This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.
Deficiencies of folic acid and vitamin B12 are associated with high reproductive risks ranging from infertility to fetal structural defects. The current study examines the effect of preconceptional omega 3 fatty acid supplementation (EPA +DHA) to a micronutrient deficient diet on reproductive cycles in Wistar rats. Female rats were divided into 5 groups, 1) control 2) folic acid deficient (FD) 3) vitamin B12 deficient (BD) 4) folic acid deficient + omega 3 fatty acid (FDO) and 5) vitamin B12 deficient + omega 3 fatty acid (BDO) from birth and throughout pregnancy. Dams were dissected at gestation day 20. Maternal micronutrient deficiency (both FD and BD) leads to abnormal estrous cyclicity (p<0.001) while omega 3 fatty acid supplementation restored the estrous cycle to normal. The number of corpora lutea was lower in ovaries of rats fed a FD diet. There was an absence of lactating ducts in the mammary glands of rats fed both micronutrient deficient diets. Our findings indicate for the first time that maternal micronutrient deficiency affects the estrous cycle and morphology of the ovary and mammary gland. Omega 3 fatty acid supplementation ameliorated above effects. This may have implications for infertility and pregnancy outcome.
Nutrition during the preconceptional period affects the development of the oocytes, embryo and plays a crucial role in the onset and development of pregnancy [Ashworth et al. 2009, Cetin et al. 2010]. Micronutrients like folate and vitamin B12 play an important role in the maturation and quality of the oocyte and have a major impact on fertility [Ebisch et al. 2007, Reznikoff-Etiévant et al. 2002, Ronnenberg et al. 2007]. Ovulatory disturbances are known to result in polycystic ovary syndrome (PCOS) and infertility [Teede et al. 2010]. In PCOS patients, lower vitamin B12 and higher homocysteine concentrations in follicular fluid were observed to be associated with poor oocyte and embryo qualities [Berker et al. 2009]. However, no studies have examined the effect of micronutrient deficiency on the estrous cycle.
Folate and vitamin B12 are the major determinants of one carbon metabolism. Folate functions as a co-enzyme in single-carbon transfers in the metabolism of amino acids and nucleic acids and is needed for homocysteine re-methylation to methionine [Reviewed by Cetin et al. 2010]. Vitamin B12 is required as a co-factor for the transfer of methyl group from 5-methyl tetra hydro folate (MTHF) to homocysteine and results in the synthesis of methionine. Methionine is converted into S-adenosyl-L-methionine (SAM) which acts as a universal methyl donor. Methyl groups from SAM are transferred by phosphatidyl ethanolamine-N-methyltransferase (PEMT) to ethanolamine-Docosahexaenoic acid (DHA) in a series of steps that convert it to phosphatidylcholine-DHA and produce homocysteine [Umhau et al 2005]. Thus, any alteration in the key constituents of one-carbon cycle will result into hyperhomocysteinemia and has been extensively discussed by us earlier [Kale et al 2010, Kulkarni et al 2011a].
Animal studies carried out by us and others indicate that micronutrients like folate and vitamin B12 influence the long chain polyunsaturated fatty acid (LCPUFA) metabolism [Pita & Delgado 2000, Rao et al. 2006, Wadhwani et al. 2012, Sable et al. 2012, Roy et al. 2011]. A series of our earlier studies have well established the role of omega 3 fatty acids in infertility and pregnancy [Mehendale et al. 2007, Mehendale et al. 2009, Kilari et al 2009, Kilari et al 2010, Kilari et al 2011, Dhobale et al 2011]. We have also provided evidence for the associations of maternal omega-3 fatty acids especially DHA and resultant homocysteine concentrations in women with preeclampsia [Kulkarni et al. 2011a]. We have recently reported in animals that an imbalance in maternal micronutrients like folic acid and vitamin B12 reduces breast milk volume [Dangat et al. 2011] suggestive of possible changes in the morphology of the mammary gland. Further, studies in animals have adequately demonstrated the beneficial effects of maternal omega 3 fatty acids to a micronutrient imbalanced diet on placental global methylation levels and brain neurotrophins [Kulkarni et al. 2011b, Sable et al. 2012].
The current study for the first time examines the effect of deficiency of micronutrients like vitamin B12 and folic acid during the period leading up conception as well as during pregnancy on number and phases of the estrous cycle, morphology of the mammary gland and ovaries in rats. Furthermore, the current study also aims to understand whether omega 3 fatty acid supplementation ameliorates the above effects.
2. Materials and Methods:
All experimental procedures were in accordance with guidelines of Institutional Animal Ethics Committee (IAEC) (2/2011/CPCSEA). The institute is recognized to undertake experiments on animals as per the committee for the purpose of control and supervision of experimental animals, Government of India. All assessments were carried out by personnel blind to the experimental conditions.
Wistar albino rats (15 Females, 10 Males) of average weight 45gms were obtained from animal house of National Toxicology Centre, Pune, India. In the F1 generation all the progeny were culled to maintain a litter size of 8 pups per dam. This helps in reducing the litter size-induced variability in the growth and development of pups during the postnatal period and increases the sensitivity to detect treatment related effects [Agnish and Keller 1997]. Hence instead of using the F0 generation directly (F0) for the experimental protocol it was thought appropriate to use their progeny i.e. F1 generation. Fig 1 shows the study design. All the rats were maintained at 22°C on a controlled 12-hr light and 12-hr dark cycle with appropriate ventilation system. Rats were marked with picric acid as Head (H), Back (B), Tail (T) etc. for identification.
These young adults (F0) were then put for breeding after attaining weight of 200gms. Males were housed individually prior to mating to acquire cage dominance. Virgin female rats were allowed to breed (sex ratio 1:3). On the following morning the vaginal smears were taken to confirm mating. Vaginal smears were taken on a clean microscope slide using a cotton bud dipped in saline. The slides were examined under a microscope at 10X magnification. The sperm positive smear was considered a result of successful mating and considered day 0 of gestation. The pregnant dams were housed individually (in polypropylene cages of 29x22x14 cm dimensions containing rice husk as bedding material) and allowed to deliver normally. Female pups born were separated on day 21 and were included in the study while male pups were not a part of the study. The female pups (F1) were distributed randomly in 5 different groups (n=8 for each group).
The composition of the control and the treatment diets (Table 1) was as per AIN 93-G purified diets for laboratory rodents [Reeves et al. 1993]. Protein level in the control and treatment diets was 18%. Five isocaloric treatment diets were formulated. Two diets were formulated to examine the effect of folic acid deficiency and the effect of vitamin B12 deficiency. In addition, 2 more diets were formulated to examine the effects of omega 3 fatty acid (DHA+EPA) supplementation on both the micronutrient deficient groups. The supplementation of omega 3 fatty acids was from Maxepa (fish oil, Merck)) and contained a combination of DHA (120mg) and eicosapentaenoic acid (EPA) (180mg).
Folic acid deficiency and vitamin B12 deficiency was obtained exclusively through dietary means. Vitamin free casein was used for all treatment diets. Rats receiving vitamin B12 and folate deficient diets were kept in special cages to prevent coprophagy. According to the guidelines of AIN-93G, there are recommendations made for both folic acid and vitamin B12. Thus, the diet for the deficiency group was made by omitting these vitamins from the vitamin mixture. Accordingly, the amount of these vitamins was made up with sucrose. Diet was prepared on alternate days in the laboratory and was stored in the refrigerator. It was given fresh to rats every day. Also, the different vitamin mixtures and the omega 3 fatty acids used for supplementation were stored at 4oC. All diets contained tertiarybutylhydroquinone (TBHQ) to prevent oxidation [Fritsche and Johnston 1988, Gonzalez et al 1992, Reeves et al. 1993]. The ingredients were weighed on a Schimadzu electronic balance with least count 0.001g, mixed thoroughly and moulded into cylindrical pellets. To ensure that there was no loss of vitamins at high temperature the diets were dried in an oven at 50oC. All the rats had free access to food and water.
Thus there were a total of 5 groups Control - Normal folate, normal vitamin B12; FD - normal vitamin B12, folate deficient; BD - normal folate, vitamin B12 deficient; FDO - folate deficient + omega 3 fatty acid supplementation and BDO - vitamin B12 deficient + omega 3 fatty acid supplementation.
2.3. Estrous Cycle Monitoring:
The young female adults (F1) were monitored for cyclicity for 15 days prior to breeding (i.e. approximately 8 weeks of age) by a method described by [Marcondes et. al., 2002]. Vaginal smear was collected every morning between 8.00 am - 9.00 am, with a pipette filled with 20µl of normal saline (NaCl 0.8%). The vaginal fluid was placed on clean glass slide. The unstained slide was observed under a light microscope at 10X and 40X magnifications. The three types of cells recognized were epithelial, cornified and leucocytes cells. The proportions among these cells were used to determine the estrous phases as described earlier [Long and Evans 1922].
2.4. Breeding and Sacrifice:
The young female adults (F1) after attaining the weight of 200gms i.e. approximately 10 weeks age were further mated with males and pregnancy was confirmed by method described above. Time taken for mating was different for females in different groups. In general, females of control, BDO and FDO groups mated within one week. However, females of BD and FD group took longer time (i.e. approximately 10 days) for mating. All dams were delivered by Caesarean section on d20 of gestation (GD20). Blood was collected in EDTA containing tubes and plasma was separated. Plasma samples of dams were stored at -80oC until further use. Organs like ovaries and mammary gland were removed and snap frozen in liquid nitrogen and stored at -80oC until further use.
2.5. Observations recorded
Daily dietary intake was recorded during pre-pregnancy and pregnancy. During pre-pregnancy period, weekly weights were recorded. During pregnancy, dam weights were recorded at 0, 7, 14 & 20 d of gestation to obtain weight gains. On d20 of gestation the litter weight, litter size and pup weight was recorded in each group.
2.6. Folate and Vitamin B12 estimations:
Folate and vitamin B12 levels were estimated from dam plasma on GD20. Folate and vitamin B12 were estimated by the Chemiluminiscent Microparticle Immunoassay (CMIA) technology (Abbott Diagnostics, Abbott Park, Illinois, USA). Briefly, 100 µl of plasma was used for analysis of folate and vitamin B12. The folate and vitamin B12 assay was a two-step assay with an automated sample pre-treatment, for determining the presence of vitamin B12 and folate in rat plasma. The detection limits for plasma vitamin B12 assay was 187 - 883 pg/mL while for plasma folate assay it was 2.34 - 17.56 ng/mL.
2.7. Hormonal Levels:
Hormonal assessments were made from the plasma of dam which was dissected by caesarean section on GD20. Plasma estrogen levels were determined by a commercially available kit (Estradiol, Abbott AXSYM systems, Abbott Park, USA). The quantity of plasma used for estradiol analysis was 194µL. Plasma progesterone levels were determined by a commercially available kit (Progesterone, Abbott AXSYM systems, Abbott Park, USA) and the quantity of plasma used for progesterone analysis was 100µL. Both the assay procedures were based on Microparticle Enzyme Immunoassay (MEIA) technology.
2.8. Histology of mammary gland and ovary:
Assessments of mammary glands were conducted on the dams dissected on GD20. Dissected ovaries and mammary glands of the dams (n=3/group) were fixed in 10% formalin. Tissues were processed for routine paraffin embedding and cut at 4µ size for haematoxylene eosin staining. The slides were analysed by an experienced histopathologist on Leica microscope (Leica Microsystems Wetzlae GmbH, Type 020-519.011DMLB 100S with Watec (WAT202B) camera attachment). Images from each case were grabbed with the help of above microscope and Intel Pentium III Cerelon computer and image grabber software called ASUS P3B-F. Images were captured on 10X magnification and 20X magnification. The software, Image Drafter (developed using "NET framework 2.0") was used for morphometric analysis to attain accuracy, repeatability and standardization. Randomly 3 tissues were analysed for morphology of mammary gland and ovary from all the groups and the evaluations made were qualitative.
2.9. Statistical methods
Means of 8 dams per group were used as the unit of analysis. Values are mean ± SD. The data was analysed using SPSS/PC+ package (Version 11.0, Chicago IL). Mean values of the estimates of various parameters for the treatment groups were compared with those of control group at conventional levels of significance (p<0.05 or p<0.01), using least significance difference estimated from one way analysis of variance (ANOVA).
Feed intakes of rats were recorded during pre-pregnancy and pregnancy period. Animals in the BDO group had higher feed intake as compared to the control group (Table 2). The feed conversion ratios for the different groups were as follows: Control (0.142), FD (0.142), BD (0.137), FDO (0.118) and BDO (0.163).
3.2. Estrous cycle:
The number of estrous cycles observed during the 15 days period prior to breeding was significantly reduced in both FD groups (1.40 ± 0.55) (p<0.001) and BD (0.25 ± 0.50) (p<0.001) as compared to control group (2.80 ± 0.45). Supplementation of omega 3 fatty acid to these groups improved the number of estrous cycles. Number of estrous cycles was increased in FDO group (2.20 ± 0.48) (p<0.05) as compared with FD group and in BDO group (2.83 ± 0.41) (p<0.001) as compared with BD group (Fig 2). . An alteration in the stages of estrous cycle was also observed in BD and FD groups as compared to control while supplementation of omega 3 fatty acid showed normal estrous cycle (Fig 3). The figure is intended to be representative of 8 animals per group.
3.3. Reproductive performance:
Total weight gain of dams during pregnancy was lower BD group. However, the difference observed was not statistically significant. Weight gain of dams in FDO group (136.25 ± 16.50g) was significantly increased as compared to FD (109.50 ± 14.63g) (p<0.01) and control group (106.50 ± 19.97g) (p<0.01). Weight gain of BD (105.50 ± 12.27g) and BDO group (114.87 ± 30.24g) was comparable to control. No difference was observed in the litter size in different groups. Litter weight of FD group (33.53 ± 10.35g) was comparable to control. Litter weight was significantly lowered in BD group (33.29 ± 4.10g) as compared to control (42.93 ± 12.90g) (p<0.05). Omega 3 fatty acid supplementation in folate deficient i.e. FDO group (43.00 ± 5.93g) as well as vitamin B12 deficient i.e. BDO group (40.21 ± 10.58g) improved the litter weights however they were not statistically significant.
3.4. Weights of ovary and mammary gland:
There was no difference observed in the weight of mammary glands of dams between different groups. Weight of the left ovary in the FD group (0.06 ± 0.01g) and BD group (0.06 ± 0.01g) was similar to that of the control (0.07 ± 0.02g). Omega 3 fatty acid supplementation to these deficient groups i.e. in FDO group (0.1 ± 0.02g) increased the weight of left ovary as compared to FD group (p<0.001) as well as compared to control (p<0.01). Also in BDO group (0.08 ± 0.02g), weight of left ovary was increased as compared to BD group (p<0.05). No difference was observed in the weight of right ovary between different groups.
3.5. Estimations of Folate and Vitamin B12:
Dam plasma folic acid and vitamin B12 levels were estimated on GD20. Levels of plasma folate were decreased in FD (6.68 ± 7.73 ng/ml) (p<0.001), BD (16.13 ± 3.26 ng/mL) (p<0.05) and FDO group (3.15 ± 1.07 ng/mL) (p<0.001) as compared to control group (26.00 ± 14.48 ng/mL). However, the levels of folic acid in BDO group (19.65 ± 2.60 ng/mL) were lower than that of control group, but were not statistically significant. Levels of plasma vitamin B12 were lower in the FD group (228.50 ± 82.28 pg/mL) (p<0.05) and FDO group (220.87 ± 19.39 pg/mL) (p<0.001) as compared to control (287.62 ± 56.32 pg/mL). It was below the detection limits in both the BD group and BDO group i.e. <83pg/mL. Thus, the deficiencies of both folic acid and vitamin B12 were reflected in the plasma levels of dam.
3.6. Hormonal Analysis:
Plasma progesterone levels were higher in the FD (83.15 ± 14.50 ng/mL) (p<0.001) and BD groups (71.07 ± 20.05 ng/mL) (p<0.01) as compared to control group (49.53 ± 26.87 ng/mL). Omega 3 fatty acid supplementation to the folic acid deficient diet i.e. in FDO group (58.71 ± 13.00 ng/mL) decreased the level of progesterone significantly as compared to FD group (p<0.01). In case of BDO group (67.77 ± 9.21 ng/mL) the levels of progesterone were similar to that of the BD group. Plasma estradiol levels were higher in the FD (14.50 ± 9.61 pg/mL) and BD groups (17.50 ± 10.41 pg/mL) as compared to control group (9.75 ± 9.59 pg/mL). Lowering of estradiol levels were observed in FDO (5.63 ± 1.77 pg/mL) (p<0.05) as compared to FD group. Lowering of estradiol levels was not significant in case of BDO group (10.00 ± 8.02 pg/mL) as compared to the BD group.
3.7. Histological analysis of Mammary gland:
The evaluations made for the morphology of mammary gland were qualitative in nature. Mammary gland of a control rat showed presence of acini, forming lactating ducts (Fig 4a). These acini were absent in mammary gland of both FD and BD group rats (Fig 4b, Fig 4c). Thus, there was absence of lactating ducts in these groups. Partial increase in the number of acini was observed in FDO and BDO groups (Fig 4d, Fig 4e).
3.8. Histological analysis of Ovary:
In case of ovaries, control rat showed presence of healthy ovary with primordial follicles, corpus luteum and Graafian follicles (Fig 5a) and the data is qualitative in nature. The number of corpora lutea was decreased in FD group while it was normal in BD group (Fig 5b, Fig5c). In both FDO and BDO groups, the number of corpora lutea was increased even more than control (Fig 5d, Fig 5e).
To our knowledge, this is the first report which has examined the effect of folic acid and vitamin B12 deficiency on the estrous cycle, levels of hormones like estradiol and progesterone and morphology of ovaries and mammary gland. The current study also examined the role of omega 3 fatty acid supplementation in these deficient diets. Our results indicate 1) Folic acid and vitamin B12 deficiency a) reduced the number of estrous cycles and altered the phases of the estrous cycle b) resulted in abnormal hormonal levels c) altered the morphology of mammary gland in vitamin B12 deficient animals while folic acid deficiency altered the morphology of both mammary gland and ovary 2) Omega 3 fatty acid supplementation ameliorated the above effects caused as a result of folic acid and vitamin B12 deficiency.
Irregularity of estrous cycle is characterized by being in the same phase during 4-5 days. Cycles, in which the alternation among the phases don't follow the sequence of being in the proestrus, estrous, metestrus and diestrus (or intermediates), stages are considered irregular [Marcondes et al. 2002]. In the vitamin B12 deficient group, proestrus stage was observed on the vaginal smear for 4 days. Similar effects of food restriction have been reported to alter the estrous cycle [Tropp and Markus 2001]. Early studies by Guilbert et al. reported that protein restriction causes the cessation of estrous or results in long and irregular estrous cycle [Guilbert and Goss 1932]. There are reports of energy restriction in addition to intense physical training altering the estrous cycle [Santos et al. 2011]. Also, irregular menstrual cycles are observed when rhesus monkeys consume a folate-restricted diet [Mohanty and Das 1982]. In humans a more prolonged vitamin B12 deficiency has been reported to result in infertility by possibly causing changes in ovulation or development of the ovum or changes leading to defective implantation [Bennett 2001].
The current study for the first time reports that the number and phases of the estrous cycle in rats can be reduced and altered as a result of deficiency of both folic acid and vitamin B12. A decrease in reproductive cycle has been reported to reduce the ovulatory period and eventually may lead to the reduction of fertility [Nah et al. 2011]. Thus, negative influences on the estrous cycle are suggestive of negative influences on the reproductive health of animals [Oluyemi et al. 2007]. The data showing poor reproductive performance in terms of reduced litter weight in BD group could be attributed to disturbed estrous cycle in that group.
Omega 3 fatty acid supplementation to these vitamin deficiency groups restored the estrous cycle comparable to that of a control rat. It may be possible that this effect could be attributed to eicosapentanoic acid, an omega 3 fatty acid. This fatty acid is a precursor for eicosanoids like prostaglandins, thromboxanes, leukotrienes and hydroxy-fatty acids which are important regulators of reproductive processes [Sales & Jabbour 2003]. Prostaglandin injections given to cows have been reported to synchronize the estrous cycle by regressing the corpus luteum [Floyd and Gimenez 1997]. Also, omega 3 fatty acid supplementation has been reported to improve the reproductive function and fertility in animals [Burke et al. 1996, Staples et al. 1998].
We have reported both in humans and animals that folate, vitamin B12 and omega 3 fatty acid are interlinked in the one carbon cycle. In fact we have extensively demonstrated that the adverse effects of vitamin B12 deficiency in terms of reduced brain fatty acids, increased oxidative stress, reduced gastric milk fatty acids and reduced placental global methylation levels can all be ameliorated by omega 3 fatty acid supplementation [Dangat et al. 2011, Kulkarni et al. 2011b, Roy et al. 2012, Wadhwani et al. 2012]. Our data additionally indicates the beneficial effects of omega 3 fatty acid in improving the estrous cycle in the absence of maternal micronutrients like folic acid and vitamin B12.
During pregnancy, DHA and arachidonic acid (AA) cross the placenta to the fetus while postnatally, these fatty acids are supplied through breast milk [Crawford 2000]. Mammary gland is the organ developed to deliver essential nutrients in the form of rich proteinaceous and lipid fluid termed milk to the new born offspring. The initial formation of the mammary bud and primitive mammary epithelial tree occurs during fetal life. The development of these structures appears dependent upon the estrogenic environment of pregnancy [Anbazhagan et al. 1991]. In the mammary gland the major development during puberty is the formation of ducts that will eventually convey milk to the breast's nipple [Prall et al. 1998]. With each estrous cycle, cyclic proliferation and involution occurs as a result of which there is side branching of the ducts and development of alveoli[Reviewed by Brisken and O'Maley, 2010].
We have previously reported that maternal micronutrients like folate and vitamin B12 play a key role in determining both the quantity and quality of milk [Dangat et al. 2011]. Breast development is a multistep process characterized by complex mesenchymal-epithelia interactions. In the current study the absence of lactating ducts observed in the morphology of mammary gland indicates that vitamin B12 deficiency may result in altered breast development. This alteration in the morphology may possibly contribute to reduced litter weight of the pups.
In the current study there was an alteration in the structure of the ovary as a result of folic acid deficiency. The ovarian follicle plays an important role in the maturation and release of a fertilizable oocyte, promotes and maintains implantation of the embryo [Channing et al. 1978]. The corpus luteum plays a central role in the regulation of the estrous cycle and in the maintenance of pregnancy which is carried out largely by progesterone [Stocco et al. 2007]. Our data shows reduced number of corpora lutea and increased progesterone levels. These increased levels of progesterone are consistent with the findings reported by Kechrid et al., 2006 wherein they have shown increase in the levels of progesterone in a micronutrient i.e. zinc deficient diet in pregnancy [Kechrid et al. 2006]. Omega 3 fatty acid supplementation to the folic acid deficient diet increased the number of corpora lutea. Fish meal supplementation has been reported to increase the luteal omega 3 fatty acid content and reduces available arachidonic acid content, the precursor for prostaglandinF2Î± resulting in increased fertility in cattle [White et al. 2012].
Some limitations of the current study are that the differences in the vaginal opening/sexual maturity, evaluations on the degree of fertility and ductal branching were not undertaken. Also, the quantitative analysis regarding morphology of mammary glands and ovaries was not undertaken. The current study is more of phenomena based; and further studies are ongoing in the lab to understand the mechanistic aspects. Future studies are also planned to undertake quantitative methods for morphology.
To summarize, current study for the first time demonstrate that maternal vitamin B12 and folate deficiency alter the estrous cycle. Supplementation with omega 3 fatty acid to these deficient diets restored the number and phases of the estrous cycle. These findings are of significance since the nutritional intake of childbearing-age women has been suggested to be inadequate during the preconceptional period in terms of nutrition [deWeerd et al. 2003, Mouratidou et al. 2006], especially micronutrients [Ronnenberg et al. 2007]. Therefore, supplementation with omega 3 fatty acids along with micronutrients may help in regularising the menstrual cycle ultimately leading to improved fertility. However there is need for future studies to examine the molecular and epigenetic changes as a consequence of omega 3 fatty acid supplementation to these micronutrient deficient diets.
Acknowledgments: We acknowledge the help of Mr. Ravindra Mulik who took care of the animals.
Funding: Author, AM received 'INSPIRE fellowship' from 'Department of Science and Technology', Government of India.
Conflict of Interest: There is no conflict of interest to declare.