The mammary fat pad (MFP) contributes to the successful growth and maturation of the mammary epithelium, but the elements that differentiate this tissue matrix from other adipose depots are poorly defined, especially in the bovine. The bovine MFP is a heterogenous tissue matrix consisting of adipocytes, blood vessels, lymphatics, fibroblasts, and various immune cells. The MFP can respond to key endocrine factors known to stimulate mammary growth and morphogenesis. Geiger et al. (Reference Geiger, Parsons and Akers2016) reported that estradiol administration can increase the MFP mass in 10-week-old dairy calves, while simultaneously increasing the mass of the mammary parenchyma. This complements the fact that approximately a third of adipocytes and fibroblasts in the MFP are positive for estradiol receptor α in growing heifer calves (Meyer et al., Reference Meyer, Capuco, Boisclair and Van Amburgh2006). This coincides with the fact that the mammary stroma, which the MFP would be classified as, is known to produce factors, notably IGF-I, that promote epithelial cell proliferation and affect epithelial growth and morphogenesis (Akers et al., Reference Akers, Ellis and Berry2005).
Fatty acids have been implicated in having a role affecting mammary growth and development in mice (Berryhill et al., Reference Berryhill, Miszewski, Trott, Kraft, Lock and Hovey2017), but this has been less explored in the bovine. McFadden et al. (Reference McFadden, Daniel and Akers1990) noted that feeding a diet enriched in polyunsaturated fatty acids increased mammary parenchyma growth in prepubertal lambs. It is also appreciated that the fatty acid profile of different adipose tissues can be unique (May et al., Reference May, Sturdivant, Lunt, Miller and Smith1993), and that the profile of fatty acid might be associated with the function of the tissue (Palmquist, Reference Palmquist1994). Given the uniqueness of the MFP in responding to known mammary epithelial cell mitogens, we hypothesized that the fatty acid profile of the MFP and the subcutaneous adipose tissue (SCA) would differ and that these adipose depots would be differentially affected by estradiol administration. The objective of this investigation was to delineate the fatty acid profile of the MFP and the SCA tissue and determine if estradiol administration differentially affects the fatty acid profile of these adipose depots.
Materials and methods
Experimental design
The tissues examined were collected from an experiment previously detailed by Hardy et al. (Reference Hardy, Enger, Hanson, Eastridge, Moraes and Enger2021), of which experimental procedures were approved by The Ohio State Institutional Animal Care and Use Committee (Protocol 2019A-00000018). Briefly, 12 prepubertal Holstein heifer calves were purchased from a single commercial dairy at 7 d of age and reared on a common ration for the duration of the trial, which lasted for approximately 11 weeks. Calves were weaned at 8 weeks of age (7 weeks into the trial). At 10 weeks of age (9 weeks into the trial), calves were blocked by BW and randomly assigned to 1 of 3 estradiol treatments. All calves, irrespective of treatment, received a total of 12 treatment injections. Injections were administered once daily during the 12 d immediately preceding euthanasia and tissue collection. Calves assigned to the control treatment (CON; n = 4) received daily injections of only corn oil. Calves assigned to the short-term estradiol treatment (SHORT; n = 4) first received 9 sequential injections of corn oil followed by 3 injections of estradiol. Calves assigned to the long-term estradiol treatment (LONG; n = 4) received daily injections of estradiol throughout the injection administration period. All calves received their final injection precisely 5 h prior to euthanasia and tissue collection; the calves also received their final meal at this time. All calves grew at similar rates as calf BW was not different among treatment groups throughout the trial (P = 0.62) and the average daily gain of CON, SHORT, and LONG calves were 0.80, 0.81, and 0.79 ± 0.04 kg/d, respectively.
Injectable preparation and administration
All treatment injections were administered subcutaneously on alternating sides of the neck. Because estradiol is lipid soluble, it was first dissolved in an absolute ethanol and benzyl benzoate mixture and then combined with corn oil, yielding a final injectable solution of 10% absolute ethanol, 20% benzyl benzoate, and 70% corn oil by volume as previously detailed (Hardy et al., Reference Hardy, Enger, Hanson, Eastridge, Moraes and Enger2021). 17β estradiol (Sigma-Aldrich Co., St. Louis, MO; cat no. E1024) was used as the estradiol source and administered at a dosage of 0.1 mg/kg of BW. Sterile, autoclaved corn oil was administered at a volume comparable to the estradiol injectable.
Tissue collection, preservation, and fatty acid extraction and analysis
Calves were stunned by captive bolt and euthanized via exsanguination. The whole udder was removed and adipose tissue was collected from the center mass of the mammary fat pad (MFP; 0.8 to 1.5 g). Subcutaneous adipose (SCA; 0.8 to 1.5 g) was collected from the rump area as described previously by us (Coleman et al., Reference Coleman, Murphy and Relling2018). All adipose tissues were snap frozen using liquid nitrogen and stored at −80°C until further analysis. The fatty acid composition of the collected adipose tissues was determined using 100 to 150 mg of tissue using the extraction and methylation procedure described by O'Fallon et al. (Reference O'Fallon, Busboom, Nelson and Gaskins2007) with the modifications detailed by Coleman et al. (Reference Coleman, Murphy and Relling2018). Fatty acid methyl esters were separated by gas–liquid chromatography using a CP-SIL88 capillary column (100-m × 0.25-mm × 0.2-μm film thickness; Varian Inc., Palo Alto, CA).
Statistical analysis
The relative abundance of the different fatty acids (percentage, %) were designated as the dependent variables in separate models and analyzed using PROC MIXED in SAS 9.4 (SAS Institute Inc., Cary NC). A model structure like that described by Hardy et al. (Reference Hardy, Enger, Hanson, Eastridge, Moraes and Enger2021) was used to analyze the resulting percentages, which included the fixed effects of estradiol treatment (n = 3), adipose tissue depot (n = 2), and their interaction. Calf BW block, and the interactive effect of BW block with estradiol treatment were specified as random effects. Resulting least squares means were separated using a PROC MIXED macro (Saxton, 1998). Differences were considered significant if P ≤ 0.05. Interactive effects were examined if P ≤ 0.15.
Results and discussion
The relative abundance of the MFP and SCA fatty acids are presented in Table 1. Overall, the MFP contained a greater abundance of saturated fatty acids than SCA tissues (47.8 vs. 44.5% ± 0.7%; P < 0.01), complementing a reduced abundance of mono-unsaturated fatty acids in the MFP than SCA (34.5 vs. 38.6% ± 0.9%; P < 0.01). The relative abundance of poly-unsaturated fatty acids was similar between the two adipose depots (P = 0.48). There was a markedly greater abundance of steric acid (18:0) in the MFP than SCA tissues (P < 0.01). No difference existed in the abundance of C18:2, C9 and T11 between the MFP and SCA tissues (P = 0.99). Conversely, many other 18 carbon fatty acid isoforms differed in their relative abundance between the MFP and SCA. The significance of each of these isoforms remains to be determined, but the observed differences in abundance indicate that there is either preferential deposition, or divergent metabolism, of these fatty acids among the adipose depots. Others have previously demonstrated that dietary inclusion of key conjugated linoleic acids, particularly c18:2 t10 c12, can affect morphogenesis of the mammary epithelium in the mouse when included in the ration at ≥0.5% of the feed intake (Foote et al., Reference Foote, Giesy, Bernal-Santos, Bauman and Boisclair2010; Berryhill et al., Reference Berryhill, Gloviczki, Trott, Aimo, Kraft, Cardiff, Paul, Petrie, Lock and Hovey2012). While it is well appreciated that diet can affect the fatty acid profile of adipose tissues (May et al., Reference May, Sturdivant, Lunt, Miller and Smith1993), the calves in our study received a common ration, and the differences in the relative abundance of fatty acids between the adipose depots indicates that tissue specific attributes are responsible for the resulting fatty acid profiles. Clearly, each adipose depot has specific attributes that are almost unequivocally tied to the function of the tissue.
Table 1. Overall fatty acid profile of tissues collected from subcutaneous adipose (SCA) and the mammary fat pad (MFP) in Holstein heifer calves that received daily estradiol treatment (Trt) injections for 0, 3 d, or 12 d immediately preceding tissue collection
Although only a single individual fatty acid was significantly affected by treatment, a number did show a numerical difference that approached significance. Others appeared to have been affected based on the interactive effect of adipose depot and estradiol treatment demonstrating P ≤ 0.15. These fatty acids are presented and contrasted in Table 2. The extended duration of estradiol administration resulted in a reduced abundance of C18:2 t10 c12 in the SCA relative to MFP tissues of LONG calves (P < 0.05); this effect was absent for CON and SHORT calves (P > 0.05). This result is associated with the fact that LONG calves had significantly more mammary epithelial growth than that of CON and SHORT calves (Hardy et al., Reference Hardy, Enger, Hanson, Eastridge, Moraes and Enger2021). Further, C18:1 t10 was significantly differentially affected (P = 0.01) and C18:1 t11 demonstrated a numerical difference (P = 0.08) by the interaction of estradiol treatment and adipose depot. When these results are taken together it would suggest that C18 fatty acids have important implications for the growth and development of the mammary fat pad, and that C18: t10 c12 appears to have MFP specific functions as its abundance is maintained in the MFP even when other tissue depots experience a reduced abundance of this fatty acid. Future work should investigate and confirm these observations and investigate estradiol's specific effects on fatty acid metabolism in the MFP of the growing dairy heifer.
Table 2. Profile of specific fatty acids that were affected either significantly or by numerical difference by estradiol treatment alone, or by the effect of estradiol treatment and adipose tissue on fatty acid abundance
Adipose tissues were collected from subcutaneous adipose (SCA) and the mammary fat pad (MFP) of Holstein heifer calves that had been administered daily estradiol injections for 0 (CON), 3 d (SHORT), or 12 d (LONG) immediately preceding tissue collection.
A–C Means not sharing the same superscripted letter within a row were significantly different (P ≤ 0.05).
1 Largest observed sem for the resulting least squares means.
In conclusion, the results of this study indicate that the fatty acid profile of the MFP and SCA differ in Holstein heifer calves, and that these adipose depots respond differentially to the potent mammary mitogen estradiol, as indicated by unique changes in fatty acid profile. The present experiment does not describe a cause-and-effect relationship between ‘functional’ fatty acids and degree of parenchymal growth, but instead identifies fatty acid responses that are associated with mammary parenchymal growth and estradiol administration. Accordingly, these fatty acids, and their proceeding and subsequent fatty acid metabolism pathways, may have pertinence for mammary fat pad and parenchymal growth and development. Future studies are needed to explore this notion.
Acknowledgments
This work was supported by a competitive Ohio Agricultural Research and Development Center SEEDS grant awarded to B. D. Enger (grant no. 2019-121) using state and federal funds appropriated to The Ohio State University, College of Food, Agriculture and Environmental Sciences.
