Results of this study demonstrate a direct relationship between feeding regimen,
biofluids, oocyte and embryo lipid composition and oocyte developmental potential in
female cattle. Diet supplementation of heifers with rumen-protected fatty acids
affected the lipid composition of biofluids (blood and oocyte microenviroment), SCAP
expression and the lipid profile in immature and in vitro matured oocytes, suggesting
a direct link between lipid metabolism and reproduction which could provide new clues for treatment of obesity related infertility in humans. Human and animal studies have shown that all stages of oocyte maturation and preimplantation embryo development can be affected by parental nutrition [48-54].
Diverse analytical strategies have been used in this work. The fatty acids profiles in the circulation and in follicular fluid were determined by GC and the most prominent differences were found only after feeding the high concentration of CLA and SA (200 g/d). However, differences in the lipid profiles were already observed in oocytes collected by OPU from heifers supplemented with 100 g/d of rumen-protected concentrates with the more sensitive DESI-MS. This demonstrates that female gametes have a dynamic metabolism able to respond even to minor changes in nutrition and points towards detrimental effects of overfeeding on fertility. To what extent the altered lipid profile is reverted with a normal diet needs further study. The trans10,cis12-CLA concentration in milk fat has been previously reported to be increased after 100 g/d of rumen-protected CLA supplementation of lactating German Holstein cows [55], showing clear effects of fatty acid diet supplementation on milk fatty acid profile.
In vitro matured oocytes collected from heifers supplemented with 100 g/d SA
displayed high concentrations of plasmalogens (ions of m/z 738 and m/z 752) as
determined by DESI-MS. Plasmalogens constitute ~18 % of the total phospholipids in
humans and belong to a class of phospholipids characterized by a vinyl ether bond in
sn-1 and an ester bond in sn-2 position of the glycerol backbone [56]. An abnormal
accumulation of plasmalogens was originally found to be associated with
neurodegeneration, but recent studies have discovered a direct relationship between
high concentrations of plasmalogens and common diseases like cardiac failure, type
II diabetes, obesity, inflammation and cancer [57]. Furthermore, the higher
abundance of the unsaturated TAG (52:3) in CLA-fed animals compared to the
higher abundance of TAG (48:1) and (50:2) in SA-derived immature oocytes
indicates that CLA was incorporated in the TAG content of immature oocytes. The
negative ion mode DESI-MS analysis revealed a higher abundance of palmitic acid
(C16:0) in in vitro matured oocytes collected from heifers after 100 g/d of SA in
comparison to the CLA supplemented counterparts. Aberrant accumulation of
saturated FFA, specifically palmitic and stearic acid, has been recently observed when single-embryo DESI-MS lipid profiling was applied to bovine in vitro produced blastocysts in comparison with their in vivo produced counterparts [18]. Recent findings with regard to the cellular mechanism underlying the impaired oocyte development have identified endoplasmic reticulum stress response-induced lipotoxicity as a critical factor [58]. Indeed, abnormal intracellular accumulation of triglycerides and FFA causes damage of the membranes of the mitochondria, endoplasmic reticulum and other organelles, resulting in accumulation of intracellular reactive oxygen species and the misfolding of endoplasmic reticulum protein, followed by protein degradation, caspase activation and activation of apoptosis [59, 60].
Along this line, another intriguing finding with regard to the effects of feeding on fertility was that stearic acid supplementation obviously had a detrimental effect on the capacity of oocytes to reach the blastocyst stage in a conventional in vitro embryo production system. Blastocyst development of fertilized oocytes collected from heifers supplemented with 100 g/d of the rumen-protected fatty acids was different between CLA (22.4 %) and SA (13.8 %) groups.
Gene expression analysis revealed significant down-regulation of SCAP, which encodes a key chaperone protein within the intracellular sterol biosynthesis pathway.
This SCAP down-regulation was observed irrespective of CLA or SA
supplementation in in vitro matured oocytes compared to their in vivo matured
counterparts. The fact that mRNA expression levels of IGF1R, GJA1, FASN and
SREBP1 were similar in in vitro matured oocytes derived from CLA and SA
supplemented donors and in vivo matured oocytes from non-supplemented animals
is intriguing. This suggests that the effects of fatty acid supplementation on oocyte
developmental capacity are not associated with changes at the transcriptional level of
genes thought to be critically involved in embryo development. Accordingly, no
differences in the gene expression pattern were observed in immature oocytes
collected from fatty acid supplemented and non-supplemented heifers. On the other
hand, we recently found a significant up-regulation of transcripts encoding cholesterol
element binding protein 1 (SREBP1) in day-8 in vitro produced blastocysts in
comparison to in vivo produced blastocysts [18]. We attribute this up-regulation to effects of the in vitro embryo culture system on intracellular lipid metabolism.
The experimental animals in this study were non-lactating heifers, and the supplementary diets (CLA or SA) were calculated on top of an isocaloric mixture that allowed us to change the composition of the diet (i.e., rumen-protected fatty supplements) without changing the energy content of the diet. The higher cholesterol plasma concentrations found ten weeks later after onset of feeding 200 g/d of rumen-protected CLA is thought to be a specific effect of the high dose of rumen-rumen-protected CLA on the energy status of supplemented animals.
In conclusion, here we have used a broad array of advanced analytical technologies,
to unravel the effects of specific fatty acid diet supplementation on oocyte
microenvironment, the female gametes, early embryos and in blood using heifers as
physiological model system. Results clearly show dramatic changes in lipid profiles in
these different physiological compartments (i.e., blood system, follicular fluid,
“Intra-oocyte”) induced by the diet supplement. Furthermore, the developmental capacity of
oocytes collected from supplemented heifers differed according to the type of
supplement. This experimental model could contribute to the further elucidation of
infertility related to metabolic disorders in cattle and at the same time supports the
use of the bovine species as experimental model for understanding metabolic-related
fertility disorders in humans. This is particularly intriguing in view of the observed
long-term effects of parental feeding during around conception and during early
embryonic development that become apparent in the adult life [58]
