Use of water turnover method to measure mother’s milk flow in a rat model: Application to dams receiving a low protein diet during gestation and lactation

Abstract

Assessment of milk production is of utmost relevance for pediatricians and scientists interested in early life nutrition. The weight-suckle-weight (WSW) method, which consists of weighing babies before and after they suckle their mother, uses the difference in body weight as an estimate of milk intake. However, this is prone to many sources of error. In the current study, we used for the first time the water turnover method and compartmental analysis with deuterated water (D2O) as a non-toxic tracer to quantify in vivo milk production in a rat model. We assessed the effect of a nutritional intervention presumed to affect milk production, a maternal dietary protein restriction during gestation and lactation, which results in the birth of pups with intrauterine growth restriction. The specific aim of this study was to determine milk production with the body water turnover method in rat dams receiving during gestation and lactation, either a control diet (NP) or an iso-caloric low-protein diet (LP). In NP dams, mass of dam’s total body water, output flow constant from dam to litter (K21) and median milk flow, calculated between days 11 to 14 after pup birth, were 282.1 g, 0.0122 h-1 and 3.30 g/h for NP dams, respectively. Maternal dietary protein restriction (-59%) during perinatal period led to a 34% reduction in milk flow (NP versus LP). With the WSW method, milk flow varied from 1.96 g/h to 2.37 g/h between days 11 to 14 for NP dams. The main advantage of the D20 method compared to the WSW method stems from its higher precision, as attested by the narrowest range of measured values of milk flow ([2.90; 3.75] and [0.98; 6.85] g/h, respectively) for NP group. This method could be suitable for testing the effectiveness of candidate galactologue molecules presumed to enhance milk production in the lactating rat model.

Fenugreek Stimulates the Expression of Genes Involved in Milk Synthesis and Milk Flow through Modulation of Insulin/GH/IGF-1 Axis and Oxytocin Secretion

“…In Xp1 and Xp2, milk flow between dams and their pups was recorded between L11 and L18 using the deuterated water enrichment method and milk sample was collected at L18. In the Xp1, as previously described [ 14 ], pups were weaned at L20 to start the involution phase for dams’ mammary glands. At post-natal day 21, also assimilated to first involution day (Inv1), dams were fasted for 4 h before sacrifice and sampling of plasma and tissues.…”

“…Milk flow was measured by the water turnover method as previously described [ 14 , 15 ]. Briefly, at L11, after 4%, isoflurane anesthesia, mothers received an injection of 4.92 ± 0.10 g/kg body weight D 2 O (99.9 mole% D 2 -enrichment, Sigma-Aldrich, Lyon, France) into a tail vein.…”

“…The D 2 O enrichment of both plasma and urine samples was measured using the Fourier Transform infrared spectrophotometer α II ® (Brucker, Rheinstetten, Germany). Milk flow calculation was performed using a bi-compartmental model [ 15 ] as no significant intra-litter sexual dimorphism was observed in previous experiments [ 14 ]. Flow constants of the model (h −1 ) were then determined with the SAAM II ® software (The Epsilon Group, Charlottesville, Virginia, US) and the dams’ total body water (g) using the intercept of dams D 2 O concentration curve.…”

“…G, L, PND and Inv correspond to the day of gestation, lactation, post-natal and involution days, respectively. Milk flow was measured by the deuterated water enrichment method [ 14 , 15 ] between L11 and L18. Tissues sampled were mammary and pituitary glands.…”

“…Fenugreek is the most consumed, accounting for 50% of the plant extracts taken during breastfeeding [ 11 ], although strong evidence of its effectiveness and safety is scarce [ 11 , 13 ]. In a recent study using the deuterium oxide enrichment method to accurately measure milk secretion in a model of rat nursing large litters [ 14 ], we found that dietary fenugreek at a dose of 1 g/kg body weight/day increased milk production by 16% and offspring growth by 11% without any evidence for adverse metabolic effect on either dams or offspring [ 15 ], demonstrating a promising potential of fenugreek in the treatment of breastfeeding deficiency. However, the mechanisms underlying its effect remain poorly understood [ 16 ] and deciphering the targets of fenugreek in the lactation process should allow initiating careful support to mothers with lactation difficulties.…”