Introduction:

The limited ability to induce classical vitamin D deficiencies, and challenges to identify traits which adequately assess vitamin D requirements, such as measurements of body tissue and serum concentrations, complicate recommendations for dietary fortification and lead to speculative hype (Crenshaw et al, 2014). These constraints and limited research efforts are especially noted for recommendations in sow gestation and lactation diets. Likewise, research efforts to define vitamin D requirements for neonatal and growing pigs are also limited. The definitive guidelines for recommendations of vitamin D fortification of swine diets require establishment of functional biological endpoints for assessments of requirements.

Vitamin D: its role in productivity and animal well-being - Image 1

Figure 1. Growing pigs displaying kyphosis (Rortvedt and Crenshaw, 2012).

In a series of experiments we have established a critical role for carryover effects of vitamin D supplementation in sow diets on skeletal development of growing pigs (Amundson et al., 2016; and Amundson, 2016). The initial observations that led to these experiments involved an association of vitamin D concentrations in sow diets with the incidence of kyphosis in growing pigs (Rortvedt and Crenshaw, 2012). Kyphosis is defined as an idiopathic disease characterized by an outward spinal curvature. In swine, kyphosis is commonly referred to as a “hump-back” pig (Figure 1). In these experiments the incidence of kyphosis has been consistently reproduced in offspring of sows fed diets with no supplemental vitamin D during gestation and lactation. Kyphosis was not visible until pigs were 9 to 13 weeks of age, but the incidence of kyphosis associated with maternal diets was not prevented by nutrient supplementation in nursery diets. Further characterization of bone abnormalities detected in these pigs has provided insights into nutritional inputs that affect cellular signals involved in endochondral ossification with implications about osteochondrosis-like lesions commonly found in growing pigs.

Endochondral ossification describes the process of bone formation in which a cartilage matrix is coupled with replacement by mineralized tissue during bone growth. A primary biological function of vitamin D involves regulation of the cellular signals critical for coupling of endochondral ossification. Assessments of these signals offer potential insights in establishment of vitamin D requirements for prevention of skeletal abnormalities, such as osteochondrosis, and illustrate the importance of maternal diets on skeletal development.

Methods:

Experiments summarized in this review involve a common design for maternal diets. In both experiments gilts were fed corn-soybean meal diets with either 0, 325, or 1,750 IU/kg vitamin D3 from breeding through lactation. Results reported herein will focus on pig responses to the effects of maternal dietary treatments. The diets fed in the nursery phase of the two experiments were slightly altered but primary responses were attributed to maternal diets. At weaning (3 weeks) pigs were fed diets with 0 or 280 IU vitamin D3/kg and relatively minor modifications to dietary Ca and P (dependent on the experiment) to exacerbate responses to dietary vitamin D supplements through the nursery phase.

Assessments of pig responses included weight gain, dual energy x-ray absorptiometry (DXA) scans to determine whole body bone mineral content (BMC, g/pig) and bone mineral density (BMD, g/cm2). Tissues collected for additional assessments included: the femur for mechanical test and computed tomography scans (CT); the femur, vertebrae, kidney, and intestine for gene expression assessment; and serum, liver, muscle, and fat tissues from piglets and colostrum and day 18 milk samples for measurements of vitamin D concentrations. Quantitative real-time polymerase chain reaction (qPCR) analysis were completed on tissues to assess relative-fold expression of mRNA for genes involved in vitamin D homeostasis and cellular signals involved in endochondral ossification.

Results and Discussion:

A 25 to 50% incidence of kyphosis was observed in 13-week old pigs produced by sows fed diets with no supplemental vitamin D (Amundson, 2016). A lower incidence, 0 to 10%, (dependent on nursery diet) was observed in pigs produced by sows fed diets with 325 IU vitamin D/kg, but only one pig displayed symptoms of kyphosis if sows were fed diets with 1,750 IU vitamin D/kg. Kyphosis was not the only apparent skeletal abnormality induced in the hypovitaminosis D kyphotic pig model. Classical symptoms of rickets, such as bowed limbs, were not visible. Preliminary histological assessments of the affected vertebrae were consistent with osteochondrosis-like lesions in kyphotic pigs compared to vertebrae from control pigs. We elected to assess expression of specific genes for proteins known to be involved in endochondral ossification.

In two experiments (Amundson, 2016) with a total of 60 litters, differences were not detected in the number of live births, still births, mummified fetuses, or total births (defined as live births plus still births) among the 3 maternal dietary treatments. The average number of live births (13.3, SEM = 0.70) was a respectable average for first parity females compared with current commercial swine industry standards.

At the end of the nursery phase in one experiment (Amundson et al., 2016), pig traits that were affected by maternal diets included an 11% reduction in growth, a 25% reduction in whole body BMD, a 15% reduction in femur bending load and a 42% reduction in femur modulus of elasticity properties. Visual gross abnormalities were observed in the growth plates of femurs and vertebrae at birth, weaning, and the end of the 4-week nursery growth phase. Bone and soft tissue responses to maternal and nursery dietary D were not explained by serum or tissue 25- OH-D3 concentrations (Amundson, 2016). Maternal and nursery diet effects on mRNA expression of genes involved in D homeostasis (1α-hydroxylase and 24-hydroxylase) and bone metabolism (matrix metalloproteinases and fibroblast growth factor 23) were evident. In future experiments we hope to ultimately identify a biomarker for early detection of bone abnormalities prior to final lesion manifestation.

Our research with vitamin D supplements in sow gestation and lactation diets and the carry-over effects of these diets on development of skeletal abnormalities in growing pigs provides a range of vitamin D concentrations with levels that clearly create deficiencies, levels that are marginally adequate, and levels that provide a safety margin for supplements in diets for healthy, productive sows in confinement. The research project also demonstrates the importance of vitamin D in maternal diets on induction of skeletal lesions in offspring. Characterization of these skeletal abnormalities in the offspring and the cellular signals which are mediated by vitamin D will provide insights into chronic skeletal disorders such as osteochondrosis-like lesions which are observed in the spinal column and femur.

Conclusions:

Pig growth, whole body skeletal ash, femur ash, and femur mechanical strength properties were affected by vitamin D supplements of sow gestation and lactation diets with limited to no responses in pig tissue vitamin D stores or serum 25-OH D concentrations. Changes were detected in expression of the classical vitamin D homeostatic regulators and in cellular and tissue signals. Skeletal lesions in 9-week old pigs which developed in response to sow diets have been partially characterized. Key changes were detected in MMP9, MMP13 and VGEF expression in tissues sub-sampled from bones where lesions are grossly evident. These changes as well as visible presentations of kyphosis were, for most traits, not evident in pigs at birth or weaning, but first appeared by visual observation in pigs at 8 to 13 weeks of age. The kyphosis effects were mainly attributed to sow gestation and lactation diets, with limited responses due to changes in nursery dietary vitamin D, Ca, or P concentrations.

Implications:

Sow diets are often neglected, especially the gestation diet. The sow is assumed to deplete maternal stores to meet the needs of the developing fetus and nursing pig. Vitamin D supplements in sow diets clearly affected the incidence of kyphosis in the offspring. The skeletal abnormalities appear to be a consequence of uncoupled endochondral ossification, which results in osteochondrosis-like lesions. We have characterized specific matrix metalloproteinases, known to be involved in the growth plate development. The maternal dietary vitamin D supplements affected the relative expression of these factors at different ages, but most notably after the pigs were weaned. The effects of sow diets appear to have a greater influence on responses than the nursery diets. Stated another way, nutrient supplements in the nursery phase were not sufficient to overcome the negative consequences carried over from vitamin D insufficiencies of the sow gestation and lactation diets. Overall our research with vitamin D supplements in sow diets illustrates the important role that nutritional supplementation of vitamin D has on the developing fetus and neonatal pig. The classical parameters used to measure vitamin D sufficiency, such as serum 25-OH vitamin D concentrations, failed to accurately predict the incidence of kyphosis and other bone abnormalities described in these studies.

Presented at CLANA 2016, Cancun, Mexico.

References:

Amundson LA. 2016. Ph.D. Dissertation. University of Wisconsin-Madison. Amundson LA et al. 2016. Br. J. Nutr. 116:771-787. Crenshaw, TD et al. 2014 J. Anim. Sci. 92:883-886. Rortvedt LA and Crenshaw TD. 2012. J. Anim. Sci. 90:4905-4915.

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