of AA supplementation and the need for continued research into understanding how and when utilizing a gestational dietary Arg strategy can optimize fetal development and sow performance.