Weaning transition, but not the administration of probiotic candidate Kazachstania slooffiae, shaped the gastrointestinal bacterial and fungal communities in nursery piglets.

As in-feed antibiotics are phased out of swine production, producers are seeking alternatives to facilitate improvements in growth typically seen from this previously common feed additive. Kazachstania slooffiae is a prominent commensal fungus in the swine gut that peaks in relative abundance shortly after weaning and has beneficial interactions with other bacteriome members important for piglet health. In this study, piglets were supplemented with K. slooffiae to characterize responses in piglet health as well as fungal and bacterial components of the microbiome both spatially (along the entire gastrointestinal tract and feces) and temporally (before, during, and after weaning). Litters were assigned to one of four treatments: no K. slooffiae (CONT); one dose of K. slooffiae 7 days before weaning (day 14; PRE); one dose of K. slooffiae at weaning (day 21; POST); or one dose of K. slooffiae 7 days before weaning and one dose at weaning (PREPOST). The bacteriome and mycobiome were analyzed from fecal samples collected from all piglets at day 14, day 21, and day 49, and from organ samples along the gastrointestinal (GI) tract at day 21 and day 49. Blood samples were taken at day 14 and day 49 for cytokine analysis, and fecal samples were assayed for antimicrobial resistance. While some regional shifts were seen in response to K. slooffiae administration in the mycobiome of the GI tract, no remarkable changes in weight gain or health of the animals were observed, and changes were more likely due to sow and the environment. Ultimately, the combined microbiome changed most considerably following the transition from suckling to nursery diets. This work describes the mycobiome along the piglet GI tract through the weaning transition for the first time. Based on these findings, K. slooffiae administered at this concentration may not be an effective tool to hasten colonization of K. slooffiae in the piglet GI tract around the weaning transition nor support piglet growth, microbial gut health, or immunity. However, diet and environment greatly influence microbial community development.

The impact of undernutrition on KNDy (kisspeptin/neurokinin B/dynorphin) neurons in female lambs.

Undernutrition limits reproduction through inhibition of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) secretion. Because KNDy neurons coexpress neuropeptides that play stimulatory (kisspeptin and neurokinin B [NKB]) and inhibitory (dynorphin) roles in pulsatile GnRH/LH release, we hypothesized that undernutrition would inhibit kisspeptin and NKB expression at the same time as increasing dynorphin expression. Fifteen ovariectomized lambs were either fed to maintain pre-study body weight (controls) or feed-restricted to lose 20% of pre-study body weight (FR) over 13 weeks. Blood samples were collected and plasma from weeks 0 and 13 were assessed for LH by radioimmunoassay. At week 13, animals were killed, and brain tissue was processed for assessment of KNDy peptide mRNA or protein expression. Mean LH and LH pulse amplitude were lower in FR lambs compared to controls. We observed lower mRNA abundance for kisspeptin within KNDy neurons of FR lambs compared to controls with no significant change in mRNA for NKB or dynorphin. We also observed that FR lambs had fewer numbers of arcuate nucleus kisspeptin and NKB perikarya compared to controls. These findings support the idea that KNDy neurons are important for regulating reproduction during undernutrition in female sheep.

Kisspeptin, Neurokinin B, and Dynorphin Expression during Pubertal Development in Female Sheep.

The neural mechanisms underlying increases in gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion that drive puberty onset are unknown. Neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin, i.e., KNDy neurons, are important as kisspeptin and NKB are stimulatory, and dynorphin inhibitory, to GnRH secretion. Given this, we hypothesized that kisspeptin and NKB expression would increase, but that dynorphin expression would decrease, with puberty. We collected blood and hypothalamic tissue from ovariectomized lambs implanted with estradiol at five, six, seven, eight (puberty), and ten months of age. Mean LH values and LH pulse frequency were the lowest at five to seven months, intermediate at eight months, and highest at ten months. Kisspeptin and NKB immunopositive cell numbers did not change with age. Numbers of cells expressing mRNA for kisspeptin, NKB, or dynorphin were similar at five, eight, and ten months of age. Age did not affect mRNA expression per cell for kisspeptin or NKB, but dynorphin mRNA expression per cell was elevated at ten months versus five months. Thus, neither KNDy protein nor mRNA expression changed in a predictable manner during pubertal development. These data raise the possibility that KNDy neurons, while critical, may await other inputs for the initiation of puberty.

Evidence that pubertal status impacts kisspeptin/neurokinin B/dynorphin neurons in the gilt†.

Puberty onset is a complex physiological process, which enables the capacity for reproduction through increased gonadotropin-releasing hormone and subsequently luteinizing hormone secretion. While cells that coexpress kisspeptin, neurokinin B (NKB), and dynorphin in the hypothalamic arcuate nucleus are believed to govern the timing of puberty, the degree to which kisspeptin/NKB/dynorphin (KNDy) neurons exist and are regulated by pubertal status remains to be determined in the gilt. Hypothalamic tissue from prepubertal and postpubertal, early follicular phase gilts was used to determine the expression of kisspeptin, NKB, and dynorphin within the arcuate nucleus. Fluorescent in situ hybridization revealed that the majority (>74%) of arcuate nucleus neurons that express mRNA for kisspeptin coexpressed mRNA for NKB and dynorphin. There were fewer arcuate nucleus cells that expressed mRNA for dynorphin in postpubertal gilts compared to prepubertal gilts (P < 0.05), but the number of arcuate nucleus cells expressing mRNA for kisspeptin or NKB was not different between groups. Within KNDy neurons, mRNA abundance for kisspeptin, NKB, and dynorphin of postpubertal gilts was the same as, less than, and greater than, respectively, prepubertal gilts. Immunostaining for kisspeptin did not differ between prepubertal and postpubertal gilts, but there were fewer NKB immunoreactive fibers in postpubertal gilts compared to prepubertal gilts (P < 0.05). Together, these data reveal novel information about KNDy neurons in gilts and support the idea that NKB and dynorphin play a role in puberty onset in the female pig.

Undernutrition reduces kisspeptin and neurokinin B expression in castrated male sheep.

Undernutrition impairs reproductive success through suppression of gonadotropin-releasing hormone (GnRH), and subsequently luteinizing hormone (LH), secretion. Given that kisspeptin and neurokinin B (NKB) neurons in the arcuate nucleus (ARC) of the hypothalamus are thought to play key stimulatory roles in the generation of GnRH/LH pulses, we hypothesized that feed restriction would reduce the ARC mRNA abundance and protein expression of kisspeptin and NKB in young, male sheep. Fourteen wethers (castrated male sheep five months of age) were either fed to maintain (FM; n = 6) pre-study body weight or feed-restricted (FR; n = 8) to lose 20% of pre-study body weight over 13 weeks. Throughout the study, weekly blood samples were collected and assessed for LH concentration using RIA. At Week 13 of the experiment, animals were killed, heads were perfused with 4% paraformaldehyde, and brain tissue containing the hypothalamus was collected, sectioned, and processed for detection of mRNA (RNAscope) and protein (immunohistochemistry) for kisspeptin and NKB. Mean LH was significantly lower and LH inter-pulse interval was significantly higher in FR wethers compared to FM wethers at the end of the experiment (Week 13). RNAscope analysis revealed significantly fewer cells expressing mRNA for kisspeptin and NKB in FR wethers compared to FM controls, and immunohistochemical analysis revealed significantly fewer immunopositive kisspeptin and NKB cells in FR wethers compared to FM wethers. Taken together, this data supports the idea that long-term feed restriction regulates GnRH/LH secretion through central suppression of kisspeptin and NKB in male sheep. LAY SUMMARY: While undernutrition is known to impair reproduction at the level of the brain, the components responsible for this in the brain remain to be fully understood. Using male sheep we examined the effect of undernutrition on two stimulatory molecules in the brain critical for reproduction: kisspeptin and neurokinin B. Feed restriction for several weeks resulted in decreased luteinizing hormone in the blood indicating reproductive function was suppressed. In addition, undernutrition also reduced both kisspeptin and neurokinin B levels within a region of the brain involved in reproduction, the hypothalamus. Given that they have stimulatory roles in reproduction, we believe that undernutrition acts in the brain to reduce kisspeptin and neurokinin B levels leading to the reduction in luteinizing hormone secretion. In summary, long-term undernutrition inhibits reproductive function in sheep through suppression of kisspeptin and neurokinin B within the brain.

Lipidome profiles of postnatal day 2 vaginal swabs reflect fat composition of gilt's postnatal diet.

We hypothesized that postnatal development of the vagina is impacted by early nutritional environment. Our objective was to determine if lipid profiles of vaginal swabs were different between postnatal gilts suckled by sow or fed milk replacer the first 48 h after birth, with or without a lard-based fat supplement. Gilts (>1.3 kg) were selected at birth across 8 litters and assigned to one of four treatments: 1) suckled by sow (S, n = 8); 2) suckled by sow plus administration of a fat supplement (SF, n = 5); 3) bottle-fed solely milk replacer (B, n = 8); or 4) bottle-fed solely milk replacer plus administration of a fat supplement (BF, n = 7). At 48 h postnatal, vaginal swabs of gilts were taken with a cytology brush, and lipids were extracted for analysis using multiple reaction monitoring (MRM)-profiling. Lipids extracted from serum collected at 48 h from gilts, milk collected at 24 h from sows, and milk replacer were also analyzed with MRM-profiling. Receiver operating characteristic curve analysis found 18 lipids recovered from vaginal swabs that highly distinguished between S and B gilts [area-under-the-curve (AUC) > 0.9], including phosphatidylethanolamine with 34 carbons and four unsaturations in the fatty acyl residues [PE (34:4)]. Twelve lipids from vaginal swabs highly correlated (r > 0.6; p < 0.01) with nutrition source. Lipids with greater abundance in milk replacer drove association. For example, mean intensity of PE (34:4) was 149-fold higher in milk replacer than colostrum. Consequently, PE (34:4) was found to have 1.6- and 2.12-fold higher levels in serum and vaginal swab samples (p < 0.001), respectively, of B gilts as compared to S gilts. Findings support that vaginal swabs can be used to noninvasively study effects of perinatal nutrition on tissue composition.

Temporal analysis of vaginal proteome reveals developmental changes in lower reproductive tract of gilts across the first two weeks postnatal.

In swine the upper reproductive tract undergoes early postnatal development, however little is known about the lower reproductive tract. Our objective was to measure cytology and proteome of vaginal swab samples taken on postnatal day (PND) 2 and 16 in gilts to determine if temporal changes occurred in cell and protein profiles during the first two weeks after birth. The posterior vagina was swabbed using a cytology brush on PND 0, 2 and 16 and slides were prepared. The proportion of anuclear and superficial cells increased and parabasal decreased (P < 0.05) from PND 0 to 16. Proteins isolated from vaginal swabs taken on PND 2 and 16 from six gilts across three litters were measured using LC-MS/MS. Over 1500 proteins were identified, with 881 differentially expressed (P-adj < 0.05) between PND 2 and 16. One-third of proteins upregulated between days were categorized as secreted, including lipocalins. Categories enriched by downregulated proteins included cell-cell adherens junction, translation and ER to Golgi vesicle-mediated transport, and reflected increased cornification of stratified epithelium and thus mirrored changes in cytology. Changes in cytology and proteome over the first two weeks after birth support that the porcine vagina continues to develop postnatal.

Diet Impacts Pre-implantation Histotroph Proteomes in Beef Cattle.

In ruminants, the period from fertilization to implantation is relatively prolonged, and the survival of embryos depends on uterine secretions known as histotroph. Our objective was to determine if the pre-breeding diet affected histotroph proteomes in beef cattle. Cows were assigned to one of four diets: a control diet (CON), a high-protein diet (PROT), a high-fat diet (OIL), or a high-protein and high-fat diet (PROT + OIL). After 185 days on these diets, an intravaginal progesterone implant (CIDR) was inserted for 7 days. At 9 days after CIDR removal, animals with a corpus luteum were selected ( n = 16; 4 per treatment). Proteins were isolated from the histotroph collected by uterine lavage and analyzed with liquid chromatography-tandem mass spectrometry. Over 2000 proteins were expressed ( n ≥ 3 cows per treatment), with 1239 proteins being common among all of the groups. There were 20, 37, 85, and 123 proteins unique to CON, PROT + OIL, PROT, and OIL, respectively. Relative to CON, 23, 14, and 51 proteins were differentially expressed in PROT + OIL, PROT, and OIL, respectively. Functional analysis found that 53% of histotroph proteins were categorized as extracellular exosome, 3.28% as cell-cell adhesion, and 17.4% in KEGG metabolic pathways. Differences in proteomes among treatments support the idea that pre-breeding diet affects histotroph. Understanding the impact of diet on histotroph proteins may help improve conception rates.