# Effects of an Immunomodulatory Supplement and Evaporative Cooling on Immune Status, Mammary Gland Microstructure, and Gene Expression of Cows Exposed to Heat Stress During the Dry Period

## TL;DR

This study shows that an immunomodulatory supplement and evaporative cooling can help reduce the negative effects of heat stress on dairy cows' immune function and mammary development during the dry period.

## Contribution

The study demonstrates that OMN supplementation during heat stress can mimic the beneficial effects of evaporative cooling on mammary gene expression and development.

## Key findings

- Heat stress reduces alveoli number and alters gene expression related to apoptosis and autophagy.
- OMN supplementation increases cell apoptosis and proliferation rates, similar to cooling effects.
- OMN helps maintain hormone dynamics and mammary gland turnover during heat stress.

## Abstract

Heat stress during the dry period reduces immune function, mammary growth, and subsequent milk yield. Mammary involution and regrowth are linked with immune function. Therefore, supplementation with an immunomodulator during involution may reverse some negative effects of heat stress on mammary development. Our previous study demonstrated that supplementing OmniGen-AF® (OMN) before, during, and after the dry period to heat-stressed dairy cows lowers respiration rate and rectal temperature and improves milk yield in the subsequent lactation. In this study, OMN supplementation during the dry period produced effects on mammary gene expression, regrowth, and hormonal shifts that were comparable to those observed with the cooling of heat-stressed dairy cows.

Nutritional and cooling strategies to abate the negative effects of heat stress during the dry period have been used to improve the performance of dairy cattle. The objective of this study was to evaluate the effects of feeding an immunomodulatory supplement (OmniGen-AF®, OMN) before, during, and after exposure to either heat stress or active cooling during the dry period on immune function and mammary development in dairy cows. During late lactation (at least 60 d before dry off), cows were provided with evaporative cooling systems (shade, fans, and soakers) and assigned to two groups: placebo (56 g/d of AB20® top-dressed; CON) or OmniGen-AF® (OMN, 56 g/d top-dressed). Cows were dried off ~46 d before the expected calving date and further split into evaporative cooling (shade, fans, and soakers; CL) or heat stress (only shade; HT) pens. Thus, after dry off, there were four treatment groups: heat stress with placebo (HT, n = 17), HT with OMN supplementation (HT + OMN, n = 19), CL with placebo (CL, n = 16), and CL with OMN supplementation (CL + OMN, n = 11). From a subset of cows (n = 6–8 per group), four blood samples were collected during the dry period (−43, −39, −32, and −21 d relative to calving) to evaluate neutrophil function and blood hematology. In addition, mammary biopsies (4–6 cows/treatment) were collected at −43, −39, −32, and −21 d relative to calving to evaluate mammary gland gene expression and histology, i.e., Tdt dUTP nick-end labeling (TUNEL) and Ki67. Genes related to autophagy, apoptosis, and cell proliferation were analyzed by qRT-PCR. Relative to CL, HT downregulated the expression of beclin-2 (BECN2) but upregulated the expression of beclin-1 (BECN1) on days −43 and −39 relative to calving, respectively. Also, relative to CL, HT upregulated the expression of BAX and FAS on day −39 relative to calving. These differences in gene expression were followed by HT cows having a lower total cell apoptosis rate during involution relative to CL cows. Further to these effects, HT leads to a lower alveoli number relative to CL cows. As in the CL treatment, OMN cows have a higher total cell apoptosis rate and alveoli number relative to CON cows. In addition, OMN cows have higher total cell proliferation relative to CON. Prolactin (PRL) and cortisol concentrations were evaluated during the dry period at days −45, −26, −3, and −1 relative to calving. Relative to CL, HT cows had higher PRL at day −45 but lower PRL on day −1 relative to calving, and a similar trend was observed for cortisol concentrations. In summary, HT impacts mammary gland gene expression, compromises mammary involution, reduces alveoli number, and alters hormone dynamics throughout the dry period. Following the same trends as the CL treatment, OMN increases mammary gland turnover by having a higher cell apoptosis and cell proliferation rate and lower connective tissue relative to CON cows.